Thirukkumaran OM, Kluba M, Hofkens J, Mizuno H.
Autophosphorylation of EGFR at Y954 facilitated homodimerization and enhanced downstream signals.
Biophys J. 2020; 119(10): 2127-2137. PMCID: PMC7732767Asymmetric dimer formation of epidermal growth factor receptor (EGFR) is crucial for EGF-induced receptor activation. Even though autophosphorylation is important for activation, its role remains elusive in the context of regulating dimers. In this study, employing overlapping time series analysis to raster image correlation spectroscopy (RICS), we observed time-dependent transient dynamics of EGFR dimerization and found EGFR kinase activity to be essential for dimerization. As a result of which, we hypothesized that phosphorylation could influence dimerization. Evaluating this point, we observed that one of the tyrosine residues (Y954) located in the C-terminal lobe of the activator kinase domain was important to potentiate dimerization. Functional imaging to monitor Ca2+ and ERK signals revealed a significant role of Y954 in influencing downstream signaling cascade. Crucial for stabilization of EGFR asymmetric dimer is a "latch" formed between kinase domains of the binding partners. Because Y954 is positioned adjacent to the latch binding region ... [truncated at 150 words]
de Luca G, Galparsoroa DF, Sancataldo G, Leone M, Foderà V, Vetri V.
Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly.
J Colloid Interface Sci. 2020; 574: 229-240.Ensembles of protein aggregates are characterized by a nano- and micro-scale heterogeneity of the species. This diversity translates into a variety of effects that protein aggregates may have in biological systems, both in connection to neurodegenerative diseases and immunogenic risk of protein drug products. Moreover, this naturally occurring variety offers unique opportunities in the field of protein-based biomaterials. In the above-mentioned fields, the isolation and structural analysis of the different amyloid types within the same ensemble remain a priority, still representing a significant experimental challenge. Here we address such complexity in the case of insulin for its relevance as biopharmaceutical and its involvement in insulin-derived amyloidosis. By combining Fourier Transform Infrared Microscopy (micro-FTIR) and fluorescence lifetime imaging microscopy (FLIM) we show the occurrence, within the same ensemble of insulin protein aggregates, of a variable β-structure architecture and content not only dependent on the species analyzed (spherulites or fibrils), but also ... [truncated at 150 words]
Boutant E, Bonzi J, Anton H, Nasim MB, Cathagne R, Réal E, Dujardin D, Carl P, Didier P, Paillart JC, Marquet R, Mély Y, de Rocquigny H, Bernacchi S.
Zinc fingers in HIV-1 Gag precursor are not equivalent for gRNA recruitment at the plasma membrane.
Biophys J. 2020; 119(2): 419-433. PMCID: PMC7376094The human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the ... [truncated at 150 words]
OsamaGarwain, Yerramilli VS, Romero K, Scarlata S.
The Gαq/phospholipase Cβ signaling system represses tau aggregation.
Cell Signal. 2020; 71, 109620. PMCID: PMC7255494Alzheimer's disease is typified by calcium dysfunction and neurofibrillary tangles of tau aggregates along with mitotic proteins. Using PC12 cells as a model system, we determined whether the Gαq/PLCβ/ calcium signaling pathway impacts the manifestation of Alzheimer's disease. Down-regulating PLCβ significantly increases tau protein expression and causes a large increase in tau aggregation. Stimulating Gαq to activate PLCβ results in a modest reduction in tau aggregation while inhibiting PLCβ activity results in a modest enhancement of tau aggregation. These results suggest that PLCβ may effect tau aggregation by an additional mechanism that is independent of its ability to transduce calcium signals. To this end, we found that a cytosolic population of PLCβ binds to a mitotic protein found in neurofibrillary tangles, CDK18, which promotes tau phosphorylation and aggregation. Taken together, our studies show that the loss of PLCβ1 can promote Alzheimer's disease by a combination of its catalytic activity and ... [truncated at 150 words]
Zhu Y, Qiu Y, Chen W, Nie Q, Lander AD.
Scaling a Dpp morphogen gradient through feedback control of receptors and co-receptors.
Dev Cell. 2020; 53(6): 724-739. PMCID: PMC7437929Gradients of decapentaplegic (Dpp) pattern Drosophila wing imaginal discs, establishing gene expression boundaries at specific locations. As discs grow, Dpp gradients expand, keeping relative boundary positions approximately stationary. Such scaling fails in mutants for Pentagone (pent), a gene repressed by Dpp that encodes a diffusible protein that expands Dpp gradients. Although these properties fit a recent mathematical model of automatic gradient scaling, that model requires an expander that spreads with minimal loss throughout a morphogen field. Here, we show that Pent's actions are confined to within just a few cell diameters of its site of synthesis and can be phenocopied by manipulating non-diffusible Pent targets strictly within the Pent expression domain. Using genetics and mathematical modeling, we develop an alternative model of scaling driven by feedback downregulation of Dpp receptors and co-receptors. Among the model's predictions is a size beyond which scaling fails-something we observe directly in wing discs.
Bourges AC, Lazarev A, Declerck N, Rogers KL, Royer CA.
Quantitative high-resolution imaging of live microbial cells at high hydrostatic pressure.
Biophys J. 2020; 118(11): 2670-2679. PMCID: PMC7264842The majority of the Earth’s microbial biomass exists in the deep biosphere, in the deep ocean, and within the Earth’s crust. Although other physical parameters in these environments, such as temperature or pH, can differ substantially, they are all under high pressures. Beyond emerging genomic information, little is known about the molecular mechanisms underlying the ability of these organisms to survive and grow at pressures that can reach over 1000-fold the pressure on the Earth’s surface. The mechanisms of pressure adaptation are also important in food safety, with the increasing use of high-pressure food processing. Advanced imaging represents an important tool for exploring microbial adaptation and response to environmental changes. Here, we describe implementation of a high-pressure sample chamber with a two-photon scanning microscope system, allowing for the first time, to our knowledge, quantitative high-resolution two-photon imaging at 100 MPa of living microbes from all three kingdoms of life. We ... [truncated at 150 words]
Mieskes F, Wehnekamp F, Plucińska G, Thong R, Misgeld T, Lamb DC.
Trajectory data of antero- and retrograde movement of mitochondria in living Zebrafish larvae.
Data Brief. 2020; 29, 105280. PMCID: PMC7068625Recently, a large number of single particle tracking (SPT) approaches have been developed. Generally, SPT techniques can be split into two groups: ex post facto approaches where trajectory extraction is carried out after data acquisition and feedback based approaches that perform particle tracking in real time [1]. One feedback approach is 3D Orbital Tracking, where the laser excitation beam is rotated in a circle about the object, generating a so called orbit [2,3]. By calculating the particle position from the detected intensity after every orbit in relation to its center, this method allows the microscope to follow a single object in real time. The high spatiotemporal resolution of this method and the potential to optically manipulate the followed object during the measurement promises to yield new deep insights into biological systems [4–7]. By upgrading this approach in a way that the specimen is recentered by a xy-stage on the center ... [truncated at 150 words]
Ward KE, Sengupta R, Ropa JP, Amiar S, Stahelin RV.
The cytosolic Phospholipase A2α N-terminal C2 domain binds and oligomerizes on membranes with positive curvature.
Biomolecules. 2020; 10(4), 647. PMCID: PMC7226022Group IV phospholipase A2α (cPLA2α) regulates the production of prostaglandins and leukotrienes via the formation of arachidonic acid from membrane phospholipids. The targeting and membrane binding of cPLA2α to the Golgi involves the N-terminal C2 domain, whereas the catalytic domain produces arachidonic acid. Although most studies of cPLA2α concern its catalytic activity, it is also linked to homeostatic processes involving the generation of vesicles that traffic material from the Golgi to the plasma membrane. Here we investigated how membrane curvature influences the homeostatic role of cPLA2α in vesicular trafficking. The cPLA2α C2 domain is known to induce changes in positive membrane curvature, a process which is dependent on cPLA2α membrane penetration. We showed that cPLA2α undergoes C2 domain-dependent oligomerization on membranes in vitro and in cells. We found that the association of the cPLA2α C2 domain with membranes is limited to membranes with positive curvature, and enhanced C2 domain oligomerization ... [truncated at 150 words]
Wang X, Wang Y, Zhang Z, Huang M, Fei Y, Ma J, Mi L.
Discriminating different grades of cervical intraepithelial neoplasia based on label-free phasor fluorescence lifetime imaging microscopy.
Biomed Opt Express. 2020; 11(4): 1977-1990. PMCID: PMC7173885This study proposed label-free fluorescence lifetime imaging and phasor analysis methods to discriminate different grades of cervical intraepithelial neoplasia (CIN). The human cervical tissue lesions associated with cellular metabolic abnormalities were detected by the status changes of important coenzymes in cells and tissues, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD). Fluorescence lifetime imaging microscopy (FLIM) was used to study human cervical tissues, human cervical epithelial cells, and standard samples. Phasor analysis was applied to reveal the interrelation between the metabolic changes and cancer development, which can distinguish among different stages of cervical lesions from low risk to high risk. This approach also possessed high sensitivity, especially for healthy sites of CIN3 tissues, and indicated the dominance of the glycolytic pathway over oxidative phosphorylation in high-grade cervical lesions. This highly adaptive, sensitive, and rapid diagnostic tool exhibits a great potential for cervical precancer diagnosis.
Huang M, Liang X, Zhang Z, Wang J, Fei Y, Ma J, Qu S, Mi L.
Carbon dots for intracellular pH sensing with fluorescence lifetime imaging microscopy.
Nanomaterials (Basel). 2020; 10(4), 604. PMCID: PMC7221822The monitoring of intracellular pH is of great importance for understanding intracellular trafficking and functions. It has various limitations for biosensing based on the fluorescence intensity or spectra study. In this research, pH-sensitive carbon dots (CDs) were employed for intracellular pH sensing with fluorescence lifetime imaging microscopy (FLIM) for the first time. FLIM is a highly sensitive method that is used to detect a microenvironment and it can overcome the limitations of biosensing methods based on fluorescence intensity. The different groups on the CDs surfaces changing with pH environments led to different fluorescence lifetime values. The CDs aqueous solution had a gradual change from 1.6 ns to 3.7 ns in the fluorescence lifetime with a pH range of 2.6–8.6. Similar fluorescence lifetime changes were found in pH buffer-treated living cells. The detection of lysosomes, cytoplasm, and nuclei in living cells was achieved by measuring the fluorescence lifetime of CDs. In ... [truncated at 150 words]
Verneri P, Echegaray CV, Oses C, Stortz M, Guberman A, Levi V.
Dynamical reorganization of the pluripotency transcription factors Oct4 and Sox2 during early differentiation of embryonic stem cells.
Sci Rep. 2020; 10(1), 5195. PMCID: PMC7089971Pluripotency maintenance requires transcription factors (TFs) that induce genes necessary to preserve the undifferentiated state and repress others involved in differentiation. Recent observations support that the heterogeneous distribution of TFs in the nucleus impacts on gene expression. Thus, it is essential to explore how TFs dynamically organize to fully understand their role in transcription regulation. Here, we examine the distribution of pluripotency TFs Oct4 and Sox2 in the nucleus of embryonic stem (ES) cells and inquire whether their organization changes during early differentiation stages preceding their downregulation. Using ES cells expressing Oct4-YPet or Sox2-YPet, we show that Oct4 and Sox2 partition between nucleoplasm and a few chromatin-dense foci which restructure after inducing differentiation by 2i/LIF withdrawal. Fluorescence correlation spectroscopy showed distinct changes in Oct4 and Sox2 dynamics after differentiation induction. Specifically, we detected an impairment of Oct4-chromatin interactions whereas Sox2 only showed slight variations in its short-lived, and probably more ... [truncated at 150 words]
Shi W, Koo DES, Kitano M, Chiang HJ, Trinh LA, Turcatel G, Steventon B, Arnesano C, Warburton D, Fraser SE, Cutrale F.
Pre-processing visualization of hyperspectral fluorescent data with spectrally encoded enhanced representations.
Nat Commun. 2020; 11(1), 726. PMCID: PMC7002680Hyperspectral fluorescence imaging is gaining popularity for it enables multiplexing of spatio-temporal dynamics across scales for molecules, cells and tissues with multiple fluorescent labels. This is made possible by adding the dimension of wavelength to the dataset. The resulting datasets are high in information density and often require lengthy analyses to separate the overlapping fluorescent spectra. Understanding and visualizing these large multi-dimensional datasets during acquisition and pre-processing can be challenging. Here we present Spectrally Encoded Enhanced Representations (SEER), an approach for improved and computationally efficient simultaneous color visualization of multiple spectral components of hyperspectral fluorescence images. Exploiting the mathematical properties of the phasor method, we transform the wavelength space into information-rich color maps for RGB display visualization. We present multiple biological fluorescent samples and highlight SEER’s enhancement of specific and subtle spectral differences, providing a fast, intuitive and mathematical way to interpret hyperspectral images during collection, pre-processing and analysis.
Suárez H, López-Martín S, Toribio V, Zamai M, Hernández-Riquer MV, Genís L, Arroyo AG, Yáñez-Mó M.
Regulation of MT1-MMP activity through its association with ERMs.
Cells. 2020; 9(2), 348. PMCID: PMC7072721Membrane-bound proteases play a key role in biology by degrading matrix proteins or shedding adhesion receptors. MT1-MMP metalloproteinase is critical during cancer invasion, angiogenesis, and development. MT1-MMP activity is strictly regulated by internalization, recycling, autoprocessing but also through its incorporation into tetraspanin-enriched microdomains (TEMs), into invadopodia, or by its secretion on extracellular vesicles (EVs). We identified a juxtamembrane positively charged cluster responsible for the interaction of MT1-MMP with ERM (ezrin/radixin/moesin) cytoskeletal connectors in breast carcinoma cells. Linkage to ERMs regulates MT1-MMP subcellular distribution and internalization, but not its incorporation into extracellular vesicles. MT1-MMP association to ERMs and insertion into TEMs are independent phenomena, so that mutation of the ERM-binding motif in the cytoplasmic region of MT1-MMP does not preclude its association with the tetraspanin CD151, but impairs the accumulation and coalescence of CD151/MT1-MMP complexes at actin-rich structures. Conversely, gene deletion of CD151 does not impact on MT1-MMP colocalization with ERM ... [truncated at 150 words]
Zhang S, Hinde E, Schneider MP, Jans DA, Bogoyevitch MA.
Nuclear bodies formed by polyQ-ataxin-1 protein are liquid RNA/protein droplets with tunable dynamics.
Sci Rep. 2020; 10(1), 1557. PMCID: PMC6994494A mutant form of the ataxin-1 protein with an expanded polyglutamine (polyQ) tract is the underlying cause of the inherited neurodegenerative disease spinocerebellar ataxia 1 (SCA1). In probing the biophysical features of the nuclear bodies (NBs) formed by polyQ-ataxin-1, we defined ataxin-1 NBs as spherical liquid protein/RNA droplets capable of rapid fusion. We observed dynamic exchange of the ataxin-1 protein into these NBs; notably, cell exposure to a pro-oxidant stress could trigger a transition to slower ataxin-1 exchange, typical of a hydrogel state, which no longer showed the same dependence on RNA or sensitivity to 1,6-hexanediol. Furthermore, we could alter ataxin-1 exchange dynamics either through modulating intracellular ATP levels, RNA helicase inhibition, or siRNA-mediated depletion of select RNA helicases. Collectively, these findings reveal the tunable dynamics of the liquid RNA/protein droplets formed by polyQ-ataxin-1.
Pooley JR, Rivers CA, Kilcooley MT, Paul SN, Cavga AD, Kershaw YM, Muratcioglu S, Gursoy A, Keskin O, Lightman SL.
Beyond the heterodimer model for mineralocorticoid and glucocorticoid receptor interactions in nuclei and at DNA.
PLoS One. 2020; 15(1), e0227520. PMCID: PMC6953809Glucocorticoid (GR) and mineralocorticoid receptors (MR) are believed to classically bind DNA as homodimers or MR-GR heterodimers to influence gene regulation in response to pulsatile basal or stress-evoked glucocorticoid secretion. Pulsed corticosterone presentation reveals MR and GR co-occupy DNA only at the peaks of glucocorticoid oscillations, allowing interaction. GR DNA occupancy was pulsatile, while MR DNA occupancy was prolonged through the inter-pulse interval. In mouse mammary 3617 cells MR-GR interacted in the nucleus and at a chromatin-associated DNA binding site. Interactions occurred irrespective of ligand type and receptors formed complexes of higher order than heterodimers. We also detected MR-GR interactions ex-vivo in rat hippocampus. An expanded range of MR-GR interactions predicts structural allostery allowing a variety of transcriptional outcomes and is applicable to the multiple tissue types that co-express both receptors in the same cells whether activated by the same or different hormones.
Brown JWP, Bauer A, Polinkovsky ME, Bhumkar A, Hunter DJB, Gaus K, Sierecki E, Gambin Y.
Single-molecule detection on a portable 3D-printed microscope.
Nat Commun. 2019; 10(1), 5662. PMCID: PMC6906517Single-molecule assays have, by definition, the ultimate sensitivity and represent the next frontier in biological analysis and diagnostics. However, many of these powerful technologies require dedicated laboratories and trained personnel and have therefore remained research tools for specialists. Here, we present a single-molecule confocal system built from a 3D-printed scaffold, resulting in a compact, plug and play device called the AttoBright. This device performs single photon counting and fluorescence correlation spectroscopy (FCS) in a simple format and is widely applicable to the detection of single fluorophores, proteins, liposomes or bacteria. The power of single-molecule detection is demonstrated by detecting single α-synuclein amyloid fibrils, that are currently evaluated as biomarkers for Parkinson’s disease, with an improved sensitivity of >100,000-fold over bulk measurements.
Clark NM, Buckner E, Fisher AP, Nelson EC, Nguyen TT, Simmons AR, de Balaguer MAL, Butler-Smith T, Sheldon PJ, Bergmann DC, Williams CM, Sozzani R.
Stem-cell-ubiquitous genes spatiotemporally coordinate division through regulation of stem-cell-specific gene networks.
Nat Commun. 2019; 10(1), 5574. PMCID: PMC6897965Stem cells are responsible for generating all of the differentiated cells, tissues, and organs in a multicellular organism and, thus, play a crucial role in cell renewal, regeneration, and organization. A number of stem cell type-specific genes have a known role in stem cell maintenance, identity, and/or division. Yet, how genes expressed across different stem cell types, referred to here as stem-cell-ubiquitous genes, contribute to stem cell regulation is less understood. Here, we find that, in the Arabidopsis root, a stem-cell-ubiquitous gene, TESMIN-LIKE CXC2 (TCX2), controls stem cell division by regulating stem cell-type specific networks. Development of a mathematical model of TCX2 expression allows us to show that TCX2 orchestrates the coordinated division of different stem cell types. Our results highlight that genes expressed across different stem cell types ensure cross-communication among cells, allowing them to divide and develop harmonically together.
Martin EW, Chakraborty S, Presman DM, Ardori FT, Oh KS, Kaileh M, Tessarollo L, Sung MH.
Assaying homodimers of NF-κB in live single cells.
Front Immunol. 2019; 10, 2609. PMCID: PMC6853996NF-κB is a family of heterodimers and homodimers which are generated from subunits encoded by five genes. The predominant classical dimer RelA:p50 is presumed to operate as “NF-κB” in many contexts. However, there are several other dimer species which exist and may even be more functionally relevant in specific cell types. Accurate characterization of stimulus-specific and tissue-specific dimer repertoires is fundamentally important for understanding the downstream gene regulation by NF-κB proteins. In vitro assays such as immunoprecipitation have been widely used to analyze subunit composition, but these methods do not provide information about dimerization status within the natural intracellular environment of intact live cells. Here we apply a live single cell microscopy technique termed Number and Brightness to examine dimers translocating to the nucleus in fibroblasts after pro-inflammatory stimulation. This quantitative assay suggests that RelA:RelA homodimers are more prevalent than might be expected. We also found that the relative proportion ... [truncated at 150 words]
Rajah A, Boudreau CG, Ilie A, Wee TL, Tang K, Borisov AZ, Orlowski J, Brown CM.
Paxillin S273 phosphorylation regulates adhesion dynamics and cell migration through a common protein complex with PAK1 and βPIX.
Sci Rep. 2019; 9(1), 11430. PMCID: PMC6686007Cell migration is an important biological phenomenon involved in many homeostatic and aberrant physiological processes. Phosphorylation of the focal adhesion adaptor protein, paxillin, on serine 273 (S273) has been implicated as a key regulator of cell migration. Here, it is shown that phosphorylation on paxillin S273 leads to highly migratory cells with small dynamic adhesions. Adhesions at protrusive edges of the cell were more dynamic than adhesions at retracting edges. Temporal image correlation microscopy revealed that these dynamic adhesions undergo rapid binding of paxillin, PAK1 and βPIX. We identified membrane proximal adhesion subdomains in protrusive regions of the cell that show rapid protein binding that is dependent on paxillin S273 phosphorylation, PAK1 kinase activity and phosphatases. These dynamic adhesion subdomains corresponded to regions of the adhesion that also show co-binding of paxillin/PAK1 and paxillin/βPIX complexes. It is likely that parts of individual adhesions are more dynamic while others are less ... [truncated at 150 words]
Ghosh RP, Shi Q, Yang L, Reddick MP, Nikitina T, Zhurkin VB, Fordyce P, Stasevich TJ, Chang HY, Greenleaf WJ, Liphardt JT.
Satb1 integrates DNA binding site geometry and torsional stress to differentially target nucleosome-dense regions.
Nat Commun. 2019; 10(1), 3221. PMCID: PMC6642133The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.
Dobrinskikh E, Al-Juboori SI, Shabeka U, Reisz JA, Zheng C, Marwan AI.
Heterogeneous pulmonary response after tracheal occlusion: clues to fetal lung growth.
J Surg Res. 2019; 239: 242-252.BACKGROUND: Understanding inconsistent clinical outcomes in infants with severe congenital diaphragmatic hernia (CDH) after tracheal occlusion (TO) is a crucial step for advancing neonatal care. The objective of this study is to explore the heterogeneous airspace morphometry and the metabolic landscape changes in fetal lungs after TO.
METHODS: Fetal lungs on days 1 and 4 after TO were examined using mass spectrometry-based metabolomics, fluorescence lifetime imaging microscopy (FLIM), the number of airspaces, and tissue-to-airspace ratio (TAR).
RESULTS: Two morphometric areas were identified in TO lungs compared with controls (more small airspaces at day 1 and a higher number of enlarged airspaces at day 4). Global metabolomics analysis revealed a significant upregulation of glycolysis and a suppression of the tricarboxylic acid cycle in day 4 TO lungs compared with day 1 TO lungs. In addition, there was a significant increase in polyamines involved in cell growth and proliferation. Locally, FLIM analysis on day ... [truncated at 150 words]
Iachina I, Antonescu IE, Dreier J, Sørensen JA, Brewer JR.
The nanoscopic molecular pathway through human skin.
Biochim Biophys Acta Gen Subj. 2019; 1863(7): 1226-1233.Background: Knowledge regarding the barrier properties of human skin is important for understanding skin pathology, developing of transdermal drug delivery systems and computational skin absorption models; however, the molecular pathways through human skin remains to be fully investigated on a nanoscopic level. In particular the nanoscopic pathway of molecules passing the intercellular lipid bilayers separating the corneocytes in the stratum corneum (SC) is not fully elucidated.
Methods: Using stimulated emission depletion microscopy (STED) and Förster resonance energy transfer (FRET) the molecular pathways through the SC, the main barrier of the skin, are determined for lipophilic and water-soluble molecules at a nanoscopic resolution.
Results: Using STED and confocal microscopy, water-soluble dyes, were observed to be present in both the corneocytes and in the intercellular lipid matrix, whereas the lipophilic dyes were predominately in the intercellular lipid bilayers. FRET was observed in the SC between the lipophilic and water-soluble dyes, the existence of a ... [truncated at 150 words]
Whiteside MD, Werner GDA, Caldas VEA, Padje Av, Dupin SE, Elbers B, Bakker M, Wyatt GAK, Klein M, Hink MA, Postma M, Vaitla B, Noë R, Shimizu TS, West SA, Kiers T.
Mycorrhizal fungi respond to resource inequality by moving phosphorus from rich to poor patches across networks.
Curr Biol. 2019; 29(12), 2043–2050.e8. PMCID: PMC6584331The world’s ecosystems are characterized by an unequal distribution of resources [1]. Trade partnerships between organisms of different species—mutualisms—can help individuals cope with such resource inequality [2, 3, 4]. Trade allows individuals to exchange commodities they can provide at low cost for resources that are otherwise impossible or more difficult to access [5, 6]. However, as resources become increasingly patchy in time or space, it is unknown how organisms alter their trading strategies [7, 8]. Here, we show how a symbiotic fungus mediates trade with a host root in response to different levels of resource inequality across its network. We developed a quantum-dot-tracking technique to quantify phosphorus-trading strategies of arbuscular mycorrhizal fungi simultaneously exposed to rich and poor resource patches. By following fluorescent nanoparticles of different colors across fungal networks, we determined where phosphorus was hoarded, relocated, and transferred to plant hosts. We found that increasing exposure to inequality stimulated ... [truncated at 150 words]
Nguyen H, Ward WS, James NG.
Spatial and temporal resolution of mORC4 fluorescent variants reveals structural requirements for achieving higher order self-association and pronuclei entry.
Methods Appl Fluoresc. 2019; 7(3), 035002. PMCID: PMC6636821The Origin Replication Complex (ORC), which is a multi-subunit protein complex composed of six proteins ORC1–6, is essential for initiating licensing at DNA replication origins. We have previously reported that ORC4 has an alternative function wherein it forms a cage surrounding the extruded chromatin in female meiosis and is required for polar body extrusion (PBE). As this is a highly unexpected finding for protein that normally binds DNA, we tested whether ORC4 can actually form larger, higher order structures, which would be necessary to form a cage-like structure. We generated two fluorescent constructs of mouse ORC4, mORC4-EGFP and mORC4-FlAsH, to examine its spatial dynamics during oocyte activation in live cells. We show that both constructs were primarily monomeric throughout the embryo but self-association into larger units was detected with both probes. However, mORC4-FlAsH clearly showed higher order self-association and unique spatial distribution while mORC4-EGFP failed to form large structures during ... [truncated at 150 words]
Rao E, Foderà V, Leone M, Vetria V.
Direct observation of alpha-lactalbumin, adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures.
Biochim Biophys Acta Gen Subj. 2019; 1863(5): 784-794.The interaction between proteins and membranes is of great interest in biomedical and biotechnological research for its implication in many functional and dysfunctional processes. We present an experimental study on the interaction between model membranes and alpha-lactalbumin (α-La). α-La is widely studied for both its biological function and its anti-tumoral properties. We use advanced fluorescence microscopy and spectroscopy techniques to characterize α-La-membrane mechanisms of interaction and α-La-induced modifications of membranes when insertion of partially disordered regions of protein chains in the lipid bilayer is favored. Moreover, using fluorescence lifetime imaging, we are able to distinguish between protein adsorption and insertion in the membranes. Our results indicate that, upon addition of α-La to giant vesicles samples, protein is inserted into the lipid bilayer with rates that are concentration-dependent. The formation of heterogeneous hybrid protein-lipid co-aggregates, paralleled with protein conformational and structural changes, alters the membrane structure and morphology, leading to an ... [truncated at 150 words]
Chen WQ, Drapek C, Li DX, Xu ZH, Benfey PN, Baia SN.
Histone deacetylase HDA19 affects root cortical cell fate by interacting with SCARECROW.
Plant Physiol. 2019; 180: 276-288. PMCID: PMC6501111The Arabidopsis (Arabidopsis thaliana) root epidermis is a simple model for investigating cell fate specification and pattern formation. In addition to regulatory networks consisting of transcription factors, histone deacetylases are also involved in the formation of cellular patterns. Here, we report thatHistone Deacetylase19 (HDA19) affects the root epidermal cellular pattern through regulation of cortical cell fate by interacting with SCARECROW (SCR). HDA19 binds to the DNA sequence upstream of SCR, as well as to those of several of SCR’s target genes, and regulates their expression. Mutant lines of several SCR target genes show impaired patterns of epidermal differentiation and cortical cell division, similar to that of hda19. This work presents HDA19 and SCR as two further players in the regulation of cortical and epidermal cell specification and describes an additional function for SCR.
Montford JR, Bauer C, Dobrinskikh E, Hopp K, Levi M, Weiser-Evans M, Nemenoff R, Furgeson SB.
Inhibition of 5-lipoxygenase decreases renal fibrosis and progression of chronic kidney disease.
Am J Physiol Renal Physiol. 2019; 316(4): F732–F742. PMCID: PMC6483031In inflammatory diseases, the 5-lipoxygenase (5-LO) pathway contributes to epithelial damage and fibrosis by catalyzing the production of leukotrienes (LTs). Antagonists of the 5-LO pathway are currently approved for use in patients and are well tolerated. We found that expression of 5-LO is strongly induced in three models of chronic kidney disease: unilateral ureteral obstruction (UUO), folate nephropathy, and an orthologous mouse model of polycystic kidney disease. Immunohistochemistry showed that macrophages are the dominant source of 5-LO. Zileuton, a US Food and Drug Administration-approved antagonist of 5-LO, significantly reduced fibrosis at 7 and 14 days after UUO; these findings were confirmed using a genetically modified [5-LO-associated protein-knockout (Alox5ap−/−)] mouse strain. Inhibition of 5-LO did not appear to change infiltration of leukocytes after UUO as measured by flow cytometry. However, fluorescence-lifetime imaging microscopy showed that 5-LO inhibitors reversed the glycolytic switch in renal tubular epithelial cells after UUO. Two downstream enzymes ... [truncated at 150 words]
Pazin WM, Vilanova N, Voets IK, Soares AEE, Ito AS.
Effects of artepillin C on model membranes displaying liquid immiscibility.
Braz J Med Biol Res. 2019; 52(3), e8281. PMCID: PMC6437936It has been hypothesized that the therapeutic effects of artepillin C, a natural compound derived from Brazilian green propolis, are likely related to its partition in the lipid bilayer component of biological membranes. To test this hypothesis, we investigated the effects of the major compound of green propolis, artepillin C, on model membranes (small and giant unilamelar vesicles) composed of ternary lipid mixtures containing cholesterol, which display liquid-ordered (lo) and liquid-disordered (ld) phase coexistence. Specifically, we explored potential changes in relevant membrane parameters upon addition of artepillin C presenting both neutral and deprotonated states by means of small angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and confocal and multiphoton excitation fluorescence microscopy. Thermotropic analysis obtained from DSC experiments indicated a loss in the lipid cooperativity of lo phase at equilibrium conditions, while at similar conditions spontaneous formation of unilamellar vesicles from SAXS experiments showed that deprotonated artepillin C preferentially ... [truncated at 150 words]
di Bona M, Mancini MA, Mazza D, Vicidomini G, Diaspro A, Lanzanò L.
Measuring mobility in chromatin by intensity-sorted FCS.
Biophys J. 2019; 116(6): 987-999. PMCID: PMC6428914The architectural organization of chromatin can play an important role in genome regulation by affecting the mobility of molecules within its surroundings via binding interactions and molecular crowding. The diffusion of molecules at specific locations in the nucleus can be studied by fluorescence correlation spectroscopy (FCS), a well-established technique based on the analysis of fluorescence intensity fluctuations detected in a confocal observation volume. However, detecting subtle variations of mobility between different chromatin regions remains challenging with currently available FCS methods. Here, we introduce a method that samples multiple positions by slowly scanning the FCS observation volume across the nucleus. Analyzing the data in short time segments, we preserve the high temporal resolution of single-point FCS while probing different nuclear regions in the same cell. Using the intensity level of the probe (or a DNA marker) as a reference, we efficiently sort the FCS segments into different populations and obtain average ... [truncated at 150 words]
Guimarães AJ, de Cerqueira MD, Zamith-Miranda D, Lopez PH, Rodrigues ML, Pontes B, Viana NB, DeLeon-Rodriguez CM, Rossi DCP, Casadevall A, Gomes AMO, Martinez LR, Schnaar RL, Nosanchuk JD, Nimrichter L.
Host membrane glycosphingolipids and lipid microdomains facilitate Histoplasma capsulatum internalization by macrophages.
Cell Microbiol. 2019; 21(3), e12976. PMCID: PMC6805151Recognition and internalisation of intracellular pathogens by host cells is a multifactorial process, involving both stable and transient interactions. The plasticity of the host cell plasma membrane is fundamental in this infectious process. Here, the participation of macrophage lipid microdomains during adhesion and internalisation of the fungal pathogen Histoplasma capsulatum (Hc) was investigated. An increase in membrane lateral organisation, which is a characteristic of lipid microdomains, was observed during the first steps of Hc-macrophage interaction. Cholesterol enrichment in macrophage membranes around Hc contact regions and reduced levels of Hc-macrophage association after cholesterol removal also suggested the participation of lipid microdomains during Hc-macrophage interaction. Using optical tweezers to study cell-to-cell interactions, we showed that cholesterol depletion increased the time required for Hc adhesion. Additionally, fungal internalisation was significantly reduced under these conditions. Moreover, macrophages treated with the ceramide-glucosyltransferase inhibitor (P4r) and macrophages with altered ganglioside synthesis (from B4galnt1-/- mice) showed a ... [truncated at 150 words]
Marwan AI, Shabeka U, Reisz JA, Zheng C, Serkova NJ, Dobrinskikh E.
Unique heterogeneous topological pattern of the metabolic landscape in rabbit fetal lungs following tracheal occlusion.
Fetal Diagn Ther. 2019; 45(3): 145-154. PMCID: NIHMS995357Fetal tracheal occlusion (TO) is currently an experimental approach to drive accelerated lung growth. It is stimulated by mechanotransduction that results in increased cellular proliferation and growth. However, it is currently unknown how TO affects the metabolic landscape of fetal lungs.
Akhunzada MJ, D’Autilia F, Chandramouli B, Bhattacharjee N, Catte A, di Rienzo R, Cardarelli F, Brancato G.
Interplay between lipid lateral diffusion, dye concentration and membrane permeability unveiled by a combined spectroscopic and computational study of a model lipid bilayer.
Sci Rep. 2019; 9(1), 1508. PMCID: PMC6365552Lipid lateral diffusion in membrane bilayers is a fundamental process exploited by cells to enable complex protein structural and dynamic reorganizations. For its importance, lipid mobility in both cellular and model bilayers has been extensively investigated in recent years, especially through the application of time-resolved, fluorescence-based, optical microscopy techniques. However, one caveat of fluorescence techniques is the need to use dye-labeled variants of the lipid of interest, thus potentially perturbing the structural and dynamic properties of the native species. Generally, the effect of the dye/tracer molecule is implicitly assumed to be negligible. Nevertheless, in view of the widespread use of optically modified lipids for studying lipid bilayer dynamics, it is highly desirable to well assess this point. Here, fluorescence correlation spectroscopy (FCS) and molecular dynamics (MD) simulations have been combined together to uncover subtle structural and dynamic effects in DOPC planar membranes enriched with a standard Rhodamine-labeled lipid. Our findings ... [truncated at 150 words]
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Autofluorescence lifetime imaging of cellular metabolism: Sensitivity towards cell density, pH, intracellular and intercellular heterogeneity.
Cytometry A. 2019; 95(1): 56-69. PMCID: PMC6329636Autofluorescence imaging (AFI) has greatly accelerated in the last decade, way past its origins in detecting endogenous signals in biological tissues to identify differences between samples. There are many endogenous fluorescence sources of contrast but the most robust and widely utilized have been those associated with metabolism. The intrinsically fluorescent metabolic cofactors Nicotinamide adenine dinucleotide (NAD+/NADH) and Flavin adenine dinucleotide (FAD/FADH2) have been utilized in a number of AFI applications including basic research, clinical and pharmaceutical studies. Fluorescence lifetime imaging microscopy (FLIM) has emerged as one of the more powerful AFI tools for NADH and FAD characterization due to its unique ability to non-invasively detect metabolite bound and free states and quantitate cellular redox ratio. However, despite this widespread biological use, many standardization methods are still needed to extend FLIM based AFI into a fully robust research and clinical diagnostic tools. FLIM is sensitive to a wide range of factors ... [truncated at 150 words]
Tan FH, Putoczki TL, Lou J, Hinde E, Hollande F, Giraud J, Stylli SS, Paradiso L, Zhu HJ, Sieber OM, Luwor RB.
Ponatinib inhibits multiple signaling pathways involved in STAT3 signaling and attenuates colorectal tumor growth.
Cancers (Basel). 2018; 10(12), 526. PMCID: PMC6316865Signal transducer and activator of transcription 3 (STAT3) signaling is a major driver of colorectal cancer (CRC) growth, however therapeutics, which can effectively target this pathway, have so far remained elusive. Here, we performed an extensive screen for STAT3 inhibitors among a library of 1167 FDA-approved agents, identifying Ponatinib as a lead candidate. We found that Ponatinib inhibits STAT3 activity driven by EGF/EGFR, IL-6/IL-6R and IL-11/IL-11R, three major ligand/receptor systems involved in CRC development and progression. Ponatinib was able to inhibit CRC migration and tumor growth in vivo. In addition, Ponatinib displayed a greater ability to inhibit STAT3 activity and mediated superior anti-proliferative efficacy compared to five FDA approved SRC and Janus Kinase (JAK) inhibitors. Finally, long-term exposure of CRC cells to Ponatinib, Dasatinib and Bosutinib resulted in acquired resistance to Dasatinib and Bosutinib occurring within six weeks. However, acquired resistance to Ponatinib was observed after long-term exposure of >4 ... [truncated at 150 words]
Almendro-Vedia VG, García C, Ahijado-Guzmán R, Fuente-Herreruela Dl, Muñoz-Úbeda M, Natale P, Viñas MH, Albuquerque RQ, Guerrero-Martínez A, Monroy F, Lillo MP, López-Montero I.
Supramolecular zippers elicit interbilayer adhesion of membranes producing cell death.
Biochim Biophys Acta Gen Subj. 2018; 1862(12): 2824-2834. PMCID: PMC6202437BACKGROUND: The fluorescent dye 10-N-nonyl acridine orange (NAO) is widely used as a mitochondrial marker. NAO was reported to have cytotoxic effects in cultured eukaryotic cells when incubated at high concentrations. Although the biochemical response of NAO-induced toxicity has been well identified, the underlying molecular mechanism has not yet been explored in detail.
METHODS: We use optical techniques, including fluorescence confocal microscopy and lifetime imaging microscopy (FLIM) both in model membranes built up as giant unilamellar vesicles (GUVs) and cultured cells. These experiments are complemented with computational studies to unravel the molecular mechanism that makes NAO cytotoxic.
RESULTS: We have obtained direct evidence that NAO promotes strong membrane adhesion of negatively charged vesicles. The attractive forces are derived from van der Waals interactions between anti-parallel H-dimers of NAO molecules from opposing bilayers. Semi-empirical calculations have confirmed the supramolecular scenario by which anti-parallel NAO molecules form a zipper of bonds at the contact ... [truncated at 150 words]
Rubio SG, Pastor NM, García C, Almendro-Vedia VG, Ferrer I, Natale P, Paz-Ares L, Lillo MP, López-Montero I.
Enhanced cytotoxic activity of mitochondrial mechanical effectors in human lung carcinoma H520 cells: pharmaceutical implications for cancer therapy.
Front Oncol. 2018; 8, 514. PMCID: PMC6242888Cancer cell mitochondria represent an attractive target for oncological treatment as they have unique hallmarks that differ from their healthy counterparts, as the presence of a stronger membrane potential that can be exploited to specifically accumulate cytotoxic cationic molecules. Here, we explore the selective cytotoxic effect of 10-N-nonyl acridine orange (NAO) on human lung carcinoma H520 cells and compare them with healthy human lung primary fibroblasts. NAO is a lipophilic and positively charged molecule that promotes mitochondrial membrane adhesion that eventually leads to apoptosis when incubated at high micromolar concentration. We found an enhanced cytotoxicity of NAO in H520 cancer cells. By means Fluorescence lifetime imaging microscopy (FLIM) we also confirmed the formation of H-dimeric aggregates originating from opposing adjacent membranes that interfere with the mitochondrial membrane structure. Based on our results, we suggest the mitochondrial membrane as a potential target in cancer therapy to mechanically control the cell proliferation ... [truncated at 150 words]
Li Y, Junge JA, Arnesano C, Gross GG, Miner JH, Moats R, Roberts RW, Arnold DB, Fraser SE.
Discs large 1 controls daughter-cell polarity after cytokinesis in vertebrate morphogenesis.
Proc Natl Acad Sci U S A. 2018; 115(46): E10859-E10868. PMCID: PMC6243286An integrative approach is presented for studying cell biology in vivo, assessing protein dynamics and cell behavior, and offering in situ analyses of cytokinesis, daughter-cell polarity, and stereotyped tissue morphogenesis. Tagging endogenous Discs Large 1 (Dlg1) in cartilage using intrabody technology permits in situ 3D time-lapse imaging and reveals Dlg1 enrichment at the midbody during cytokinesis. Functional significance is tested by disrupting Dlg1 multimerization and its midbody localization by using an ablating intrabody, DLGE3. Building on prior work on Dlg1 in epithelia, our work reveals that Dlg1 propagates cell polarity in proliferative mesenchymal tissues and suggests that multiple mechanisms act in concert at distinct phases of the cell cycle to transmit and maintain cell polarity.
Sediqi H, Wray A, Jones C, Jones M.
Application of Spectral Phasor analysis to sodium microenvironments in myoblast progenitor cells.
PLoS One. 2018; 13(10), e0204611. PMCID: PMC6209149Sodium ions (Na+) are key regulators of molecular events in many cellular processes, yet the dynamics of this ion remain poorly defined. Developing approaches to identify and characterise Na+ microenvironments will enable more detailed elucidation of the mechanisms of signal transduction. Here we report the application of Spectral Phasor analysis to the Na+ fluorophore, CoroNa Green, to identify and spatially map spectral emissions that report Na+ microenvironments. We use differentiating stem cells where Na+ fluxes were reported as an antecedent. Myoblast stem cells were induced to differentiate by serum starvation and then fixed at intervals between 0 and 40-minutes of differentiation prior to addition of CoroNa Green. The fluorescent intensity was insufficient to identify discrete Na+ microenvironments. However, using Spectral Phasor analysis we identified spectral shifts in CoroNa Green fluorescence which is related to the Na+ microenvironment. Further, spectral-heterogeneity appears to be contingent on the distance of Na+ from the ... [truncated at 150 words]
Goo BMSS, Sanstrum BJ, Holden DZY, Yu Y, James NG.
Arc/Arg3.1 has an activity-regulated interaction with PICK1 that results in altered spatial dynamics.
Sci Rep. 2018; 8(1), 14675. PMCID: PMC6168463Activity-regulated cytoskeleton-associated protein (Arc; also known as Arg3.1) is an immediate early gene product that is transcribed in dendritic spines and, to date, has been best characterized as a positive regulator of AMPAR endocytosis during long-term depression (LTD) through interaction with endocytic proteins. Here, we show that protein interacting with C terminal kinase 1 (PICK1), a protein known to bind to the GluA2 subunit of AMPARs and associated with AMPAR trafficking, was pulled-down from brain homogenates and synaptosomes when using Arc as immobilized bait. Fluctuation and FLIM-FRET-Phasor analysis revealed direct interaction between these proteins when co-expressed that was increased under depolarizing conditions in live cells. At the plasma membrane, Arc-mCherry oligomerization was found to be concentration dependent. Additionally, co-expression of Arc-mCherry and EGFP-PICK1 followed by depolarizing conditions resulted in significant increases in the number and size of puncta containing both proteins. Furthermore, we identified the Arc binding region to be ... [truncated at 150 words]
Ferri G, Bugliani M, Marchetti P, Cardarelli F.
Probing the light scattering properties of insulin secretory granules in single live cells.
Biochem Biophys Res Commun. 2018; 503(4): 2710-2714.Light scattering was recently demonstrated to serve as an intrinsic indicator for pancreatic islet cell mass and secretion. The insulin secretory granule (ISG), in particular, was proposed to be a reasonable candidate as the main intracellular source of scattered light due to the densely-packed insulin semi-crystal in the granule lumen. This scenario, if confirmed, would in principle open new perspectives for label-free single-granule imaging, tracking, and analysis. Contrary to such expectations, here we demonstrate that ISGs are not a primary source of scattering in primary human β-cells, as well as in immortalized β-like cells, quantitatively not superior to other intracellular organelles/structures, such as lysosomes and internal membranes. This result is achieved through multi-channel imaging of scattered light along with fluorescence arising from selectively-labelled ISGs. Co-localization and spatiotemporal cross-correlation analysis is performed on these signals, and compared among different cell lines. Obtained results suggest a careful re-thinking of the possibility to ... [truncated at 150 words]
Ferri G, Digiacomo L, D’Autilia F, Durso W, Caracciolo G, Cardarellia F.
Time-lapse confocal imaging datasets to assess structural and dynamic properties of subcellular nanostructures.
Sci Data. 2018; 5, 180191. PMCID: PMC6142892Time-lapse optical microscopy datasets from living cells can potentially afford an enormous amount of quantitative information on the relevant structural and dynamic properties of sub-cellular organelles/structures, provided that both the spatial and temporal dimensions are properly sampled during the experiment. Here we provide exemplary live-cell, time-lapse confocal imaging datasets corresponding to three sub-cellular structures of the endo-lysosomal pathway, i.e. early endosomes, late endosomes and lysosomes, along with detailed guidelines to produce analogous experiments. Validation of the datasets is conducted by means of established analytical tools to extract the structural and dynamic properties at the sub-cellular scale, such as Single Particle Tracking (SPT) and imaging derived Mean Square Displacement (iMSD) analyses. In our aim, the present work would help other researchers in the field to reuse the provided datasets for their own scopes, and to combine their creative approaches/analyses to similar acquisitions.
Pham XQ, Jonusauskaite L, Kumar ADN, Lefevre JP, Perrier A, Ha-Thi MH, Leray I.
New water-soluble fluorescent sensors based on calix[4]arene biscrown-6 for selective detection of cesium.
J Photochem Photobiol A. 2018; 364: 355-362.Two new fluorescent chemosensors based on calix[4]arene bis(crown-6) bearing coumarin units have been synthesized for the detection of cesium ions in water. The photophysical and complexing properties of these sensors with cesium were investigated using absorption and fluorescence spectroscopies as well as DFT calculations. The coordination of Cs+ by the these ligands induces a better charge transfer between the donor and acceptor groups of the coumarin, resulting in a bathochromic shift in absorption spectra and an enhancement of emission spectra. Both the ligands display an excellent selectivity for Cs+ over other potentially interfering cations such as Na+, Li+, K+, Mg2+, Ca2+ and Sr2+ ions.
Drapek C, Sparks EE, Marhavy P, Taylor I, Andersen TG, Hennacy JH, Geldner N, Benfey PN.
Minimum requirements for changing and maintaining endodermis cell identity in the Arabidopsis root.
Nat Plants. 2018; 4(8): 586-595. PMCID: PMC6135099Changes in gene regulation during differentiation are governed by networks of transcription factors. The Arabidopsis root endodermis is a tractable model to address how transcription factors contribute to differentiation. We used a bottom-up approach to understand the extent to which transcription factors required for endodermis differentiation can confer endodermis identity to a non-native cell-type. Our results show the transcription factors SHORTROOT and MYB36 alone have limited ability to induce ectopic endodermal features in the absence of additional cues. The stele-derived signaling peptide CIF2 stabilizes SHORTROOT-induced endodermis identity acquisition. The outcome is a partially impermeable barrier deposited in the sub-epidermal cell layer, which has a transcriptional signature similar to the endodermis. These results demonstrate other root cell-types can be forced to differentiate into endodermis and highlights a previously unappreciated role for receptor-kinase signaling in maintaining endodermis identity.
Martin JL, Mendonça LM, Marusinec R, Zuczek J, Angert I, Blower RJ, Mueller JD, Perilla JR, Zhang W, Mansky LM.
Critical role of the human T-Cell leukemia virus type 1 capsid N-terminal domain for Gag-Gag interactions and virus particle assembly.
J Virol. 2018; 92(14), e00333-18. PMCID: PMC6026748The retroviral Gag protein is the main structural protein responsible for virus particle assembly and release. Like human immunodeficiency virus type 1 (HIV-1) Gag, human T-cell leukemia virus type 1 (HTLV-1) has a structurally conserved capsid (CA) domain, including a β-hairpin turn and a centralized coiled-coil-like structure of six α helices in the CA amino-terminal domain (NTD), as well as four α-helices in the CA carboxy-terminal domain (CTD). CA drives Gag oligomerization, which is critical for both immature Gag lattice formation and particle production. The HIV-1 CA CTD has previously been shown to be a primary determinant for CA-CA interactions, and while both the HTLV-1 CA NTD and CTD have been implicated in Gag-Gag interactions, our recent observations have implicated the HTLV-1 CA NTD as encoding key determinants that dictate particle morphology. Here, we have conducted alanine-scanning mutagenesis in the HTLV-1 CA NTD nucleotide-encoding sequences spanning the loop regions and ... [truncated at 150 words]
O’Lexy R, Kasai K, Clark N, Fujiwara T, Sozzani R, Gallagher KL.
Exposure to heavy metal stress triggers changes in plasmodesmatal permeability via deposition and breakdown of callose.
J Exp Bot. 2018; 69(15), 3715–3728. PMCID: PMC6022669Both plants and animals must contend with changes in their environment. The ability to respond appropriately to these changes often underlies the ability of the individual to survive. In plants, an early response to environmental stress is an alteration in plasmodesmatal permeability with accompanying changes in cell to cell signaling. However, the ways in which plasmodesmata are modified, the molecular players involved in this regulation, and the biological significance of these responses are not well understood. Here, we examine the effects of nutrient scarcity and excess on plasmodesmata-mediated transport in the Arabidopsis thaliana root and identify two CALLOSE SYNTHASES and two β-1,3-GLUCANASES as key regulators of these processes. Our results suggest that modification of plasmodesmata-mediated signaling underlies the ability of the plant to maintain root growth and properly partition nutrients when grown under conditions of excess nutrients.
Stortz M, Angiolini J, Mocskos E, Wolosiuk A, Pecci A, Levi V.
Mapping the dynamical organization of the cell nucleus through fluorescence correlation spectroscopy.
Methods. 2018; 140-141: 10-22.The hierarchical organization of the cell nucleus into specialized open reservoirs and the nucleoplasm overcrowding impose restrictions to the mobility of biomolecules and their interactions with nuclear targets. These properties determine that many nuclear functions such as transcription, replication, splicing or DNA repair are regulated by complex, dynamical processes that do not follow simple rules. Advanced fluorescence microscopy tools and, in particular, fluorescence correlation spectroscopy (FCS) provide complementary and exquisite information on the dynamics of fluorescent labeled molecules moving through the nuclear space and are helping us to comprehend the complexity of the nuclear structure. Here, we describe how FCS methods can be applied to reveal the dynamical organization of the nucleus in live cells. Specifically, we provide instructions for the preparation of cellular samples with fluorescent tagged proteins and detail how FCS can be easily instrumented in commercial confocal microscopes. In addition, we describe general rules to set the ... [truncated at 150 words]
Nolan R, Iliopoulou M, Alvarez L, Padilla-Parra S.
Detecting protein aggregation and interaction in live cells: A guide to number and brightness.
Methods. 2018; 140-141: 172-177.The possibility to detect and quantify protein-protein interactions with good spatial and temporal resolutions in live cells is crucial in biology. Number and brightness is a powerful approach to detect both protein aggregation/desegregation dynamics and stoichiometry in live cells. Importantly, this technique can be applied in commercial set ups: both camera based and laser scanning microscopes. It provides pixel-by-pixel information on protein oligomeric states. If performed with two colours, the technique can retrieve the stoichiometry of the reaction under study. In this review, we discuss the strengths and weaknesses of the technique, stressing which are the correct acquisition parameters for a given microscope, the main challenges in analysis, and the limitations of the technique.
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Dissecting single-cell molecular spatiotemporal mobility and clustering at focal adhesions in polarised cells by fluorescence fluctuation spectroscopy methods.
Methods. 2018; 140–141: 85-96.Quantitative fluorescence fluctuation spectroscopy from optical microscopy datasets is a very powerful tool to resolve multiple spatiotemporal cellular and subcellular processes at the molecular level. In particular, raster image correlation spectroscopy (RICS) and number and brightness analyses (N&B) yield molecular mobility and clustering dynamic information extracted from real-time cellular processes. This quantitative information can be inferred in a highly flexible and detailed manner, i.e. 1) at the localisation level: from full-frame datasets and multiple regions of interest within; and 2) at the temporal level: not only from full-frame and multiple regions, but also intermediate temporal events. Here we build on previous research in deciphering the molecular dynamics of paxillin, a main component of focal adhesions. Cells use focal adhesions to attach to the extracellular matrix and interact with their local environment. Through focal adhesions and other adhesion structures, cells sense their local environment and respond accordingly; due to this continuous ... [truncated at 150 words]
Thattikota Y, Tollis S, Palou R, Vinet J, Tyers M, D’Amours D.
Cdc48/VCP promotes chromosome morphogenesis by releasing condensin from self-entrapment in chromatin.
Mol Cell. 2018; 69(4): 664-676.e5.The morphological transformation of amorphous chromatin into distinct chromosomes is a hallmark of mitosis. To achieve this, chromatin must be compacted and remodeled by a ring-shaped enzyme complex known as condensin. However, the mechanistic basis underpinning condensin's role in chromosome remodeling has remained elusive. Here we show that condensin has a strong tendency to trap itself in its own reaction product during chromatin compaction and yet is capable of interacting with chromatin in a highly dynamic manner in vivo. To resolve this apparent paradox, we identified specific chromatin remodelers and AAA-class ATPases that act in a coordinated manner to release condensin from chromatin entrapment. The Cdc48 segregase is the central linchpin of this regulatory mechanism and promotes ubiquitin-dependent cycling of condensin on mitotic chromatin as well as effective chromosome condensation. Collectively, our results show that condensin inhibition by its own reaction product is relieved by forceful enzyme extraction from chromatin.
Scipioni L, di Bona M, Vicidomini G, Diaspro A, Lanzanò L.
Local raster image correlation spectroscopy generates high-resolution intracellular diffusion maps.
Commun Biol. 2018; 1, 10.Raster image correlation spectroscopy (RICS) is a powerful method for measuring molecular diffusion in live cells directly from images acquired on a laser scanning microscope. However, RICS only provides single average diffusion coefficients from regions with a lateral size on the order of few micrometers, which means that its spatial resolution is mainly limited to the cellular level. Here we introduce the local RICS (L-RICS), an easy-to-use tool that generates high resolution maps of diffusion coefficients from images acquired on a laser scanning microscope. As an application we show diffusion maps of a green fluorescent protein (GFP) within the nucleus and within the nucleolus of live cells at an effective spatial resolution of 500 nm. We find not only that diffusion in the nucleolus is slowed down compared to diffusion in the nucleoplasm, but also that diffusion in the nucleolus is highly heterogeneous.
Sant'Anna-Silva ACB, Santos GC, Campos SPC, Gomes AMO, Pérez-Valencia JA, Rumjanek FD.
Metabolic profile of oral squamous carcinoma cell lines relies on a higher demand of lipid metabolism in metastatic cells.
Front Oncol. 2018; 8, 13. PMCID: PMC5801303Tumor cells are subjected to a broad range of selective pressures. As a result of the imposed stress, subpopulations of surviving cells exhibit individual biochemical phenotypes that reflect metabolic reprograming. The present work aimed at investigating metabolic parameters of cells displaying increasing degrees of metastatic potential. The metabolites present in cell extracts fraction of tongue fibroblasts and of cell lines derived from human tongue squamous cell carcinoma lineages displaying increasing metastatic potential (SCC9 ZsG, LN1 and LN2) were analyzed by 1H NMR (nuclear magnetic resonance) spectroscopy. Living, intact cells were also examined by the non-invasive method of fluorescence lifetime imaging microscopy (FLIM) based on the auto fluorescence of endogenous NADH. The cell lines reproducibly exhibited distinct metabolic profiles confirmed by Partial Least-Square Discriminant Analysis (PLS-DA) of the spectra. Measurement of endogenous free and bound NAD(P)H relative concentrations in the intact cell lines showed that ZsG and LN1 cells displayed high ... [truncated at 150 words]
Descalzo AB, Xu HJ, Shen Z, Rurack K.
Influence of the meso-substituent on strongly red emitting phenanthrene-fused boron–dipyrromethene (BODIPY) fluorophores with a propeller-like conformation.
J Photochem Photobiol A. 2018; 352: 98-105.Highly emissive phenanthrene-fused boron–dipyrromethene (PBDP) dyes have been spectroscopically characterized in a series of solvents. The influence of different substituents (−H, −I, −CN, −DMA or a 15C5-crown ether) in the para-position of a phenyl ring attached to the meso-position of the BODIPY core is discussed. This family of dyes has an intense emission at λ ≥ 630 nm, with fluorescence quantum yields between 0.7 and 1.0 in all solvents studied, except in the case of the dimethylamino-substituted derivative, PBDP-DMA, which undergoes excited-state intramolecular charge transfer (CT), leading to broadband dual fluorescence in highly polar solvents. Introduction of a weaker electron donor such as a benzocrown to the meso-position is not able to trigger a second (charge or electron transfer) process and, interestingly, heavy atom (iodine, PBDP-I derivative) substitution at that moiety does also not have a relevant influence on the photophysics, i.e., enhanced intersystem crossing was not observed. Electrochemical studies ... [truncated at 150 words]
Friedman JE, Dobrinskikh E, Alfonso-Garcia A, Fast A, Janssen RC, Soderborg TK, Anderson AL, Reisz JA, D'Alessandro A, Frank DN, Robertson CE, de Houssaye BAl, Johnson LK, Orlicky DJ, Wang XX, Levi M, Potma EO, Kasmi KCE, Jonscher KR.
Pyrroloquinoline quinone prevents developmental programming of microbial dysbiosis and macrophage polarization to attenuate liver fibrosis in offspring of obese mice.
Hepatol Commun. 2018; 2(3): 313-328. PMCID: PMC5831029Increasingly, evidence suggests that exposure to maternal obesity creates an inflammatory environment in utero, exerting long‐lasting postnatal signatures on the juvenile innate immune system and microbiome that may predispose offspring to development of fatty liver disease. We found that exposure to a maternal Western‐style diet (WD) accelerated fibrogenesis in the liver of offspring and was associated with early recruitment of proinflammatory macrophages at 8‐12 weeks and microbial dysbiosis as early as 3 weeks of age. We further demonstrated that bone marrow‐derived macrophages (BMDMs) were polarized toward an inflammatory state at 8 weeks of age and that a potent antioxidant, pyrroloquinoline quinone (PQQ), reversed BMDM metabolic reprogramming from glycolytic toward oxidative metabolism by restoring trichloroacetic acid cycle function at isocitrate dehydrogenase. This resulted in reduced inflammation and inhibited collagen fibril formation in the liver at 20 weeks of age, even when PQQ was withdrawn at 3 weeks of age. Beginning at ... [truncated at 150 words]
García C, Losada A, Sacristán MA, Martínez-Leal JF, Galmarini CM, Lillo MP.
Dynamic cellular maps of molecular species: Application to drug-target interactions.
Sci Rep. 2018; 8(1), 1140. PMCID: PMC5773516The design of living cell studies aimed at deciphering the mechanism of action of drugs targeting proteins with multiple functions, expressed in a wide range of concentrations and cellular locations, is a real challenge. We recently showed that the antitumor drug plitidepsin (APL) localizes sufficiently close to the elongation factor eEF1A2 so as to suggest the formation of drug-protein complexes in living cells. Here we present an extension of our previous micro-spectroscopy study, that combines Generalized Polarization (GP) images, with the phasor approach and fluorescence lifetime imaging microscopy (FLIM), using a 7-aminocoumarin drug analog (APL*) as fluorescence tracer. Using the proposed methodology, we were able to follow in real time the formation and relative distribution of two sets of APL-target complexes in live cells, revealing two distinct patterns of behavior for HeLa-wt and APL resistant HeLa-APL-R cells. The information obtained may complement and facilitate the design of new experiments and ... [truncated at 150 words]
Cielecki PP, Sobolewska EK, Kostiučenko O, Leißner T, Tamulevičius T, Tamulevičius S, Rubahn HG, Adam J, Fiutowski J.
Plasmon-organic fiber interactions in diamond-like carbon coated nanostructured gold films.
Opt Commun. 2017; 402: 635-640.Gold is the most commonly used plasmonic material, however soft and prone to mechanical deformations. It has been shown that the durability of gold plasmonic substrates can be improved by applying a protective diamond-like carbon (DLC) coating. In this work, we investigate the influence of such protective layers on plasmonic interactions in organic–plasmonic hybrid systems. We consider systems, consisting of 1-Cyano-quaterphenylene nanofibers on top of gold nano-square plasmonic arrays, coated with protective layers of varying thickness. We numerically investigate the spectral position of surface plasmon polariton resonances and electric field intensity, as a function of protective layer thickness, using the finite-difference time-domain method. To confirm the numerically indicated field enhancement preservation on top of protective layers, we experimentally map the second harmonic response of organic nanofibers. Subsequently, we characterize the plasmonic coupling between organic nanofibers and underlying substrates, considered as one of the main loss channels for photoluminescence from nanofibers, ... [truncated at 150 words]
Sena F, Sotelo-Silveira M, Astrada S, ABotella M, Malacrida L, Borsani O.
Spectral phasor analysis reveals altered membrane order and function of root hair cells in Arabidopsis dry2/sqe1-5 drought hypersensitive mutant.
Plant Physiol Biochem. 2017; 119: 224-231.Biological membranes allow the regulation of numerous cellular processes, which are affected when unfavorable environmental factors are perceived. Lipids and proteins are the principal components of biological membranes. Each lipid has unique biophysical properties, and, therefore the lipid composition of the membrane is critical to maintaining the bilayer structure and functionality. Membrane composition and integrity are becoming the focus of studies aiming to understand how plants adapt to its environment.
In this study, using a combination of di-4-ANEPPDHQ fluorescence and spectral phasor analysis, we report that the drought hypersensitive/squalene epoxidase (dry2/sqe1-5) mutant with reduced major sterols such as sitosterol and stigmasterol in roots presented higher membrane fluidity than the wild type. Moreover, analysis of endomembrane dynamics showed that vesicle formation was affected in dry2/sqe1-5. Further analysis of proteins associated with sterol rich micro domains showed that dry2/sqe1-5 presented micro domains function altered.
Hsu K, Lee TY, Periasamy A, Kao FJ, Li LT, Lin CY, Lin HJ, Lin M.
Adaptable interaction between aquaporin-1 and band 3 reveals a potential role of water channel in blood CO2 transport.
FASEB J. 2017; 31(10): 4256-4264. PMCID: PMC6207180Human CO2 respiration requires rapid conversion between CO2 and HCO3−. Carbonic anhydrase II facilitates this reversible reaction inside red blood cells, and band 3 [anion exchanger 1 (AE1)] provides a passage for HCO3− flux across the cell membrane. These 2 proteins are core components of the CO2 transport metabolon. Intracellular H2O is necessary for CO2/HCO3− conversion. However, abundantly expressed aquaporin 1 (AQP1) in erythrocytes is thought not to be part of band 3 complexes or the CO2 transport metabolon. To solve this conundrum, we used Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging (FLIM-FRET) and identified interaction between aquaporin-1 and band 3 at a distance of 8 nm, within the range of dipole–dipole interaction. Notably, their interaction was adaptable to membrane tonicity changes. This suggests that the function of AQP1 in tonicity response could be coupled or correlated to its function in band 3-mediated CO2/HCO3− exchange. By demonstrating ... [truncated at 150 words]
Sadybekov A, Tian C, Arnesano C, Katritch V, Herring BE.
An autism spectrum disorder-related de novo mutation hotspot discovered in the GEF1 domain of Trio.
Nat Commun. 2017; 8(1), 601. PMCID: PMC5605661The Rho guanine nucleotide exchange factor (RhoGEF) Trio promotes actin polymerization by directly activating the small GTPase Rac1. Recent studies suggest that autism spectrum disorder (ASD)-related behavioral phenotypes in animal models of ASD can be produced by dysregulation of Rac1’s control of actin polymerization at glutamatergic synapses. Here, in humans, we discover a large cluster of ASD-related de novo mutations in Trio’s Rac1 activating domain, GEF1. Our study reveals that these mutations produce either hypofunctional or hyperfunctional forms of Trio in rodent neurons in vitro. In accordance with pathological increases or decreases in glutamatergic neurotransmission observed in animal models of ASD, we find that these mutations result in either reduced synaptic AMPA receptor expression or enhanced glutamatergic synaptogenesis. Together, our findings implicate both excessive and reduced Trio activity and the resulting synaptic dysfunction in ASD-related pathogenesis, and point to the Trio-Rac1 pathway at glutamatergic synapses as a possible key point ... [truncated at 150 words]
Doll F, Hassenrück J, Wittmann V, Zumbusch A.
Intracellular imaging of protein-specific glycosylation.
Methods in Enzymology. 2017; 598: 283-319.Posttranslational protein glycosylation is conserved in all kingdoms of life and implicated in the regulation of protein structure, function, and localization. The visualization of glycosylation states of designated proteins within living cells is of great importance for unraveling the biological roles of intracellular protein glycosylation. Our generally applicable approach is based on the incorporation of a glucosamine analog, Ac4GlcNCyoc, into the cellular glycome via metabolic engineering. Ac4GlcNCyoc can be labeled in a second step via inverse-electron-demand Diels–Alder chemistry with fluorophores inside living cells. Additionally, target proteins can be expressed as enhanced green fluorescent protein (EGFP)-fusion proteins. To assess the proximity of the donor EGFP and the glycan-anchored acceptor fluorophore, Förster resonance energy transfer (FRET) is employed and read out with high contrast by fluorescence lifetime imaging (FLIM) microscopy. In this chapter, we present a detailed description of methods required to perform protein-specific imaging of glycosylation inside living cells. These include ... [truncated at 150 words]
Strom AR, Emelyanov AV, Mir M, Fyodorov DV, Darzacq X, Karpen GH.
Phase separation drives heterochromatin domain formation.
Nature. 2017; 547(7622): 241-245. PMCID: PMC6022742Constitutive heterochromatin is an important component of eukaryotic genomes that has essential roles in nuclear architecture, DNA repair and genome stability1, and silencing of transposon and gene expression2. Heterochromatin is highly enriched for repetitive sequences, and is defined epigenetically by methylation of histone H3 at lysine 9 and recruitment of its binding partner heterochromatin protein 1 (HP1). A prevalent view of heterochromatic silencing is that these and associated factors lead to chromatin compaction, resulting in steric exclusion of regulatory proteins such as RNA polymerase from the underlying DNA3. However, compaction alone does not account for the formation of distinct, multi-chromosomal, membrane-less heterochromatin domains within the nucleus, fast diffusion of proteins inside the domain, and other dynamic features of heterochromatin. Here we present data that support an alternative hypothesis: that the formation of heterochromatin domains is mediated by phase separation, a phenomenon that gives rise to diverse non-membrane-bound nuclear, cytoplasmic and ... [truncated at 150 words]
Cheniour M, Brewer J, Bagatolli L, Marcillat O, Granjon T.
Evidence of proteolipid domain formation in an inner mitochondrial membrane mimicking model.
Biochim Biophys Acta. 2017; 1861(5): 969-976.Background
Mitochondrial creatine kinase (mtCK) is highly abundant in mitochondria; its quantity is equimolecular to the Adenylic Nucleotide Translocator and represents 1% of the mitochondrial proteins. It is a multitask protein localized in the mitochondria intermembrane space where it binds to the specific cardiolipin (CL) phospholipid. If mtCK was initially thought to be exclusively implicated in energy transfer between mitochondria and cytosol through a mechanism referred to as the phosphocreatine shuttle, several recent studies suggested an additional role in maintaining mitochondria membrane structure.
Methods
To further characterized mtCK binding process we used multiphoton excitation fluorescence microscopy coupled with Giant Unilamellar Vesicles (GUV) and laurdan as fluorescence probe.
Results
We gathered structural and dynamical information on the molecular events occurring during the binding of mtCK to the mitochondria inner membrane.
We present the first visualization of mtCK-induced CL segregation on a bilayer model forming micrometer-size proteolipid domains at the surface of the GUV. Those microdomains, which only ... [truncated at 150 words]
Eriksen AZ, Brewer J, Andresen TL, Urquhart AJ.
The diffusion dynamics of PEGylated liposomes in the intact vitreous of the ex vivo porcine eye: A fluorescence correlation spectroscopy and biodistribution study.
Int J Pharm. 2017; 522(1-2): 90-97.The diffusion dynamics of nanocarriers in the vitreous and the influence of nanocarrier physicochemical properties on these dynamics is an important aspect of the efficacy of intravitreal administered nanomedicines for the treatment of posterior segment eye diseases. Here we use fluorescence correlation spectroscopy (FCS) to determine liposome diffusion coefficients in the intact vitreous (DVit) of ex vivo porcine eyes using a modified Miyake-Apple technique to minimize the disruption of the vitreous fine structure. We chose to investigate whether the zeta potential of polyethylene glycol functionalized (i.e. PEGylated) liposomes altered liposome in situ diffusion dynamics in the vitreous. Non-PEGylated cationic nanocarriers have previously shown little to no diffusion in the vitreous, whilst neutral and anionic have shown diffusion. The liposomes investigated had diameters below 150 nm and zeta potentials ranging from −20 to +12 mV. We observed that PEGylated cationic liposomes had significantly lower DVit values (1.14 μm2s−1) than PEGylated neutral ... [truncated at 150 words]
Pelras T, Duong HTT, Kim BJ, Hawkett BS, Müllner M.
A 'grafting from' approach to polymer nanorods for pH-triggered intracellular drug delivery.
Polymer. 2017; 112: 244-251.We report the use of the ‘grafting from’ approach to produce inherently rod-shaped polymer nanoparticles with triggered drug release. Cylindrical polymer brushes (CPBs) can be directly used to yield functional polymer nanorods for pH-sensitive drug release of doxorubicin (DOX). Water-soluble CPBs have been produced via a straightforward one-step grafting of vinyl benzaldehyde (VBA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) comonomers, in which the VBA distributed throughout the CPBs provides a cost-effective and simple functionality for the subsequent conjugation of DOX using imine chemistry. Atomic force microscopy (AFM) underlined the rod-like conformation of the CPBs prior and after drug conjugation. Fluorescence spectroscopy studies revealed faster drug release in acidic environments (pH 5.0) compared to physiological pH conditions (pH 7.4). Fluorescence lifetime imaging microscopy (FLIM) and in vitro cell studies further highlighted the intracellular DOX release from the CPB drug carriers within MCF-7 breast cancer cells.
Modzel M, Lund FW, Wüstner D.
Synthesis and live-cell imaging of fluorescent sterols for analysis of intracellular cholesterol transport.
Cholesterol Homeostasis (Methods in Molecular Biology, Vol. 1583). By I Gelissen and A Brown (Editors). pp. 111-140, 2017. Cellular cholesterol homeostasis relies on precise control of the sterol content of organelle membranes. Obtaining insight into cholesterol trafficking pathways and kinetics by live-cell imaging relies on two conditions. First, one needs to develop suitable analogs that resemble cholesterol as closely as possible with respect to their biophysical and biochemical properties. Second, the cholesterol analogs should have good fluorescence properties. This interferes, however, often with the first requirement, such that the imaging instrumentation must be optimized to collect photons from suboptimal fluorophores, but good cholesterol mimics, such as the intrinsically fluorescent sterols, cholestatrienol (CTL) or dehydroergosterol (DHE). CTL differs from cholesterol only in having two additional double bonds in the ring system, which is why it is slightly fluorescent in the ultraviolet (UV). In the first part of this protocol, we describe how to synthesize and image CTL in living cells relative to caveolin, a structural component of caveolae. In ... [truncated at 150 words]
Jones DM, Alvarez LA, Nolan R, Ferriz M, Urruela RS, Massana-Muñoz X, Novak-Kotzer H, Dustin ML, Padilla-Parra S.
Dynamin-2 stabilizes the HIV-1 fusion pore with a low oligomeric state.
Cell Rep. 2017; 18(2): 443-453. PMCID: PMC5263234One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry, its exact role during the first steps of HIV-1 infection is not well characterized. Here, we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall, our data suggest that dynamin-2, as a tetramer, might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion.
Palominos MA, Vilches D, Bossel E, Soto-Arriazza MA.
Interaction between amphipathic triblock copolymers and L-α-dipalmitoyl phosphatidylcholine large unilamellar vesicles.
Colloids Surf B Biointerfaces. 2016; 148: 30-40.This study contributes to an understanding of how different polymeric structures, in special triblock copolymers can interact with the lipid bilayer. To study the phospholipid-copolymer vesicles system, we report the effect of two amphipathic triblock copolymers of the type BAB, i.e., hydrophobic-hydrophilic-hydrophobic triblock copolymers arranged as poly(ε-caprolactone)-poly(ethylene oxide)-poly(ε-caprolactone) (PCLn-PEOm-PCLn), where n=12 and m=45 for COP1 and n=16 and m=104 for COP2, on the dynamic and structural properties of dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles (LUVs). The interaction between the copolymers and DPPC LUVs was evaluated by means of several techniques: (a) Photographs of the dispersion for evaluation of colloidal stability; (b) Thermotropic behavior from generalized polarization of Laurdan and fluorescence anisotropy of DPH (c) Main phase transition temperature determination; (d) Order parameters and limiting anisotropy by time-resolved fluorescence anisotropy measurements; (e) Water outflow through the lipid bilayer and (f) Calcein release from DPPC LUVs. Steady-state fluorescence measurements as a function of ... [truncated at 150 words]
Wrona-Piotrowicz A, Ciechańska M, Zakrzewski J, Makal A.
Pyrene fluorophores bearing two carbonyl groups in 1,2- positions: Synthesis and photophysical properties of pyrene-1,2-dicarboximides and a pyrene-1,2-dicarboxamide.
J Photochem Photobiol A. 2016; 330: 15-21.Directed lithiation of pyrene-1-carboxamides, followed by reaction with DMF, afforded 7-hydroxy-8,9-dihydro-7H-phenaleno-[1,9-ef]isoindoles, which were oxidized with Jones reagent to the corresponding pyrene-1,2-dicarboximides. Similarly, the reaction of lithiated N-tert-butylpyrene-1-carboxamide with tert-butyl isocyanate afforded N,N’-di-tert-butylpyrene-1,2-dicarboxamide. The electronic structure and photophysical properties of these compounds were studied by means of theoretical (TD DFT) calculations and experimental (steady-state and time-resolved fluorescence) methods and were compared with those of their pyrene-1- monocarbonyl counterparts. The obtained results reveal the considerable influence of the carbonyl group at the 2-position on the structure and luminescent properties of this class of pyrenyl fluorophores.
Blackwell DJ, Zak TJ, Robia SL.
Cardiac Calcium ATPase dimerization measured by cross-linking and fluorescence energy transfer.
Biophys J. 2016; 111(6): 1192-1202. PMCID: PMC5034344The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, ... [truncated at 150 words]
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Future perspective of single-molecule FRET biosensors and intravital FRET microscopy.
Biophys J. 2016; 111(6): 1103-1111. PMCID: PMC5034264Förster (or fluorescence) resonance energy transfer (FRET) is a nonradiative energy transfer process between two fluorophores located in close proximity to each other. To date, a variety of biosensors based on the principle of FRET have been developed to monitor the activity of kinases, proteases, GTPases or lipid concentration in living cells. In addition, generation of biosensors that can monitor physical stresses such as mechanical power, heat, or electric/magnetic fields is also expected based on recent discoveries on the effects of these stressors on cell behavior. These biosensors can now be stably expressed in cells and mice by transposon technologies. In addition, two-photon excitation microscopy can be used to detect the activities or concentrations of bioactive molecules in vivo. In the future, more sophisticated techniques for image acquisition and quantitative analysis will be needed to obtain more precise FRET signals in spatiotemporal dimensions. Improvement of tissue/organ position fixation methods for ... [truncated at 150 words]
Kim YE, Hosp F, Frottin F, Ge H, Mann M, Hayer-Hartl M, Hartl FU.
Soluble oligomers of PolyQ-expanded Huntingtin target a multiplicity of key cellular factors.
Mol Cell. 2016; 63(6): 951-964.Huntington’s disease is one of several neurodegenerative disorders characterized by the aggregation of polyglutamine (polyQ)-expanded mutant protein. How polyQ aggregation leads to cellular dysfunction is not well understood. Here, we analyzed aberrant protein interactions of soluble oligomers and insoluble inclusions of mutant huntingtin using in-cell single molecule fluorescence spectroscopy and quantitative proteomics. We find that the interactome of soluble oligomers is highly complex, with an enrichment of RNA-binding proteins as well as proteins functioning in ribosome biogenesis, translation, transcription, and vesicle transport. The oligomers frequently target proteins containing extended low-complexity sequences, potentially interfering with key cellular pathways. In contrast, the insoluble inclusions are less interactive and associate strongly with protein quality control components, such as Hsp40 chaperones and factors of the ubiquitin-proteasome system. Our results suggest a “multiple hit” model for the pathogenic effects of mutant huntingtin, with soluble forms engaging more extensively in detrimental interactions than insoluble aggregates.
Celli A, Crumrine D, Meyer JM, Mauro TM.
Endoplasmic reticulum Calcium regulates epidermal barrier response and desmosomal structure.
J Invest Dermatol. 2016; 136(9): 1840-1847. PMCID: PMC5070468Ca(2+) fluxes direct keratinocyte differentiation, cell-to-cell adhesion, migration, and epidermal barrier homeostasis. We previously showed that intracellular Ca(2+) stores constitute a major portion of the calcium gradient especially in the stratum granulosum. Loss of the calcium gradient triggers epidermal barrier homeostatic responses. In this report, using unfixed ex vivo epidermis and human epidermal equivalents we show that endoplasmic reticulum (ER) Ca(2+) is released in response to barrier perturbation, and that this release constitutes the major shift in epidermal Ca(2+) seen after barrier perturbation. We find that ER Ca(2+) release correlates with a transient increase in extracellular Ca(2+). Lastly, we show that ER calcium release resulting from barrier perturbation triggers transient desmosomal remodeling, seen as an increase in extracellular space and a loss of the desmosomal intercellular midline. Topical application of thapsigargin, which inhibits the ER Ca(2+) ATPase activity without compromising barrier integrity, also leads to desmosomal remodeling and loss of ... [truncated at 150 words]
Volz P, Krause N, Balke J, Schneider C, Walter M, Schneider F, Schlesinger R, Alexiev U.
Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2.
J Biol Chem. 2016; 291: 17382-17393. PMCID: PMC5016135A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest ... [truncated at 150 words]
Chiu CL, Patsch K, Cutrale F, Soundararajan A, Agus DB, Fraser SE, Ruderman D.
Intracellular kinetics of the androgen receptor shown by multimodal Image Correlation Spectroscopy (mICS).
Sci Rep. 2016; 6: 22435. PMCID: PMC4776155The androgen receptor (AR) pathway plays a central role in prostate cancer (PCa) growth and progression and is a validated therapeutic target. In response to ligand binding AR translocates to the nucleus, though the molecular mechanism is not well understood. We therefore developed multimodal Image Correlation Spectroscopy (mICS) to measure anisotropic molecular motion across a live cell. We applied mICS to AR translocation dynamics to reveal its multimodal motion. By integrating fluorescence imaging methods we observed evidence for diffusion, confined movement, and binding of AR within both the cytoplasm and nucleus of PCa cells. Our findings suggest that in presence of cytoplasmic diffusion, the probability of AR crossing the nuclear membrane is an important factor in determining the AR distribution between cytoplasm and the nucleus, independent of functional microtubule transport. These findings may have implications for the future design of novel therapeutics targeting the AR pathway in PCa.
Wrona-Piotrowicz A, Ciechańska M, Zakrzewski J, Métivier R, Brosseau A, Makal A.
Directed lithiation of a pyrene-1-carboxamide as a route to new pyrenyl fluorophores.
Dyes and Pigments. 2016; 125: 331-338.The lithiation of N-tert-butylpyrene-1-carboxamide with nBuLi-TMEDA in THF at −78 °C afforded, after quenching with chlorosilanes, the corresponding 2-(trialkylsilyl)pyrene-1-carboxamides. When DMF and diethyl oxalate were used as quenchers compounds having 8-tert-butyl-7-hydroxy-8,9-dihydro-7H-phenaleno-[1,9-ef]isoindole skeleton were obtained. The oxidation of compound having secondary OH group with Jones’ reagent afforded 8-tert-butyl-7-hydroxy-8,9-dihydro-7H-phenaleno-[1,9-ef]isoindole-7,9(8H)dione. All of the synthesized compounds displayed fluorescence in solution (λmax = 425–451 nm; ΦF = 15.1–40.8%) and in the solid state ((λmax = 410–555 nm; ΦF = 6–40%). The silylated amides display in the solid state monomer emission whereas in the case of the of phenalenoisoindole derivatives the emitting species are π-stacked aggregates. The formation of aggregates in the crystals of the latter compounds was confirmed by a single-crystal X-ray diffraction study.
Constantine M, Liew CK, Lo V, Macmillan A, Cranfield CG, Sunde M, Whan R, Graham RM, Martinac B.
Heterologously-expressed and Liposome-reconstituted Human Transient Receptor Potential Melastatin 4 Channel (TRPM4) is a Functional Tetramer.
Sci Rep. 2016; 6: 19352. PMCID: PMC4726259Mutation, irregular expression and sustained activation of the Transient Receptor Potential Channel, type Melastatin 4 (TRPM4), have been linked to various cardiovascular diseases. However, much remains unknown about the structure of this important ion channel. Here, we have purified a heterologously expressed TRPM4-eGFP fusion protein and investigated the oligomeric state of TRPM4-eGFP in detergent micelles using crosslinking, native gel electrophoresis, multi-angle laser light scattering and electron microscopy. Our data indicate that TRPM4 is tetrameric, like other TRP channels studied to date. Furthermore, the functionality of liposome reconstituted TRPM4-eGFP was examined using electrophysiology. Single-channel recordings from TRPM4-eGFP proteoliposomes showed inhibition of the channel using Flufenamic acid, a well-established inhibitor of TRPM4, suggesting that the channels are functional upon reconstitution. Our characterisation of the oligomeric structure of TRPM4 and the ability to reconstitute functional channels in liposomes should facilitate future studies into the structure, function and pharmacology of this therapeutically relevant channel.
Dreier J, Sørensen JA, Brewer JR.
Superresolution and fluorescence dynamics evidence reveal that intact liposomes do not cross the human skin barrier.
PLoS One. 2016; 11(1): e0146514. PMCID: PMC4709185In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer ... [truncated at 150 words]
Johnson KA, Taghon GJF, Scott JL, Stahelin RV.
The Ebola Virus matrix protein, VP40, requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for extensive oligomerization at the plasma membrane and viral egress.
Sci Rep. 2016; 6: 19125. PMCID: PMC4709572VP40 is one of eight proteins encoded by the Ebola Virus (EBOV) and serves as the primary matrix protein, forming virus like particles (VLPs) from mammalian cells without the need for other EBOV proteins. While VP40 is required for viral assembly and budding from host cells during infection, the mechanisms that target VP40 to the plasma membrane are not well understood. Phosphatidylserine is required for VP40 plasma membrane binding, VP40 hexamer formation, and VLP egress, However, PS also becomes exposed on the outer membrane leaflet at sites of VP40 budding, raising the question of how VP40 maintains an interaction with the plasma membrane inner leaflet when PS is flipped to the opposite side. To address this question, cellular and in vitro assays were employed to determine if phosphoinositides are important for efficient VP40 localization to the plasma membrane. Cellular studies demonstrated that PI(4,5)P2 was an important component of VP40 assembly ... [truncated at 150 words]
Ferri G, Nucara L, Biver T, Battisti A, Signore G, Bizzarri R.
Organization of inner cellular components as reported by a viscosity-sensitive fluorescent Bodipy probe suitable for phasor approach to FLIM.
Biophys Chem. 2016; 208: 17-25.According to the recent developments in imaging strategies and in tailoring fluorescent molecule as probe for monitoring biological systems, we coupled a Bodipy-based molecular rotor (BoMe) with FLIM phasor approach to evaluate the viscosity in different intracellular domains. BoMe rapidly permeates cells, stains cytoplasmic as well as nuclear domains, and its optical properties make it perfectly suited for widely diffused confocal microscopy imaging setups. The capability of BoMe to report on intracellular viscosity was put to the test by using a cellular model of a morbid genetic pathology (Hutchinson–Gilford progeria syndrome, HGPS). Our results show that the nucleoplasm of HGPS cells display reduced viscosity as compared to normal cells. Since BoMe displays significant affinity towards DNA, as demonstrated by an in vitro essay, we hypothesize that genetic features of HGPS, namely the misassembly of lamin A protein within the nuclear lamina, modulates chromatin compaction. This hypothesis nicely agrees with literature ... [truncated at 150 words]
Macchi S, Signore G, Boccardi C, di Rienzo C, Beltram F, Cardarelli F.
Spontaneous membrane-translocating peptides: influence of peptide self-aggregation and cargo polarity.
Sci Rep. 2015; 16(5): 16914. PMCID: PMC4645181Peptides that translocate spontaneously across cell membranes could transform the field of drug delivery by enabling the transport of otherwise membrane-impermeant molecules into cells. In this regard, a 9-aminoacid-long motif (representative sequence: PLIYLRLLR, hereafter Translocating Motif 9, TM9) that spontaneously translocates across membranes while carrying a polar dye was recently identified by high-throughput screening. Here we investigate its transport properties by a combination of in cuvette physico-chemical assays, rational mutagenesis, live-cell confocal imaging and fluorescence correlation spectroscopy measurements. We unveil TM9 ability to self-aggregate in a concentration-dependent manner and demonstrate that peptide self-aggregation is a necessary –yet not sufficient– step for effective membrane translocation. Furthermore we show that membrane crossing can occur with apolar payloads while it is completely inhibited by polar ones. These findings are discussed and compared to previous reports. The present results impose a careful rethinking of this class of sequences as direct-translocation vectors suitable for delivery ... [truncated at 150 words]
Kurniawansyah F, Duonga HTT, Luu TD, Mammucari R, Vittorio O, Boyer C, Foster N.
Inhalable curcumin formulations: Micronization and bioassay.
Chem Eng J. 2015; 279: 799–808.Inhalable formulations of curcumin with hydroxypropyl-β-cyclodextrin and polyvinylpyrrolidone have been produced by a newly developed anti-solvent micronization technique based on the supercritical fluid (SCF) technology. The micronization process used is the atomized rapid injection solvent extraction process (ARISE), which utilizes high pressure carbon dioxide as the anti-solvent. The composition, particle morphology and aerodynamic performance of the samples were observed. Curcumin formulations with aerodynamic performance suitable for inhalation delivery have been produced and tested in vitro using normal and lung cancer cells (MRC-5 cells and H1299 cells, respectively). The cytotoxicity study revealed that the encapsulation of curcumin improved its biodistribution and solubility for lung cancer cells. A rapid cell uptake was observed for all formulations by confocal microscopy and flow cytometry. Fluorescence life time imaging microscopy showed a rapid release of curcumin in the cells. Results show that co-processing by ARISE preserved the anticancer properties of curcumin and generated inhalable powders ... [truncated at 150 words]
Golebiewska U, Scarlata S.
High pressure promotes alpha-synuclein aggregation in cultured neuronal cells.
FEBS Letters. 2015; 589(21): 3309-3312. PMCID: PMC4661088α-Synuclein is found in plaques associated with Parkinson’s and other neurodegenerative diseases. Changes in α-synuclein oligomerization are thought to give rise to nucleation of neurodegenerative plaques. Here, we investigated the effect of hydrostatic pressure on the aggregation of α-synuclein in cultured neuronal cells. We found that hydrostatic pressure is associated with a transition from monomeric to higher order α-synuclein aggregates. We then tested whether this aggregation is associated with the loss of binding partners, such as phospholipase Cβ. We found that increased pressure reduces the level of PLCβ1 and the amount of α-synuclein/PLCβ1 complexes. These studies suggest that pressure promotes release of α-synuclein from protein partners promoting its oligomerization.
Molina-Guijarro JM, García C, Macías A, García-Fernández LF, Moreno C, Reyes F, Martínez-Leal JF, Fernández R, Martínez V, Valenzuela C, Lillo MP, Galmarini CM.
Elisidepsin interacts directly with Glycosylceramides in the plasma membrane of tumor cells to induce necrotic cell death.
PLoS One. 2015; 10(10): e0140782. PMCID: PMC4608773Plasma membrane integrity is essential for cell life. Any major break on it immediately induces the death of the affected cell. Different molecules were described as disrupting this cell structure and thus showing antitumor activity. We have previously defined that elisidepsin (Irvalec®, PM02734) inserts and self-organizes in the plasma membrane of tumor cells, inducing a rapid loss of membrane integrity, cell permeabilization and necrotic death. Here we show that, in sensitive HCT-116 colorectal cells, all these effects are consequence of the interaction of elisidepsin with glycosylceramides in the cell membrane. Of note, an elisidepsin-resistant subline (HCT-116-Irv) presented reduced levels of glycosylceramides and no accumulation of elisidepsin in the plasma membrane. Consequently, drug treatment did not induce the characteristic necrotic cell death. Furthermore, GM95, a mutant derivative from B16 mouse melanoma cells lacking ceramide glucosyltransferase (UGCG) activity and thus the synthesis of glycosylceramides, was also resistant to elisidepsin. Over-expression of UGCG ... [truncated at 150 words]
Kluba M, Engelborghs Y, Hofkens J, Mizuno H.
Inhibition of receptor dimerization as a novel negative feedback mechanism of EGFR signaling.
PLoS One. 2015; 10(10): e0139971. PMCID: PMC4605717Dimerization of the epidermal growth factor receptor (EGFR) is crucial for initiating signal transduction. We employed raster image correlation spectroscopy to continuously monitor the EGFR monomer-dimer equilibrium in living cells. EGFR dimer formation upon addition of EGF showed oscillatory behavior with a periodicity of about 2.5 min, suggesting the presence of a negative feedback loop to monomerize the receptor. We demonstrated that monomerization of EGFR relies on phospholipase Cγ, protein kinase C, and protein kinase D (PKD), while being independent of Ca2+ signaling and endocytosis. Phosphorylation of the juxtamembrane threonine residues of EGFR (T654/T669) by PKD was identified as the factor that shifts the monomer-dimer equilibrium of ligand bound EGFR towards the monomeric state. The dimerization state of the receptor correlated with the activity of an extracellular signal-regulated kinase, downstream of the EGFR. Based on these observations, we propose a novel, negative feedback mechanism that regulates EGFR signaling via receptor ... [truncated at 150 words]
Wrona-Piotrowicz A, Plażuk D, Zakrzewski J, Métivier R, Nakatani K, Makal A.
Solution-and solid-state emitters with large Stokes shifts combining pyrene and 4-hydroxythiazole fluorophores.
Dyes and Pigments. 2015; 121: 290-298.4-Hydroxy-5-nitrophenyl-2-(pyren-1-yl)thiazole and a series of its O-substituted derivatives were synthesised. These compounds are fluorescent in solution, emitting light in the region 598–626 nm with quantum yields 0.19–0.29. Large Stokes shifts, approaching 8500 cm−1, were explained by the intramolecular charge transfer character of the lowest excited state, confirmed by TD DFT calculations. The O-substituted compounds also exhibited fluorescence in the solid state. The lack of solid-state emissive properties of the parent hydroxythiazole and the emission of its O-acetyl derivative are discussed in terms of their crystal packings.
van Maarschalkerweerd A, Vetri V, Vestergaard B.
Cholesterol facilitates interactions between α-synuclein oligomers and charge-neutral membranes.
Febs Lett. 2015; 589(19): 2661-2667.Oligomeric species formed during α-synuclein fibrillation are suggested to be membrane-disrupting agents, and have been associated with cytotoxicity in Parkinson’s disease. The majority of studies, however, have revealed that the effect of α-synuclein oligomers is only noticeable on systems composed of anionic lipids, while the more physiologically relevant zwitterionic lipids remain intact. We present experimental evidence for significant morphological changes in zwitterionic membranes containing cholesterol, induced by α-synuclein oligomers. Depending on the lipid composition, model membranes are either unperturbed, disrupt, or undergo dramatic morphological changes and segregate into structurally different components, which we visualize by 2-photon fluorescence microscopy and generalized polarization analysis using the fluorescent probe Laurdan. Our results highlight the crucial role of cholesterol for mediating interactions between physiologically relevant membranes and α-synuclein.
Biswas KH, Hartman KL, Yu Ch, Harrison OJ, Song H, Smith AW, Huang WYC, Lin WC, Guo Z, Padmanabhan A, Troyanovsky SM, Dustin ML, Shapiro L, Honig B, Zaidel-Bar R, Groves JT.
E-cadherin junction formation involves an active kinetic nucleation process.
Proc Natl Acad Sci USA. 2015; 112(35): 10932-10937. PMCID: PMC4568248Epithelial (E)-cadherin-mediated cell−cell junctions play important roles in the development and maintenance of tissue structure in multicellular organisms. E-cadherin adhesion is thus a key element of the cellular microenvironment that provides both mechanical and biochemical signaling inputs. Here, we report in vitro reconstitution of junction-like structures between native E-cadherin in living cells and the extracellular domain of E-cadherin (E-cad-ECD) in a supported membrane. Junction formation in this hybrid live cell-supported membrane configuration requires both active processes within the living cell and a supported membrane with low E-cad-ECD mobility. The hybrid junctions recruit α-catenin and exhibit remodeled cortical actin. Observations suggest that the initial stages of junction formation in this hybrid system depend on the trans but not the cis interactions between E-cadherin molecules, and proceed via a nucleation process in which protrusion and retraction of filopodia play a key role.
Nieves DJ, Li Y, Fernig DG, Lévy R.
Photothermal raster image correlation spectroscopy of gold nanoparticles in solution and on live cells.
R Soc Open Sci. 2015; 2(6): 140454. PMCID: PMC4632534Raster image correlation spectroscopy (RICS) measures the diffusion of fluorescently labelled molecules from stacks of confocal microscopy images by analysing correlations within the image. RICS enables the observation of a greater and, thus, more representative area of a biological system as compared to other single molecule approaches. Photothermal microscopy of gold nanoparticles allows long-term imaging of the same labelled molecules without photobleaching. Here, we implement RICS analysis on a photothermal microscope. The imaging of single gold nanoparticles at pixel dwell times short enough for RICS (60 μs) with a piezo-driven photothermal heterodyne microscope is demonstrated (photothermal raster image correlation spectroscopy, PhRICS). As a proof of principle, PhRICS is used to measure the diffusion coefficient of gold nanoparticles in glycerol : water solutions. The diffusion coefficients of the nanoparticles measured by PhRICS are consistent with their size, determined by transmission electron microscopy. PhRICS was then used to probe the diffusion speed of gold nanoparticle-labelled ... [truncated at 150 words]
Angiolini J, Plachta N, Mocskos E, Levi V.
Exploring the dynamics of cell processes through simulations of fluorescence microscopy experiments.
Biophys J. 2015; 108(11): 2613–2618. PMCID: PMC4457496Fluorescence correlation spectroscopy (FCS) methods are powerful tools for unveiling the dynamical organization of cells. For simple cases, such as molecules passively moving in a homogeneous media, FCS analysis yields analytical functions that can be fitted to the experimental data to recover the phenomenological rate parameters. Unfortunately, many dynamical processes in cells do not follow these simple models, and in many instances it is not possible to obtain an analytical function through a theoretical analysis of a more complex model. In such cases, experimental analysis can be combined with Monte Carlo simulations to aid in interpretation of the data. In response to this need, we developed a method called FERNET (Fluorescence Emission Recipes and Numerical routines Toolkit) based on Monte Carlo simulations and the MCell-Blender platform, which was designed to treat the reaction-diffusion problem under realistic scenarios. This method enables us to set complex geometries of the simulation space, distribute ... [truncated at 150 words]
Byers CE, Barylko B, Ross JA, Southworth DR, James NG, IV CAT, Wang L, Collins KA, Estrada A, Waung M, Tassin TC, Huber KM, Jameson DM, Albanesi JP.
Enhancement of dynamin polymerization and GTPase activity by Arc/Arg3.1.
Biochim Biophys Acta. 2015; 1850(6): 1310-1318. PMCID: PMC4398645Background: The Activity-regulated cytoskeleton-associated protein, Arc, is an immediate-early gene product implicated in various forms of synaptic plasticity. Arc promotes endocytosis of AMPA type glutamate receptors and regulates cytoskeletal assembly in neuronal dendrites. Its role in endocytosis may be mediated by its reported interaction with dynamin 2, a 100 kDa GTPase that polymerizes around the necks of budding vesicles and catalyzes membrane scission.
Methods: Enzymatic and turbidity assays are used in this study to monitor effects of Arc on dynamin activity and polymerization. Arc oligomerization is measured using a combination of approaches, including size exclusion chromatography, sedimentation analysis, dynamic light scattering, fluorescence correlation spectroscopy, and electron microscopy.
Results: We present evidence that bacterially-expressed His6-Arc facilitates the polymerization of dynamin 2 and stimulates its GTPase activity under physiologic conditions (37 °C and 100 mM NaCl). At lower ionic strength Arc also stabilizes pre-formed dynamin 2 polymers against GTP-dependent disassembly, thereby prolonging assembly-dependent GTP ... [truncated at 150 words]
Anton H, Taha N, Boutant E, Richert L, Khatter H, Klaholz B, Rondé P, Réal E, de Rocquigny H, Mély Y.
Investigating the cellular distribution and interactions of HIV-1 nucleocapsid protein by quantitative fluorescence microscopy.
PLoS One. 2015; 10(2): e0116921. PMCID: PMC4344342The nucleocapsid protein (NCp7) of the Human immunodeficiency virus type 1 (HIV-1) is a small basic protein containing two zinc fingers. About 2000 NCp7 molecules coat the genomic RNA in the HIV-1 virion. After infection of a target cell, the viral core enters into the cytoplasm, where NCp7 chaperones the reverse transcription of the genomic RNA into the proviral DNA. As a consequence of their much lower affinity for double-stranded DNA as compared to single-stranded RNAs, NCp7 molecules are thought to be released in the cytoplasm and the nucleus of infected cells in the late steps of reverse transcription. Yet, little is known on the cellular distribution of the released NCp7 molecules and on their possible interactions with cell components. Hence, the aim of this study was to identify potential cellular partners of NCp7 and to monitor its intracellular distribution and dynamics by means of confocal fluorescence microscopy, fluorescence lifetime ... [truncated at 150 words]
Sun R, Celli A, Crumrine D, Hupe M, Adame LC, Pennypacker SD, Park K, Uchida Y, Feingold KR, Elias PM, Ilic D, Mauro TM.
Lowered humidity produces human epidermal equivalents with enhanced barrier properties.
Tissue Eng Part C Methods. 2015; 21(1): 15-22. PMCID: PMC4291214Multilayered human keratinocyte cultures increasingly are used to model human epidermis. Until now, studies utilizing human epidermal equivalents (HEEs) have been limited because previous preparations do not establish a normal epidermal permeability barrier. In this report, we show that reducing environmental humidity to 50% relative humidity yields HEEs that closely match human postnatal epidermis and have enhanced repair of the permeability barrier. These cultures display low transepidermal water loss and possess a calcium and pH gradient that resembles those seen in human epidermis. These cultures upregulate glucosylceramide synthase and make normal-appearing lipid lamellar bilayers. The epidermal permeability barrier of these cultures can be perturbed, using the identical tools previously described for human skin, and recover in the same time course seen during in vivo barrier recovery. These cultures will be useful for basic and applied studies on epidermal barrier function.
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Measuring protein dynamics in live cells: protocols and practical considerations for fluorescence fluctuation microscopy.
J Biomed Opt. 2014; 19(9): 090801. PMCID: PMC4183152Quantitative analysis of protein complex stoichiometries and mobilities are critical for elucidating the mechanisms that regulate cellular pathways. Fluorescence fluctuation spectroscopy (FFS) techniques can measure protein dynamics, such as diffusion coefficients and formation of complexes, with extraordinary precision and sensitivity. Complete calibration and characterization of the microscope instrument is necessary in order to avoid artifacts during data acquisition and to capitalize on the full capabilities of FFS techniques. We provide an overview of the theory behind FFS techniques, discuss calibration procedures, provide protocols, and give practical considerations for performing FFS experiments. One important parameter recovered from FFS measurements is the relative molecular brightness that can correlate with oligomerization. Three methods for measuring molecular brightness (fluorescence correlation spectroscopy, photon-counting histogram, and number and brightness analysis) recover similar values when measuring samples under ideal conditions in vitro. However, examples are given illustrating that these different methods used for calculating molecular brightness of ... [truncated at 150 words]
Thiery S, Declairieux C, Tondelier D, Seo G, Geffroy B, Jeannin O, Métivier R, Rault-Berthelot J, Poriel C.
2-Substituted vs 4-substituted-9,9'-spirobifluorene host materials for green and blue phosphorescent OLEDs: a structure–property relationship study.
Tetrahedron. 2014; 70(36): 6337-6351.We report a structure–property relationship study of four 9,9′-spirobifluorene (SBF) derivatives (4-5Pm-SBF, 2-5Pm-SBF, 4-Ph-SBF and 2-Ph-SBF), substituted with either phenyl or pyrimidine at the C2 or C4 position of the SBF core. Structural, thermal, electrochemical and photophysical properties have been examined and correlated to theoretical calculations in order to study the influence of the nature and the position of the substituent. The emission properties of 4- versus 2-substituted SBFs are noticeably different highlighting, in the excited state, the remarkable effect of substitution in ortho position of SBF. All compounds have been used as host material for green dopant in PhOLEDs with very high performances (2-5Pm-SBF: CE>58 cd/A, PE>35 lm/W, EQE>14%). More importantly, the two 4-substituted SBFs have been used as host materials in blue PhOLEDs, displaying high performance and a decrease of VTH for 4-5Pm-SBF due to the incorporation of the electron-withdrawing pyrimidine.
Maksimov EG, Schmitt FJ, Shirshin EA, Svirin MD, Elanskaya IV, Friedrich T, Fadeev VV, Paschenko VZ, Rubin AB.
The time course of non-photochemical quenching in phycobilisomes of Synechocystis sp. PCC6803 as revealed by picosecond time-resolved fluorimetry.
Biochim Biophys Acta. 2014; 1837(9): 1540-1547.As high-intensity solar radiation can lead to extensive damage of the photosynthetic apparatus, cyanobacteria have developed various protection mechanisms to reduce the effective excitation energy transfer (EET) from the antenna complexes to the reaction center. One of them is non-photochemical quenching (NPQ) of the phycobilisome (PB) fluorescence. In Synechocystis sp. PCC6803 this role is carried by the orange carotenoid protein (OCP), which reacts to high-intensity light by a series of conformational changes, enabling the binding of OCP to the PBs reducing the flow of energy into the photosystems. In this paper the mechanisms of energy migration in two mutant PB complexes of Synechocystis sp. were investigated and compared. The mutant CK is lacking phycocyanin in the PBs while the mutant ΔPSI/PSII does not contain both photosystems. Fluorescence decay spectra with picosecond time resolution were registered using a single photon counting technique. The studies were performed in a wide range of ... [truncated at 150 words]
Montecinos-Franjola F, James NG, Concha-Marambio L, Brunet JE, Lagos R, Monasterio O, Jameson DM.
Single tryptophan mutants of FtsZ: nucleotide binding/exchange and conformational transitions.
Biochim Biophys Acta. 2014; 1844(7): 1193-1200.Cell division protein FtsZ cooperatively self-assembles into straight filaments when bound to GTP. A set of conformational changes that are linked to FtsZ GTPase activity are involved in the transition from straight to curved filaments that eventually disassemble. In this work, we characterized the fluorescence of single Trp mutants as a reporter of the predicted conformational changes between the GDP- and GTP-states of Escherichia coli FtsZ. Steady-state fluorescence characterization showed the Trp senses different environments and displays low solvent accessibility. Time-resolved fluorescence data indicated that the main conformational changes in FtsZ occur at the interaction surface between the N and C domains, but also minor rearrangements were detected in the bulk of the N domain. Surprisingly, despite its location near the bottom protofilament interface at the C domain, the Trp 275 fluorescence lifetime did not report changes between the GDP and GTP states. The equilibrium unfolding of FtsZ features an ... [truncated at 150 words]
Bianchini P, Cardarelli F, di Luca M, Diaspro A, Bizzarri R.
Nanoscale protein diffusion by STED-based pair correlation analysis.
PLoS One. 2014; 9(6): e99619. PMCID: PMC4072630We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested in systems characterized by high and low signal to noise ratio, i.e. Capsid Like Particles (CLPs) bearing several (>100) active fluorescent proteins and monomeric fluorescent proteins transiently expressed in living Chinese Hamster Ovary cells, respectively. The latter system represents the usual condition encountered in living cell studies on fluorescent protein chimeras. Spatial resolution of STED-pCF was found to be about 110 nm, with a more than twofold improvement over conventional confocal acquisition. We successfully applied our method to highlight how the proximity to nuclear envelope affects the mobility features of proteins actively imported into the nucleus in living cells. Remarkably, STED-pCF unveiled the existence of local barriers to diffusion as ... [truncated at 150 words]
Mercado J, Baylie R, Navedo MF, Yuan C, Scott JD, Nelson MT, Brayden JE, Santana LF.
Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle.
J Gen Physiol. 2014; 143(5): 559-575. PMCID: PMC4003184Transient receptor potential vanilloid 4 (TRPV4) channels are Ca2+-permeable, nonselective cation channels expressed in multiple tissues, including smooth muscle. Although TRPV4 channels play a key role in regulating vascular tone, the mechanisms controlling Ca2+ influx through these channels in arterial myocytes are poorly understood. Here, we tested the hypothesis that in arterial myocytes the anchoring protein AKAP150 and protein kinase C (PKC) play a critical role in the regulation of TRPV4 channels during angiotensin II (AngII) signaling. Super-resolution imaging revealed that TRPV4 channels are gathered into puncta of variable sizes along the sarcolemma of arterial myocytes. Recordings of Ca2+ entry via single TRPV4 channels (“TRPV4 sparklets”) suggested that basal TRPV4 sparklet activity was low. However, Ca2+ entry during elementary TRPV4 sparklets was ∼100-fold greater than that during L-type CaV1.2 channel sparklets. Application of the TRPV4 channel agonist GSK1016790A or the vasoconstrictor AngII increased the activity of TRPV4 sparklets in specific ... [truncated at 150 words]
Jameson DM, Vetromile CM, James NG.
Investigations of protein–protein interactions using time-resolved fluorescence and phasors.
Methods. 2014; 59(3): 278-286.Protein interactions are critical for biological specificity and techniques able to characterize these interactions are of fundamental importance in biochemistry and cell biology. Fluorescence methodologies have been extremely useful for studying many biological systems including protein–ligand and protein–protein interactions. In this review we focus on the application of time-resolved fluorescence approaches to macromolecular systems. We also include a detailed discussion of a relatively new time-resolved technique, the phasor method, for studying protein interactions both in vitro and in live cells.
Labilloy A, Youkera RT, Bruns JR, Kukicd I, Kiselyov K, Halftere W, Finegold D, Monte SJHd, Weisz OA.
Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease.
Mol Genet Metab. 2014; 111(2): 184-92. PMCID: PMC3946758Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin–Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of ... [truncated at 150 words]
Salvemini IL, Gau DM, Reid J, Bagatolli LA, Macmillan A, Moens PDJ.
Low PIP(2) molar fractions induce nanometer size clustering in giant unilamellar vesicles.
Chem Phys Lipids. 2014; 177: 51-63.Phosphatidylinositol (4,5) bisphosphate (PIP2) is an important signaling molecule located on the inner leaflet of the cell membrane. In order to perform its various signaling functions, it is suggested that PIP2 must be able to form localized clusters. In this study, we have used LAURDAN generalized polarization function (GP) with unlabeled PIP2 and single point fluorescence correlation spectroscopy and brightness analysis of various BODIPY labeled PIP2 to determine the presence of clusters in the membrane of giant unilamellar vesicles (GUVs) made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin and cholesterol. We determined the number of freely diffusing fluorescent BODIPY molecules in the membrane and found that in GUVs containing various amounts of labeled PIP2, this number was significantly lower than in GUVs made with the control BODIPY labeled hexadecyl phosphatidylcholine (BODIPY-HPC). Also, we noted an increase in brightness of the labeled PIP2 particles with increasing labeled PIP2 ... [truncated at 150 words]
Abdisalaam S, Davis AJ, Chen DJ, Alexandrakis G.
Scanning fluorescence correlation spectroscopy techniques to quantify the kinetics of DNA double strand break repair proteins after γ-irradiation and bleomycin treatment.
Nucleic Acids Res. 2014; 42(1): e5. PMCID: PMC3874206A common feature of DNA repair proteins is their mobilization in response to DNA damage. The ability to visualizing and quantifying the kinetics of proteins localizing/dissociating from DNA double strand breaks (DSBs) via immunofluorescence or live cell fluorescence microscopy have been powerful tools in allowing insight into the DNA damage response, but these tools have some limitations. For example, a number of well-established DSB repair factors, in particular those required for non-homologous end joining (NHEJ), do not form discrete foci in response to DSBs induced by ionizing radiation (IR) or radiomimetic drugs, including bleomycin, in living cells. In this report, we show that time-dependent kinetics of the NHEJ factors Ku80 and DNA-dependent protein kinase catalytic subunits (DNA–PKcs) in response to IR and bleomycin can be quantified by Number and Brightness analysis and Raster-scan Image Correlation Spectroscopy. Fluorescent-tagged Ku80 and DNA–PKcs quickly mobilized in response to IR and bleomycin treatments consistent ... [truncated at 150 words]
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Monitoring peripheral protein oligomerization on biological membranes.
In Receptor-Receptor Interactions (Methods in Cell Biology, Vol. 117). By PM Conn (Editors). Academic Press, pp. 359-371, 2013. ISBN: 9780124081437Peripheral proteins transiently interact with cellular membranes where they regulate important cellular events such as signal transduction. A number of peripheral proteins harbor lipid-binding modules that not only bind selectively with nanomolar affinity to biological membranes but also oligomerize on the membrane surface. In some cases, specific lipid binding or specific lipid compositions can induce peripheral protein oligomerization on cellular membranes. These oligomers serve different roles in biological signaling such as regulating protein-protein interactions, induction of membrane bending, or facilitating membrane scission. A number of technologies have been employed to study protein oligomerization with fluctuation analysis of fluorescently labeled molecules recently developed for use with commercial laser-scanning microscopes. In this chapter, the approach of raster image correlation spectroscopy coupled with number and brightness (N&B) analysis to investigate protein oligomerization on cellular membranes in live cells is presented. Important considerations are discussed for designing experiments, collecting data, and performing analysis. N&B ... [truncated at 150 words]
Soto-Arriaza MA, Olivares-Ortega C, Quina FH, Aguilar LF, Sotomayor CP.
Effect of cholesterol content on the structural and dynamic membrane properties of DMPC/DSPC large unilamellar bilayers.
Biochim Biophys Acta. 2013; 1828(11): 2763-2769.In this study, we report the effect of cholesterol content on the dynamic and structural properties of a dimyristoyl-phosphatidylcholine and distearoyl-phosphatidylcholine mixture in large unilamellar vesicles. The range of cholesterol concentrations studied varied around approximately 33.3mol%, where it has been postulated that an abrupt change in bilayer organization occurs. Steady-state fluorescence measurements demonstrated a typical behavior; at low temperatures in the main phase transition, the cholesterol concentration did not affect the gel phase, but at 37.5°C (phase coexistence) and in the liquid crystalline phase, the presence of cholesterol produced an increase in the fluorescence anisotropy of DPH and the generalized polarization of Laurdan. The greater effect was observed in the liquid crystalline phase, in which the bilayer became a mixture of fluid-like and liquid-ordered phases. The results obtained at approximately 33.3mol% of Cholesterol demonstrated that the Generalized Polarization of Laurdan, the DPH lifetime, the limiting anisotropy and the rotational correlation ... [truncated at 150 words]
Rocha-Perugini V, Zamai M, González-Granado JM, Barreiro O, Tejera E, Yañez-Mó M, Caiolfa VR, Sanchez-Madrid F.
CD81 controls sustained T cell activation signaling and defines the maturation stages of cognate immunological synapses.
Mol Cell Biol. 2013; 33(18): 3644-3658. PMCID: PMC3753866In this study, we investigated the dynamics of the molecular interactions of tetraspanin CD81 in T lymphocytes, and we show that CD81 controls the organization of the immune synapse (IS) and T cell activation. Using quantitative microscopy, including fluorescence recovery after photobleaching (FRAP), phasor fluorescence lifetime imaging microscopy-Föster resonance energy transfer (phasorFLIM-FRET), and total internal reflection fluorescence microscopy (TIRFM), we demonstrate that CD81 interacts with ICAM-1 and CD3 during conjugation between T cells and antigen-presenting cells (APCs). CD81 and ICAM-1 exhibit distinct mobilities in central and peripheral areas of early and late T cell-APC contacts. Moreover, CD81-ICAM-1 and CD81-CD3 dynamic interactions increase over the time course of IS formation, as these molecules redistribute throughout the contact area. Therefore, CD81 associations unexpectedly define novel sequential steps of IS maturation. Our results indicate that CD81 controls the temporal progression of the IS and the permanence of CD3 in the membrane contact area, ... [truncated at 150 words]
Butler CE, de Carvalho TMU, Grisard EC, Field RA, Tyler KM.
Trans-sialidase stimulates eat me response from epithelial cells.
Traffic. 2013; 14(7): 853-869. PMCID: PMC3770925Epithelial cell invasion by the protozoan parasite Trypanosoma cruzi is enhanced by the presence of an enzyme expressed on its cell surface during the trypomastigote life cycle stage. The enzyme, trans-sialidase (TS), is a member of one of the largest gene families expressed by the parasite and the role of its activity in mediating epithelial cell entry has not hitherto been understood. Here we show that the T. cruzi TS generates an eat me signal which is capable of enabling epithelial cell entry. We have utilized purified, recombinant, active (TcTS) and inactive (TcTS2V0) TS coated onto beads to challenge an epithelial cell line. We find that TS activity acts upon G protein coupled receptors present at the epithelial cell synapse with the coated bead, thereby enhancing cell entry. By so doing, we provide evidence that TS proteins bind glycans, mediate the formation of distinct synaptic domains and promote macropinocytotic uptake ... [truncated at 150 words]
Youker RT, Bruns JR, Costa SA, Rbaibi Y, Lanni F, Kashlan OB, Teng H, Weisz OA.
Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization.
Mol Biol Cell. 2013; 24(12): 1996-2007. PMCID: PMC3681702The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N- and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proximal stalk, but how this domain mediates targeting is unknown. Protein oligomerization or clustering has been suggested as a common step in the segregation of all apical proteins. Like many apical proteins, p75 forms dimers, and we hypothesized that formation of higher-order clusters mediated by p75 dimerization and interactions of the stalk facilitate its apical sorting. Using fluorescence fluctuation techniques (photon-counting histogram and number and brightness analyses) to study p75 oligomerization status in vivo, we found that wild-type p75-green fluorescent protein forms clusters in the trans-Golgi network (TGN) but not at the plasma membrane. Disruption of either the dimerization motif or the stalk domain impaired both clustering and polarized ... [truncated at 150 words]
Fiche JB, Cattoni DI, Diekmann N, Langerak JM, Clerte C, Royer CA, Margeat E, Doan T, Nöllmann M.
Recruitment, assembly, and molecular architecture of the SpoIIIE DNA pump revealed by superresolution microscopy.
PLoS Biol. 2013; 11(5): e1001557. PMCID: PMC3646729ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transport. A dramatic example of intercompartmental DNA transfer occurs during sporulation in Bacillus subtilis, in which two-thirds of a chromosome is transported across a division septum by the SpoIIIE ATPase. Here, we use photo-activated localization microscopy, structured illumination microscopy, and fluorescence fluctuation microscopy to investigate the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We find that SpoIIIE assembles into ∼45 nm complexes that are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. SpoIIIE complexes contain 47±20 SpoIIIE molecules, a majority of which are assembled into hexamers. Finally, we show that directional DNA translocation leads to the establishment of ... [truncated at 150 words]
Cabello G, Lillo L, Caro C, Soto-Arriaza MA, Chornik B, Buono-Core GE.
Evaluation on the optical properties of Ga2O3−x thin films co-doped with Tb3+ and transition metals (Mn2+, Cr3+) prepared by a photochemical route.
Ceramics International. 2013; 39(3): 2443-2450.Gallium β-diketonate complexes were studied as precursors for the photochemical deposition of amorphous thin films of gallium oxide doped with terbium and co-doped with chromium or manganese. Solutions of the inorganic complexes were spin coated on Si(100) and quartz substrates and photolyzed at room temperature using 254 nm UV light. The photolysis of these films induces the fragmentation of the complexes and the partial reduction of the metal ion together with the release of volatile organic compounds as sub-products. When the metallic complexes are irradiated under air, the products of the reactions are metal oxide thin films. The photochemical reactivity of these films was monitored by UV–vis spectroscopy, followed by a post-annealing treatment. The obtained films were characterized by X-ray photoelectron spectroscopy and X-ray diffraction. The optical properties of the films showed that these are highly transparent in the visible spectrum but decrease significantly in doped and co-doped films. Under ... [truncated at 150 words]
Bachir AI, Kubow KE, Horwitz AR.
Fluorescence fluctuation approaches to the study of adhesion and signaling.
Fluorescence Fluctuation Spectroscopy (FFS), Part B (Methods in Enzymology, Vol. 519). By SY Tetin (Editors). Academic Press, pp. 167-201, 2013. ISBN: 9780124055391Cell-matrix adhesions are large, multimolecular complexes through which cells sense and respond to their environment. They also mediate migration by serving as traction points and signaling centers and allow the cell to modify the surroucnding tissue. Due to their fundamental role in cell behavior, adhesions are germane to nearly all major human health pathologies. However, adhesions are extremely complex and dynamic structures that include over 100 known interacting proteins and operate over multiple space (nm-μm) and time (ms-min) regimes. Fluorescence fluctuation techniques are well suited for studying adhesions. These methods are sensitive over a large spatiotemporal range and provide a wealth of information including molecular transport dynamics, interactions, and stoichiometry from a single time series. Earlier chapters in this volume have provided the theoretical background, instrumentation, and analysis algorithms for these techniques. In this chapter, we discuss their implementation in living cells to study adhesions in migrating cells. Although each ... [truncated at 150 words]
di Rienzo C, Jacchetti E, Cardarelli F, Bizzarri R, Beltram F, Cecchini M.
Unveiling LOX-1 receptor interplay with nanotopography: mechanotransduction and atherosclerosis onset.
Sci Rep. 2013; 3: 1141. PMCID: PMC3555090Lectin-like ox-LDL receptors (LOX-1) play a crucial role in the ox-LDL–induced pathological transformation of vessel-wall components, a crucial early step in atherogenesis. LOX-1 dynamics is quantitatively investigated in human endothelial cells (HUVECs) exposed to environmental nanotopographies. We demonstrate distinct nanotopography-induced cell phenotypes, characterized by different morphology, LOX-1 diffusivity and oligomerization state: HUVECs on flat surfaces exhibit the behavior found in pro-atherogenic conditions, while growth on nanogratings can interfere with LOX-1 dynamics and lead to a behavior characteristic of normal, non-pathological conditions.
Lund FW, Lomholt MA, Solanko LM, Bittman R, Wüstner D.
Two-photon time-lapse microscopy of BODIPY-cholesterol reveals anomalous sterol diffusion in chinese hamster ovary cells.
BMC Biophys. 2012; 5(20). PMCID: PMC3532368BACKGROUND: Cholesterol is an important membrane component, but our knowledge about its transport in cells is sparse. Previous imaging studies using dehydroergosterol (DHE), an intrinsically fluorescent sterol from yeast, have established that vesicular and non-vesicular transport modes contribute to sterol trafficking from the plasma membrane. Significant photobleaching, however, limits the possibilities for in-depth analysis of sterol dynamics using DHE. Co-trafficking studies with DHE and the recently introduced fluorescent cholesterol analog BODIPY-cholesterol (BChol) suggested that the latter probe has utility for prolonged live-cell imaging of sterol transport.
RESULTS: We found that BChol is very photostable under two-photon (2P)-excitation allowing the acquisition of several hundred frames without significant photobleaching. Therefore, long-term tracking and diffusion measurements are possible. Two-photon temporal image correlation spectroscopy (2P-TICS) provided evidence for spatially heterogeneous diffusion constants of BChol varying over two orders of magnitude from the cell interior towards the plasma membrane, where D ~ 1.3 μm2/s. Number and brightness (N&B) ... [truncated at 150 words]
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Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) specifically induces membrane penetration and deformation by Bin/amphiphysin/Rvs (BAR) domains.
J Biol Chem. 2012; 287(41): 34078-34090. PMCID: PMC3464517Cellular proteins containing Bin/amphiphysin/Rvs (BAR) domains play a key role in clathrin-mediated endocytosis. Despite extensive structural and functional studies of BAR domains, it is still unknown how exactly these domains interact with the plasma membrane containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) and whether they function by a universal mechanism or by different mechanisms. Here we report that PtdIns(4,5)P(2) specifically induces partial membrane penetration of the N-terminal amphiphilic α-helix (H(0)) of two representative N-BAR domains from Drosophila amphiphysin (dAmp-BAR) and rat endophilin A1 (EndoA1-BAR). Our quantitative fluorescence imaging analysis shows that PtdIns(4,5)P(2)-dependent membrane penetration of H(0) is important for self-association of membrane-bound dAmp-BAR and EndoA1-BAR and their membrane deformation activity. EndoA1-BAR behaves differently from dAmp-BAR because the former has an additional amphiphilic α-helix that penetrates the membrane in a PtdIns(4,5)P(2)-independent manner. Depletion of PtdIns(4,5)P(2) from the plasma membrane of HEK293 cells abrogated the membrane deforming activity of EndoA1-BAR and dAmp-BAR. Collectively, these studies ... [truncated at 150 words]
Bloksgaard M, Bek S, Marcher AB, Neess D, Brewer J, Hannibal-Bach HK, Helledie T, Fenger C, Due M, Berzina Z, Neubert R, Chemnitz J, Finsen B, Clemmensen A, Wilbertz J, Saxtorph H, Knudsen J, Bagatolli L, Mandrup S.
The acyl-CoA binding protein is required for normal epidermal barrier function in mice.
J Lipid Res. 2012; 53(10): 2162-2174. PMCID: PMC3435549The acyl-CoA binding protein (ACBP) is a 10 kDa intracellular protein expressed in all eukaryotic species. Mice with targeted disruption of Acbp (ACBP−/− mice) are viable and fertile but present a visible skin and fur phenotype characterized by greasy fur and development of alopecia and scaling with age. Morphology and development of skin and appendages are normal in ACBP−/− mice; however, the stratum corneum display altered biophysical properties with reduced proton activity and decreased water content. Mass spectrometry analyses of lipids from epidermis and stratum corneum of ACBP+/+ and ACBP−/− mice showed very similar composition, except for a significant and specific decrease in the very long chain free fatty acids (VLC-FFA) in stratum corneum of ACBP−/− mice. This finding indicates that ACBP is critically involved in the processes that lead to production of stratum corneum VLC-FFAs via complex phospholipids in the lamellar bodies. Importantly, we show that ACBP−/− mice display ... [truncated at 150 words]
Aguilar LF, Pino JA, Soto-Arriaza MA, Cuevas FJ, Sánchez SA, Sotomayor CP.
Differential dynamic and structural behavior of lipid-cholesterol domains in model membranes.
PLoS One. 2012; 7(6): e40254. PMCID: PMC3386959Changes in the cholesterol (Chol) content of biological membranes are known to alter the physicochemical properties of the lipid lamella and consequently the function of membrane-associated enzymes. To characterize these changes, we used steady-state and time resolved fluorescence spectroscopy and two photon-excitation microscopy techniques. The membrane systems were chosen according to the techniques that were used: large unilamellar vesicles (LUVs) for cuvette and giant unilamellar vesicles (GUVs) for microscopy measurements; they were prepared from dipalmitoyl phosphatidylcholine (DPPC) and dioctadecyl phosphatidylcholine (DOPC) in mixtures that are well known to form lipid domains. Two fluorescent probes, which insert into different regions of the bilayer, were selected: 1,6-diphenyl-1,3,5-hexatriene (DPH) was located at the deep hydrophobic core of the acyl chain regions and 2-dimethylamino-6-lauroylnaphthalene (Laurdan) at the hydrophilic-hydrophobic membrane interface. Our spectroscopy results show that (i) the changes induced by cholesterol in the deep hydrophobic phospholipid acyl chain domain are different from the ones ... [truncated at 150 words]
Bok S, Korampally V, Polo-Parada L, Mamidi V, Baker GA, Gangopadhyay K, Folk WR, Dasgupta PK, Gangopadhyay S.
Confeito-like assembly of organosilicate-caged fluorophores: ultrabright suprananoparticles for fluorescence imaging.
Nanotechnology. 2012; 23(17): 175601. PMCID: PMC3360483We report ultrabright, photostable, sub-25 nm nanoparticle agglomerates (suprananoparticles) assembled from a few hundred 3.3 ± 0.9 nm units, each hosting on average a single rhodamine 6G (Rh6G) dye molecule encased in a thin organosilicate cage. These individual Rh6G-doped nanoparticle (DOSNP) units consist of a hydrophobic core containing the dye and an ultrathin, conformal silicate shell modified by CO2 plasma to confer a beneficial “cage effect” as well as surface hydrophilicity. The isolation of the dye within individual DOSNP units in the final 22 ± 5 nm agglomerate avoids dimerization and related spontaneous molecular interactions that otherwise lead to self-quenching in closely co-localized fluorophores. The resulting suprananoparticles are over 200 times brighter than the free Rh6G molecules in the same volume. There is no observable dye leaching, and the labels are 20-fold more resistant to photobleaching than free Rh6G in solution. We demonstrate the attractive features of DOSNPs as labels ... [truncated at 150 words]
Jourdan N, Jobart-Malfait A, Reis GD, Quignon F, Piolot T, Klein C, Tramier M, Coppey-Moisan M, Marechal V.
Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in ... [truncated at 150 words]
Gustavsson T, Improta R, Banyasz A, Vaya I, Markovitsi D.
The effect of methylation on the excited state dynamics of aminouracils.
J Photochem Photobiol A Chem. 2012; 234: 37-43.The excited state properties of two amino substituted uracils, 5-aminouracil (5AU) and 5-dimethylaminouracil (5DMAU) have been studied by steady-state and time-resolved fluorescence spectroscopy. In addition, the electronic transitions corresponding to photon absorption and emission were calculated using the TD-DFT/PCM method (with PBE0 and CAM-B3LYP functionals), explicitly including water molecules of the first solvent shell.
The fluorescence decays, recorded by fluorescence upconversion, reveal that the emitting states of both molecules are substantially longer-lived than that of uracil. However, while the 5AU fluorescence decays on a picosecond timescale, the 5DMAU fluorescence contains a weak, long-lived component, continuing beyond 100 ps.
Calculations show that the excited state deactivation of 5AU is governed by the existence of a minimum on the ππ* excited state potential energy surface. For 5DMAU, on the other hand, a very long-lived but weakly fluorescent intramolecular charge transfer (ICT) state, involving a significant charge transfer between the dimethylamino group and the pyrimidine ... [truncated at 150 words]
Golebiewska U, Johnston JM, Devi L, Filizola M, Scarlata S.
Differential Response to Morphine of the Oligomeric State of μ-Opioid in the Presence of δ-Opioid Receptors.
Biochemistry. 2012; 50(14): 2829-2837. PMCID: PMC3071705Prolonged morphine treatment induces extensive desensitization of the μ-opioid receptor (μOR) which is the G protein-coupled receptor that primarily mediates the cellular response to morphine. To date, the molecular mechanism underlying this process is unknown. Here, we have used live cell fluorescence imaging to investigate whether prolonged morphine treatment affects the physical environment of μOR, or its coupling with G proteins, in two neuronal cell lines. We find that chronic morphine treatment does not change the amount of enhanced yellow fluorescence protein (eYFP)-tagged μOR on the plasma membrane, and only slightly decreases its association with G protein subunits. Additionally, morphine treatment does not have a detectable effect on the diffusion coefficient of eYFP-μOR. However, in the presence of another family member, the δ–opioid receptor (δOR), prolonged morphine exposure results in a significant increase in the diffusion rate of μOR. Number and brightness measurements suggest that μOR exists primarily as a ... [truncated at 150 words]
Storti B, Bizzarri R, Cardarelli F, Beltram F.
Intact microtubules preserve transient receptor potential vanilloid 1 (TRPV1) functionality through receptor binding.
J Biol Chem. 2012; 287(10): 7803-7811. PMCID: PMC3293569The transient receptor potential cation channel subfamily V member 1 (TRPV1) is a protein currently under scrutiny as a pharmacological target for pain management therapies. Recently, the role of TRPV1-microtubule interaction in transducing nociception stimuli to cells by cytoskeletal rearrangement was proposed. In this work, we investigate TRPV1-microtubule interaction in living cells under the resting or activated state of TRPV1, as well as in presence of structurally intact or depolymerized cytoskeletal microtubules. We combined a toolbox of high resolution/high sensitivity fluorescence imaging techniques (such as FRET, correlation spectroscopy, and fluorescence anisotropy) to monitor TRPV1 aggregation status, membrane mobility, and interaction with microtubules. We found that TRPV1 is a dimeric membrane protein characterized by two populations with different diffusion properties in basal condition. After stimulation with resiniferatoxin, TRPV1 dimers tetramerize. The tetramers and the slower population of TRPV1 dimers bind dynamically to intact microtubules but not to tubulin dimers. Upon microtubule ... [truncated at 150 words]
Cebecauer M, Humpolíčková J, Rossy J.
Advanced imaging of cellular signaling events.
Imaging and Spectroscopic Analysis of Living Cells - Live Cell Imaging of Cellular Elements and Functions (Methods in Enzymology, Vol. 505). By PM Conn (Editors). Academic Press, pp. 273-289, 2012. ISBN: 9780123884480Cells continuously communicate with the surrounding environment employing variety of signaling molecules and pathways to integrate and transport the information in the cell. An example of signaling initiation is binding of extracellular ligand to its receptor at the plasma membrane. This initializes enzymatic reactions leading to the formation of bi- or multimolecular signaling complexes responsible for the regulation or progress of signal transduction. Here, we describe three imaging techniques enabling detection of individual signaling molecules, their complexes, and clusters in human cells. Described imaging techniques require only basic microscopy systems available in the majority of current biomedical research centers but apply advanced data processing. First, total internal reflection fluorescence microscopy (TIRFM) variant of wide-field fluorescence microscopy for imaging highly dynamic clusters is described. Second, superresolution localization microscopy techniques-photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)-recently enabled to achieve higher resolution with precision limit of about 20 nm in ... [truncated at 150 words]
Corti V, Ossato G, Cavallaro U, Francavilla C, Caiolfa VR, Zamai M.
TIRFM-N&B analysis of FGFR1 clustering in response to NCAM and FGF2.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 192a.Neural cell adhesion molecule (NCAM) is a nonconventional ligand for fibroblast growth factor receptor-1 (FGFR1). NCAM exerts a peculiar control on the intracellular trafficking of FGFR1, resulting in a specific cellular response, which is remarkably different from that elicited by the canonical ligand FGF2 (Francavilla et al., JCB 2009).
We studied the dynamics and oligomerization of cell-surface FGFR1 using a biologically active FGFR1-mEGFP chimera expressed in HeLa cells. FGFR1-mEGFP was shown to bind FGF2 and NCAM, and to undergo phosphorylation and internalization.
The monomer-dimer-oligomer dynamics of FGFR1 in response to NCAM and FGF2 was studied as a function of time by total internal reflection fluorescence (TIRF) microscopy combined with the number and brightness (N&B) analysis.
HeLa/FGFR1-mEGFP cells were starved overnight, and time series of TIRF images were collected up to 40-70 min after ligand-mediated stimulation. N&B analysis was carried out using SimFCS SW (E. Gratton) and EMCCD camera calibrated according to Unruh and ... [truncated at 150 words]
McCary CA, Yoon Y, Panagabko C, Cho W, Atkinson J, Cook-Mills JM.
Vitamin E isoforms directly bind PKCα and differentially regulate activation of PKCα.
Biochem J. 2012; 44(1): 189-198. PMCID: PMC3271793Vitamin E isoforms have opposing regulatory effects on leukocyte recruitment during inflammation. Furthermore, in vitro, vitamin E isoforms have opposing effects on leukocyte migration across endothelial cells by regulating vascular cell adhesion molecule (VCAM)-1 activation of endothelial cell protein kinase Cα (PKCα). However, it is not known whether tocopherols directly regulate co-factor-dependent or oxidative activation of PKCα. We report herein that co-factor-dependent activation of recombinant PKCα was increased by γ-tocopherol and was inhibited by α-tocopherol. Oxidative activation of PKCα was inhibited by α-tocopherol at a 10 fold lower concentration than γ-tocopherol. In binding studies, NBD-tagged-α-tocopherol directly bound to full-length PKCα or the PKCα-C1a domain but not PKCζ. NBD-tagged-α-tocopherol binding to PKCα or the PKCα-C1a domain was blocked by diacylglycerol, α-tocopherol, γ-tocopherol, and retinol but not by cholesterol or phosphatidylserine (PS). Tocopherols enhanced PKCα-C2 domain binding to PS-containing lipid vesicles. In contrast, the PKCα-C2 domain did not bind to lipid vesicles ... [truncated at 150 words]
Stock RP, Brewer J, Wagner K, Ramos-Cerrillo B, Duelund L, Jernshøj KD, Olsen LF, Bagatolli LA.
Sphingomyelinase D activity in model membranes: structural effects of in situ generation of ceramide-1-phosphate.
PLoS One. 2012; 7(4): e36003. PMCID: PMC3338491The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and ... [truncated at 150 words]
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Phosphatidylserine Binding Is Essential for Plasma Membrane Recruitment and Signaling Function of 3-Phosphoinositide-dependent Kinase-1.
J Biol Chem. 2011; 286(48): 41265-41272. PMCID: PMC33088393-Phosphoinositide-dependent kinase-1 (PDK1) is a ubiquitously expressed serine/threonine kinase that functions downstream of phosphoinositide 3-kinase. Although binding of 3′-phosphoinositides, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate, to the pleckstrin homology (PH) domain of PDK1 is known to be essential for its interaction with and activation of downstream kinases, the mechanism by which PDK1 is recruited to the plasma membrane remains controversial. Our surface plasmon resonance analysis of the PDK1 PH domain and selected mutants shows that the PH domain specifically binds phosphatidylserine using a site that is separate from the canonical phosphoinositide-binding site. Further cell studies show that this specific phosphatidylserine binding is important for the plasma membrane localization and signaling function of PDK1.
Salinas S, Soto-Arriaza MA, Loeb B.
New iridium cyclometallated complexes with potential application in OLED.
Polyhedron. 2011; 30(17): 2863-2869.The photophysical and electrochemical properties of cyclometallic cationic iridium complexes that also contain bipyridine type ligands have been investigated in this work. The effect on the photophysical properties of the complexes on the presence of aliphatic long chain or branched substitution was analyzed. The complexes show photoluminescence maxima in the green-blue region of the visible spectrum, with acceptable quantum yields and lifetime, showing in this way potentiality to be tested in OLED devices.
Cerreto M, Cavaliere P, Carluccio C, Amato F, Zagari A, Daniele A, Salvatore F.
Natural phenylalanine hydroxylase variants that confer a mild phenotype affect the enzyme's conformational stability and oligomerization equilibrium.
Biochim Biophys Acta. 2011; 1812(11): 1435-1445.Hyperphenylalaninemias are genetic diseases prevalently caused by mutations in the phenylalanine hydroxylase (PAH) gene. The wild-type PAH enzyme is a homotetramer regulated by its substrate, cofactor and phosphorylation. We reproduced a full-length wild-type protein and seven natural full-length PAH variants, p.I65M, p.N223Y, p.R297L, p.F382L, p.K398N, p.A403V, and p.Q419R, and analyzed their biochemical and biophysical behavior. All mutants exhibited reduced enzymatic activity, namely from 38% to 69% of wild-type activity. Biophysical characterization was performed by size-exclusion chromatography, light scattering and circular dichroism. In the purified wild-type PAH, we identified the monomer in equilibrium with the dimer and tetramer. In most mutants, the equilibrium shifted toward the dimer and most tended to form aggregates. All PAH variants displayed different biophysical behaviors due to loss of secondary structure and thermal destabilization. Specifically, p.F382L was highly unstable at physiological temperature. Moreover, using confocal microscopy with the number and brightness technique, we studied the effect ... [truncated at 150 words]
Lacoste J, Vining C, Zuo D, Spurmanis A, Brown CM.
Optimal conditions for live cell microscopy and raster image correlation spectroscopy.
Reviews in Fluorescence (Vol. 110). By CD Geddes (Editors). Springer New York, pp. 269-309, 2011. ISBN: 9781441998279Live cell microscopy is now commonplace across all fields of the life sciences, as well as, many of the physical sciences. In order to properly study physiological processing within living cells, tissues, or organisms it is crucial that viability of the sample takes the forefront as the most important aspect of the experiments. If samples are subject to high levels of light, phototoxicity can alter the very physiological processes under investigation. In order to minimize damage to the sample it is crucial to have as sensitive a microscope platform as possible so that light impact on the sample will be minimized. In order to minimize this impact, many aspects have to be kept in mind to maintain the sample and protect it from phototoxicity such as (1) keeping the cells in a favorable environment; (2) using transmitted light techniques when possible and carefully selecting fluorescent dyes; (3) using low light ... [truncated at 150 words]
Ruan YB, Maisonneuve S, Xie J.
Highly selective fluorescent and colorimetric sensor for Hg2+ based on triazole-linked NBD.
Dyes and Pigments. 2011; 90(3): 239-244.7-Nitrobenzo-2-oxa-1,3-diazole (NBD) derived compound 1 bearing triazole binding site was used as a selective fluorescent and colorimetric sensor for Hg2+ in aqueous solution. Among the metal ions examined, only Hg2+ caused significant fluorescence quenching in EtOH/HEPES (v/v = 9:1) at pH 7.4, along with a remarkable red shift in both absorption and fluorescence spectra which then facilitated naked-eye detection. 1H NMR titration and control experiments by using more rigid triazolyl NBD derivative 2 were carried out to illustrate the complexation mode and the importance of cooperation of amino acid moiety and triazole ring in improving the binding ability of 1 to Hg2+.
Ross JA, Gilmore MA, Williams D, Aoki KR, Steward LE, Jameson DM.
Characterization of Förster resonance energy transfer in a botulinum neurotoxin protease assay.
Anal Biochem. 2011; 413(1): 43-49.Our previous article described a fluorescence-based assay for monitoring the proteolytic activity of botulinum neurotoxin types A and E (BoNT/A and BoNT/E). As detailed in that article, the assay is based on depolarization due to Förster resonance energy transfer between blue fluorescent protein (BFP) and green fluorescent protein (GFP) moieties linked via residues 134-206 of SNAP-25 (synaptosome-associated protein of 25kDa), the protein substrate for BoNT/A and BoNT/E. Before cleavage of this recombinant substrate, the polarization observed for the GFP emission, excited near the absorption maximum of the BFP, is very low due to depolarization following energy transfer from BFP to GFP. After substrate cleavage and diffusion of the fluorescent proteins beyond the energy transfer distance, the polarization is high due to observation of the emission only from directly excited GFP. This change in fluorescence polarization allows an assay, termed DARET (depolarization after resonance energy transfer), that is robust and sensitive. ... [truncated at 150 words]
Mahen R, Jeyasekharan AD, Barry NP, Venkitaraman AR.
Continuous polo-like kinase 1 activity regulates diffusion to maintain centrosome self-organization during mitosis.
Proc Natl Acad Sci USA. 2011; 108(22): 9310-9315. PMCID: PMC3107272Whether mitotic structures like the centrosome can self-organize from the regulated mobility of their dynamic protein components remains unclear. Here, we combine fluorescence spectroscopy and chemical genetics to study in living cells the diffusion of polo-like kinase 1 (PLK1), an enzyme critical for centrosome maturation at the onset of mitosis. The cytoplasmic diffusion of a functional EGFP-PLK1 fusion correlates inversely with known changes in its enzymatic activity during the cell cycle. Specific EGFP-PLK1 inhibition using chemical genetics enhances mobility, as do point mutations inactivating the polo-box or kinase domains responsible for substrate recognition and catalysis. Spatial mapping of EGFP-PLK1 diffusion across living cells, using raster image correlation spectroscopy and line scanning, detects regions of low mobility in centrosomes. These regions exhibit characteristics of increased transient recursive EGFP-PLK1 binding, distinct from the diffusion of stable EGFP-PLK1–containing complexes in the cytoplasm. Chemical genetic suppression of mitotic EGFP-PLK1 activity, even after centrosome maturation, ... [truncated at 150 words]
Schmitt FJ, Maksimov EG, Suedmeyer H, Jeyasangar V, Theiss C, Paschenko VZ, Eichler HJ, Renger G.
Time resolved temperature switchable excitation energy transfer processes between CdSe/ZnS nanocrystals and phycobiliprotein antenna from Acaryochloris marina.
Phot Nano Fund Appl. 2011; 9(2): 190-195.Hybrid systems were self-assembled in solution from surface treated CdSe/ZnS quantum dots (QDs) and isolated phycobiliprotein (PBP) complexes from the cyanobacterium Acaryochloris marina. The excitation energy transfer (EET) from the QDs to attached PBPs was analyzed by time correlated single photon counting and time integrated fluorescence measurements at different temperatures. It was found:
(1) The green emission of the QDs (3.3 nm diameter of the CdSe core) in solution at 530 nm becomes strongly quenched after addition of PBPs.
(2) The functional connection between QDs and PBPs via EET interrupts at temperatures below 273 K (0 °C)
(3) This temperature dependent effect is fully reversible
(4) EET from QDs to PBPs occurs with a time constant of about 140 ps and an efficiency of 85–90% for coupled QDs/PBP hybrid complexes.
A model of the EET steps is presented which is based on data evaluation of the time integrated fluorescence emission and the time resolved measurement ... [truncated at 150 words]
Štefl M, James NG, Ross JA, Jameson DM.
Applications of phasors to in vitro time-resolved fluorescence measurements.
Anal Biochem. 2011; 410(1): 62-69. PMCID: PMC3065364The phasor method of treating fluorescence lifetime data provides a facile and convenient approach to characterize lifetime heterogeneity and to detect the presence of excited state reactions such as solvent relaxation and Förster resonance energy transfer. The method uses a plot of M sin(Φ) versus M cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. A principal advantage of the phasor method is that it provides a model-less approach to time-resolved data amenable to visual inspection. Although the phasor approach has been recently applied to fluorescence lifetime imaging microscopy, it has not been used extensively for cuvette studies. In the current study, we explore the applications of the method to in vitro samples. The phasors of binary and ternary mixtures of fluorescent dyes demonstrate the utility of the method for investigating complex mixtures. Data from excited state reactions, such as dipolar ... [truncated at 150 words]
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Widefield microscopy for live imaging of lipid domains and membrane dynamics.
Biochim Biophys Acta. 2011; 1808(3): 634-641. PMCID: PMC3048960Within the lateral organisation of plasma membranes of polarized cell types there exist heterogenous microdomains of distinct lipid composition, the small size of which (10–200 nm) makes them difficult to discern with traditional microscopic techniques, but which can be distinguished on the basis of lipid packing. These microdomains or rafts can be concentrated in larger more visible liquid-ordered regions, particularly by cross-linking of their constituents as in the immunological synapse or in features of the polarized cell such as pseudopodia or flagella. One technique, Laurdan fluorescence microscopy, has proven very useful for distinguishing such regions but has hitherto relied on 2-photon confocal microscopy. This has to some extent limited its utility to living systems and its widespread adoption in studying membrane dynamics on the surface of living cells. Here we describe and validate the adaptation of a standard widefield fluorescence microscope for live imaging of Laurdan stained cell membranes.
Kubiak J, Brewer J, Hansen S, Bagatolli LA.
Lipid lateral organization on giant unilamellar vesicles containing lipopolysaccharides.
Biophys J. 2011; 100(4): 978-986. PMCID: PMC3037713We developed a new (to our knowledge) protocol to generate giant unilamellar vesicles (GUVs) composed of mixtures of single lipopolysaccharide (LPS) species and Escherichia coli polar lipid extracts. Four different LPSs that differed in the size of the polar headgroup (i.e., LPS smooth > LPS-Ra > LPS-Rc > LPS-Rd) were selected to generate GUVs composed of different LPS/E. coli polar lipid mixtures. Our procedure consists of two main steps: 1), generation and purification of oligolamellar liposomes containing LPSs; and 2), electroformation of GUVs using the LPS-containing oligolamellar vesicles at physiological salt and pH conditions. Analysis of LPS incorporation into the membrane models (both oligolamellar vesicles and GUVs) shows that the final concentration of LPS is lower than that expected from the initial E. coli lipids/LPS mixture. In particular, our protocol allows incorporation of no more than 15 mol % for LPS-smooth and LPS-Ra, and up to 25 mol % for ... [truncated at 150 words]
Ross JA, Chen Y, Müller J, Barylko B, Wang L, Banks HB, Albanesi JP, Jameson DM.
Dimeric endophilin A2 stimulates assembly and GTPase activity of dynamin 2.
Biophys J. 2011; 100(3): 729-737. PMCID: PMC3030234Endophilin, which participates in membrane vesiculation during receptor-mediated endocytosis, is a ∼40 kDa SH3 domain-containing protein that binds to the proline/arginine-rich domain of dynamin, a ∼100 kDa GTPase that is essential for endocytic membrane scission. It has been suggested that endophilin is monomeric in the cytoplasm and dimerizes only after it binds to membranes (or perhaps to dimers or tetramers of dynamin). To clarify this issue, we studied the oligomeric state of endophilin both in vitro using analytical ultracentrifugation and fluorescence anisotropy, and in living cells using two-photon fluorescence fluctuation spectroscopy. We analyzed the fluctuation data using the Q-analysis method, which allowed us to determine the intrinsic brightness of the labeled protein complexes and hence its aggregation state in the cytoplasmic regions of the cell. Although a relatively high K(d) (∼5-15 μM) was observed in vitro, the cell measurements indicate that endophilin is dimeric in the cytoplasm, even at submicromolar ... [truncated at 150 words]
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Photophysical properties of poly(triptycene vinylene) derivatives and effect of nitrotoluene exposure.
Chem Phys Lett. 2010; 501(1-3): 54-58.The photophysical properties of two conjugated polymers containing triptycene group were studied. These systems are highly fluorescent and time-resolved fluorescent measurements show that there are different types of excited species whose relative population is correlated to a heterogeneous collection of chromophore sites. A fluorescence quenching is observed upon DNT exposure because of a dynamic electron transfer process from the polymer to the quencher. The reversibility of the quenching process has been demonstrated by a simple air flow method which allows the re-use of the sensor for cycling measurement of the nitroaromatic analyte.
Flach H, Rosenbaum M, Duchniewicz M, Kim S, Zhang SL, Cahalan MD, Mittler G, Grosschedl R.
Mzb1 protein regulates calcium homeostasis, antibody secretion, and integrin activation in innate-like B cells.
Immunity. 2010; 33(5): 723-735. PMCID: PMC3125521Marginal zone (MZ) B cells of the spleen and B1 cells, termed innate-like B cells, differ from follicular B cells by their attenuated Ca(2+) mobilization, fast antibody secretion, and increased cell adhesion. We identified and characterized Mzb1 as an endoplasmic reticulum-localized and B cell-specific protein that was most abundantly expressed in MZ B and B1 cells. Knockdown of Mzb1 in MZ B cells increased Ca(2+) mobilization and nuclear NFAT transcription factor localization, but reduced lipopolysaccharide-induced antibody secretion and integrin-mediated cell adhesion. Conversely, ectopic expression of an Lck-Mzb1 transgene in peripheral T cells resulted in attenuated Ca(2+) mobilization and augmented integrin-mediated cell adhesion. In addition to its interaction with the substrate-specific chaperone Grp94, Mzb1 augmented the function of the oxidoreductase ERp57 in favoring the expression of integrins in their activated conformation. Thus, Mzb1 helps to diversify peripheral B cell functions by regulating Ca(2+) stores, antibody secretion, and integrin activation.
Hornillos V, Tormo L, Amat-Guerri F, Acuña AU.
Synthesis and spectral properties of fluorescent linear alkylphosphocholines labeled with all-(E)-1,6-diphenyl-1,3,5-hexatriene.
J Photochem Photobiol A. 2010; 216(1): 79-84.A synthetic method has been designed to produce alkylphosphatidylcholine lipid molecules with the fluorescent group all-(E)-1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated to the end of a polymethylene chain of variable length. The resulting compounds may be viewed as single-chain fluorescent lipid probes terminated with a phosphocholine (PHC) polar head-group. The method was applied to the synthesis of two lipids with different alkyl chain length, PHC-C4-DPH (1) and PHC-C6-DPH (2), as well as the corresponding alcohols OH-C4-DPH (8) and OH-C6-DPH (9). The absorption and fluorescence properties of these compounds in fluid solution were very similar to those of the well-known lipid probe propionic acid-DPH (PA-DPH). In addition, it was observed that the alkylphosphocholines were easily incorporated into unilamellar lipid vesicles, in which the DPH group would be positioned most likely near the centre of the bilayer. In these conditions, 1 shows a fluorescence quantum yield of 0.4 ± 0.1 and a relatively simple decay ... [truncated at 150 words]
Risso VA, Primo ME, Brunet JE, Sotomayor CP, Ermácora MR.
Optical studies of single-tryptophan B. licheniformis β-lactamase variants.
Biophys Chem. 2010; 151(3): 111-118.β-lactamases (penicillinases) are important complicating factors in bacterial infections and excellent theoretical and experimental models in protein structure, dynamics and evolution. Bacillus licheniformis exo-small penicillinase (ESP) is a Class A β-lactamase with three tryptophan residues, one located in each of the two protein domains and one located in the interface between domains. To determine the tryptophan contribution to the ESP UV-absorption, circular dichroism, and steady-state and time-resolved fluorescence, four Trp → Phe mutants were prepared and characterized. The residue substitutions had little impact on the native conformation. UV-absorption and CD features were identified and ascribed to specific aromatic residues. Time-resolved fluorescence showed that most of the fluorescence decay of ESP tryptophans is due to a discrete exponential component with a lifetime of 5–6 ns. Fluorescence polarization measurements indicated that fluorescence of Trp 210 is nearly independent of the fluorescence of Trp 229 and Trp 251, whereas a substantial energy homotransfer ... [truncated at 150 words]
Weinreis SA, Ellis JP, Cavagnero S.
Dynamic fluorescence depolarization: a powerful tool to explore protein folding on the ribosome.
Methods. 2010; 52(1): 57-73. PMCID: PMC2934862Protein folding is a fundamental biological process of great significance for cell function and life-related processes. Surprisingly, very little is presently known about how proteins fold in vivo. The influence of the cellular environment is of paramount importance, as molecular chaperones, the ribosome, and the crowded medium affect both folding pathways and potentially even equilibrium structures. Studying protein folding in physiologically relevant environments, however, poses a number of technical challenges due to slow tumbling rates, low concentrations and potentially non-homogenous populations. Early work in this area relied on biological assays based on antibody recognition, proteolysis, and activity studies. More recently, it has been possible to directly observe the structure and dynamics of nascent polypeptides at high resolution by spectroscopic and microscopic techniques. The fluorescence depolarization decay of nascent polypeptides labeled with a small extrinsic fluorophore is a particularly powerful tool to gain insights into the dynamics of newly synthesized proteins. ... [truncated at 150 words]
Brewer J, de Serna JBl, Wagner K, Bagatolli LA.
Multiphoton excitation fluorescence microscopy in planar membrane systems.
Biochim Biophys Acta. 2010; 1798(7): 1301-1308.The feasibility of applying multiphoton excitation fluorescence microscopy-related techniques in planar membrane systems, such as lipid monolayers at the air-water interface (named Langmuir films), is presented and discussed in this paper. The non-linear fluorescence microscopy approach, allows obtaining spatially and temporally resolved information by exploiting the fluorescent properties of particular fluorescence probes. For instance, the use of environmental sensitive probes, such as LAURDAN, allows performing measurements using the LAURDAN generalized polarization function that in turn is sensitive to the local lipid packing in the membrane. The fact that LAURDAN exhibit homogeneous distribution in monolayers, particularly in systems displaying domain coexistence, overcomes a general problem observed when "classical" fluorescence probes are used to label Langmuir films, i.e. the inability to obtain simultaneous information from the two coexisting membrane regions. Also, the well described photoselection effect caused by excitation light on LAURDAN allows: (i) to qualitative infer tilting information of the monolayer ... [truncated at 150 words]
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Transient anomalous subdiffusion: Effects of specific and non-specific probe binding with Actin gels.
J Phys Chem B. 2010; 114(2): 959-972. PMCID: PMC2810870When signaling molecules diffuse through the cytosol they encounter a wide variety of obstacles that hinder their mobility in space and time. Some of those factors include, but are not limited to, interactions with mobile and immobile targets or obstacles. Besides finding a crowded environment inside the cell, macromolecules assemble into molecular complexes that drive specific biological functions adding additional complexity to their diffusion. Thus, simple models of diffusion often fail to explain mobility through the cell interior and new approaches are needed. Here we used fluorescent correlation spectroscopy to measure diffusion of three molecules of similar size with different surface properties diffusing in actin gels. The fluorescent probes were a) quantum dots, b) yellow-green fluorescent spheres and c) the β isoform of Ca2+ calmodulin-dependent protein kinase II tagged with green fluorescent protein. We compared various models for fitting the autocorrelation function (ACF) including single component, two-component, and anomalous diffusion. ... [truncated at 150 words]
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Using phasors in interpreting one- and two-photon fluorescence lifetime images of fruit and polymer interfaces.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 397a.Phasors prove to be an elegant way of characterizing time-resolved fluorescence images, (Digman et al., Biophys. J., 94, 1483-96, 2008). Fast Flim micro- and macro imaging (Biophys. J., 82, 502a) was applied to: 1. the pre-symptom and early detection of biotic and abiotic stress as well as surface defects and physiological disorders in fruit tissue using photosystem II Chlorophyll a fluorescence and 2. the characterization of conjugated polymer film produced under various conditions for biosensor development. Both Olympus and Zeiss imaging systems were used in conjunction with one photon 488 nm and 80 MHz, typically 15 mW two-photon illumination. For comparison overview color or transmission images were also collected. Several spots spread over the surfaces were used. Images have been analysed using phasors with Globals for Images, aka. SimFCS (LFD, UCI, CA, USA). The potential of the phasor approach as analysis tool for detection of both ageing and physiological stress ... [truncated at 150 words]
Alia B, Jabara S, Saliha W, Tamimia RKA, Attarb HA, Monkman AP.
Synthesis and spectroscopic characterization studies of low molecular weight light emitting PPV segmented copolymers.
Opt Mater. 2009; 32(2): 350-357.New tuneable segmented alternating copoly(p-phenylene vinylene) (PPV) derivatives were chemically synthesised via the Wittig polycondensation reaction. The chemical and optical properties in solution and in solid state were evaluated. The absorption and photoemission spectra show that incorporating an ethane dioxy spacer limits the conjugation length and produces a blue emitting copolymer, poly[1,2-ethane dioxy 2-methoxy-1,4-phenylene-vinyl 1,4-phenylene-vinyl 3-methoxy 1,4-phenylene] (EDO-PPV-1) 1. A red shift in the copolymer emission was achieved by adding an electron-donating group, a hexoloxy-substituted PPV segment, poly[1,2-ethane dioxy 2-methoxy-1,4-phenylene-vinyl 2,5-dihexoloxy 1,4-phenylene-vinyl 3-methoxy 1,4-phenylene] (EDO-PPV-2) 2. The photophysical properties of these block copolymers was compared with the fully conjugated copolymer poly[2-methoxy-5-(2′-ethyl-hexyloxy)-1,4-phenylene vinylene] (MEH-PPV) 3. The absorption band of the πdeloc–View the MathML sourceπdeloc∗ transition at 400–500 nm is limited in 1 and 2 indicating that the conjugation is effectively interrupted. The effect of torsional motions on the Stokes shift in these segmented copolymers is discussed. The photoluminescence (PL) efficiency and ... [truncated at 150 words]
Abu-Arish A, Kalab P, Ng-Kamstra J, Weis K, Fradin C.
Spatial distribution and mobility of the Ran GTPase in live interphase cells.
Biophys J. 2009; 97(8): 2164-2178. PMCID: PMC2764085The GTPase Ran is a key regulator of molecular transport through nuclear pore complex (NPC) channels. To analyze the role of Ran in its nuclear transport function, we used several quantitative fluorescence techniques to follow the distribution and dynamics of an enhanced yellow fluorescent protein (EYFP)-Ran in HeLa cells. The diffusion coefficient of the majority of EYFP-Ran molecules throughout the cells corresponded to an unbound state, revealing the remarkably dynamic Ran regulation. Although we observed no significant immobile Ran populations in cells, approximately 10% of the cytoplasmic EYFP-Ran and 30% of the nuclear EYFP-Ran exhibited low mobility indicative of molecular interactions. The high fraction of slow nuclear EYFP-Ran reflects the expected numerous interactions of nuclear RanGTP with nuclear transport receptors. However, it is not high enough to support retention mechanisms as the main cause for the observed nuclear accumulation of Ran. The highest cellular concentration of EYFP-Ran was detected at ... [truncated at 150 words]
Padilla-Parra S, Audugé N, Lalucque H, Mevel JC, Coppey-Moisan M, Tramier M.
Quantitative comparison of different fluorescent protein couples for fast FRET-FLIM acquisition.
Biophys J. 2009; 97(8): 2368-2376. PMCID: PMC2764072The fluorescent-protein based fluorescence resonance energy transfer (FRET) approach is a powerful method for quantifying protein-protein interactions in living cells, especially when combined with fluorescence lifetime imaging microscopy (FLIM). To compare the performance of different FRET couples for FRET-FLIM experiments, we first tested enhanced green fluorescent protein (EGFP) linked to different red acceptors (mRFP1-EGFP, mStrawberry-EGFP, HaloTag (TMR)-EGFP, and mCherry-EGFP). We obtained a fraction of donor engaged in FRET (f(D)) that was far from the ideal case of one, using different mathematical models assuming a double species model (i.e., discrete double exponential fixing the donor lifetime and double exponential stretched for the FRET lifetime). We show that the relatively low f(D) percentages obtained with these models may be due to spectroscopic heterogeneity of the acceptor population, which is partially caused by different maturation rates for the donor and the acceptor. In an attempt to improve the amount of donor protein engaged ... [truncated at 150 words]
Brunstein M, Bruno L, Desposito M, Levi V.
Anomalous dynamics of melanosomes driven by myosin-V in Xenopus laevis melanophores.
Biophys J. 2009; 97(6): 1548-1557. PMCID: PMC2749791The organization of the cytoplasm is regulated by molecular motors, which transport organelles and other cargoes along cytoskeleton tracks. In this work, we use single particle tracking to study the in vivo regulation of the transport driven by myosin-V along actin filaments in Xenopus laevis melanophores. Melanophores have pigment organelles or melanosomes, which, in response to hormones, disperse in the cytoplasm or aggregate in the perinuclear region. We followed the motion of melanosomes in cells treated to depolymerize microtubules during aggregation and dispersion, focusing the analysis on the dynamics of these organelles in a time window not explored before to our knowledge. These data could not be explained by previous models that only consider active transport. We proposed a transport-diffusion model in which melanosomes may detach from actin tracks and reattach to nearby filaments to resume the active motion after a given time of diffusion. This model predicts that organelles ... [truncated at 150 words]
Martin GL, Ross JA, Minteer SD, Jameson DM, Cooney MJ.
Fluorescence characterization of chemical microenvironments in hydrophobically modified chitosan.
Carbohydr Polym. 2009; 77(4): 695-702.In this work we use the steady state and time-resolved fluorescence of free and enzyme-bound fluorophores to characterize the binding capacity of both unmodified and hydrophobically modified chitosan polymers. Additionally, fluorescence emission is used to qualitatively characterize the extent to which hydrophobic modification of the chitosan polymer affects the relative polarity of the resultant amphiphillic micelles. In total, these results are used to describe how fluorescence techniques can be used to characterize the chemical microenvironment provided by immobilization polymers such as chitosan. Commentary is also given on how this information can be correlated to enzyme activity and spatial distribution during the immobilization processes.
Fidorra M, Heimburg T, Bagatolli LA.
Direct visualization of the lateral structure of porcine brain cerebrosides/POPC mixtures in presence and absence of cholesterol.
Biophys J. 2009; 97(1): 142-154. PMCID: PMC2711360We studied the thermal behavior of membranes composed of mixtures of natural cerebrosides (from porcine brain) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with and without cholesterol, using differential scanning calorimetry, Fourier transform infrared spectroscopy, and confocal/multiphoton fluorescence microscopy. The POPC/cerebroside mixture display solid ordered/liquid disordered phase coexistence in a broad range of compositions and temperatures in agreement with previous results reported for POPC/(bovine brain)cerebrosides. The observed phase coexistence scenario consists of elongated, micrometer-sized cerebroside-rich solid ordered domains that span the bilayer, embedded in a POPC-rich liquid disordered phase. The data obtained from differential scanning calorimetry and Fourier transform infrared spectroscopy was in line with that obtained in the microscopy experiments for the binary mixture, except at very high cerebroside molar fractions (0.8-0.9) were some differences are observed. Cholesterol incorporation exerts strong changes on the lateral organization of POPC/porcine brain cerebroside membranes. At intermediate cholesterol concentrations (10-25 mol %) the solid ordered/liquid disordered phase ... [truncated at 150 words]
Suwalsky M, Novoa V, Villena F, Sotomayor CP, Aguilar LF, Ronowska A, Szutowicz A.
Structural effects of Zn2+ on cell membranes and molecular models.
J Inorg Biochem. 2009; 103(5): 797-804.Zinc is an essential element for nutrition as well as for the proper development and function of brain cells, and its traces are present in a wide range of foods. It is a constituent of many enzyme systems and is an integral part of insulin and of the active site of intracellular enzymes. However, excessive accumulation of zinc or its release from the binding sites may become detrimental for neurons. With the aim to better understand the molecular mechanisms of the interaction of zinc ions with cell membranes, it was incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), cholinergic murine neuroblastoma cells, and molecular models of cell membranes. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, particularly that of human erythrocytes, respectively. The capacity of zinc ions to perturb the ... [truncated at 150 words]
Neubauer A, Bendig J, Rettig W.
Control of charge transfer by conformational and electronic effects: Donor-donor and donor-acceptor phenyl pyrroles.
Chem Phys. 2009; 358(3): 235-244.Derivatives of N-pyrrolobenzene with a para-donor and a para-acceptor substituent on the benzene ring are compared. It is shown that by a suitable increase of the donor strength of the pyrrolo group, CT fluorescence can be achieved even for donor–donor-substituted benzenes. The ICT emission for sterically hindered compounds is more forbidden than that of unhindered phenyl pyrroles. This suggests conformational effects which induce a narrower twist angle distribution around a perpendicular minimum in the excited state.
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Fluorescence fluctuation spectroscopy of mCherry in living cells.
Biophys J. 2009; 96(6): 2391-404. PMCID: PMC2907682The red fluorescent protein mCherry is of considerable interest for fluorescence fluctuation spectroscopy (FFS), because the wide separation in color between mCherry and green fluorescent protein provides excellent conditions for identifying protein interactions inside cells. This two-photon study reveals that mCherry exists in more than a single brightness state. Unbiased analysis of the data needs to account for the presence of multiple states. We introduce a two-state model that successfully describes the brightness and fluctuation amplitude of mCherry. The properties of the two states are characterized by FFS and fluorescence lifetime experiments. No interconversion between the two states was observed over the experimentally probed timescales. The effect of fluorescence resonance energy transfer between enhanced green fluorescent protein (EGFP) and mCherry is incorporated into the two-state model to describe protein hetero-oligomerization. The model is verified by comparing the predicted and measured brightness and fluctuation amplitude of several fusion proteins that contain ... [truncated at 150 words]
Zhao Y, Knee JL, Baranger AM.
Characterization of two adenosine analogs as fluorescence probes in RNA.
Bioorg Chem. 2008; 36(6): 271-277. PMCID: PMC2661016The fluorescence properties of two adenosine analogs, 2-(3-phenylpropyl)adenosine [A-3CPh] and 2-(4-phenylbutyl)adenosine [A-4CPh], are reported. As monomers, the quantum yields and the mean lifetimes are 0.011 and 6.22 ns for A-3CPh and 0.007 and 7.13 ns for A-4CPh, respectively. Surprisingly, the quantum yields of the two probes are enhanced 11- to 82-fold upon incorporation into RNA, while the mean lifetimes decrease 23–40%. The data suggest that a subpopulation of molecules is responsible for the fluorescence characteristics and that the distribution of emitting and non-emitting structures is altered upon incorporation of the probes into RNA. Thus, although both adenosine analogs have low quantum yields as monomers, their fluorescence signals are significantly enhanced in RNA. Thermodenaturation experiments and CD spectroscopy indicate that incorporation of the adenosine analogs into three different RNAs does not alter their global structure or stability. Therefore, these probes should be useful for probing events occurring close to the site ... [truncated at 150 words]
Carrera DC, Vermehren C, Bagatolli LA.
Pig skin structure and transdermal delivery of liposomes: a two photon microscopy study.
J Control Release. 2008; 132(1): 12-20.In this work we have characterized the architecture and physical properties of pig skin epidermis including its permeability to different liposome formulations. Autofluorescence images show that cells in the epidermis, from the basal layer to the stratum corneum, are organized in clusters that are in turn separated by particular structures we named "canyons". These canyons start in the surface as a wrinkle, eventually closing and going all the way inside the epidermis as a distinct structure that reaches the stratum basale. This structure, described previously in the epidermis of mouse skin as "intercluster pathway", was suggested to be filled with an unknown material and offer low resistance to vesicle penetration. Analysis of LAURDAN Generalized Polarization images of pig skin show that the canyons are filled with a non-polar poorly hydrated material, similar to that observed in pig skin stratum corneum. These results together with the data obtained from skin autofluorescence ... [truncated at 150 words]
Padilla-Parra S, Audugé N, Coppey-Moisan M, Tramier M.
Quantitative FRET analysis by fast acquisition time domain FLIM at high spatial resolution in living cells.
Biophys J. 2008; 95(6): 2976-2988. PMCID: PMC2527249Quantitative analysis in Förster resonance energy transfer (FRET) experiments in live cells for protein interaction studies is still a challenging issue. In a two-component system (FRET and no FRET donor species), fitting of fluorescence lifetime imaging microscopy (FLIM) data gives the fraction of donor molecules involved in FRET (f(D)) and the intrinsic transfer efficiency. But when fast FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are not reliable, even for single lifetime donors. We introduce the new concept of a minimal fraction of donor molecules involved in FRET (mf(D)), coming from the mathematical minimization of f(D). We find particular advantage in the use of mf(D) because it can be obtained without fitting procedures and it is derived directly from FLIM data. mf(D) constitutes an interesting quantitative ... [truncated at 150 words]
Alston RW, Lasagna M, Grimsley GR, Scholtz JM, Reinhart GD, Pace CN.
Peptide sequence and conformation strongly influence tryptophan fluorescence.
Biophys J. 2008; 94(6): 2280-2287. PMCID: PMC2257887This article probes the denatured state ensemble of ribonuclease Sa (RNase Sa) using fluorescence. To interpret the results obtained with RNase Sa, it is essential that we gain a better understanding of the fluorescence properties of tryptophan (Trp) in peptides. We describe studies of N-acetyl-L-tryptophanamide (NATA), a tripeptide: AWA, and six pentapeptides: AAWAA, WVSGT, GYWHE, HEWTV, EAWQE, and DYWTG. The latter five peptides have the same sequence as those surrounding the Trp residues studied in RNase Sa. The fluorescence emission spectra, the fluorescence lifetimes, and the fluorescence quenching by acrylamide and iodide were measured in concentrated solutions of urea and guanidine hydrochloride. Excited-state electron transfer from the indole ring of Trp to the carbonyl groups of peptide bonds is thought to be the most important mechanism for intramolecular quenching of Trp fluorescence. We find the maximum fluorescence intensities vary from 49,000 for NATA with two carbonyls, to 24,400 for AWA ... [truncated at 150 words]
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Applications of dual-color fluorescence cross-correlation spectroscopy in antibody binding studies.
Anal Biochem. 2008; 374(1): 182-195.Two-photon dual-color fluorescence cross-correlation spectroscopy (DC-FCCS) was applied to study the binding interactions of monoclonal antibodies (mAbs) and protein antigens. We measured the binding constant of the interaction of a 32-amino acid brain natriuretic peptide (BNP) with a mAbs and demonstrated the utility of DC-FCCS in studies of antibody sandwiches, trimolecular formations, where two different antibodies bind the same antigen simultaneously. We also show the use of DC-FCCS for monitoring competitive displacement of the labeled antibody in antibody-antigen complexes and subsequent determination of the pertinent dissociation rate (off-rate). The off-rate measurements were performed for two mAbs toward tissue inhibitor 1 of metalloproteinases (TIMP-1). From a methodological perspective, selection of the best labeling protocols and careful optimization of the FCCS instrumentation are essential to achieve the highest cross-correlation signal. When working in vitro, it is practical to generate a complete binding curve using the normalized cross-correlation signal and then fit the ... [truncated at 150 words]
Jose M, Nair DK, Altrock WD, Dresbach T, Gundelfinger ED, Zuschratter W.
Investigating interactions mediated by the presynaptic protein bassoon in living cells by Foerster's resonance energy transfer and fluorescence lifetime imaging microscopy.
Biophys J. 2008; 94(4): 1483-1496. PMCID: PMC2212683Neuronal synapses are highly specialized structures for communication between nerve cells. Knowledge about their molecular organization and dynamics is still incomplete. The large multidomain protein Bassoon plays a major role in scaffolding and organizing the cytomatrix at the active zone of neurotransmitter release in presynaptic boutons. Utilizing immunofluorescence techniques, we show that Bassoon is essential for corecruitment of its synaptic interaction partners, C-terminal binding protein 1/brefeldin A-dependent ADP-ribosylation substrate and CAZ-associated structural protein, into protein complexes upon heterologous expression in COS-7 cells. A combination of Foerster's resonance energy transfer and fluorescence lifetime imaging microscopy in the time domain was adopted to investigate the potential for the association of these proteins in the same complexes. A direct physical association between Bassoon and CtBP1 could also be observed at synapses of living hippocampal neurons. Simultaneous analysis of fluorescence decays of the donor and the acceptor probes along with their decay-associated spectra allowed ... [truncated at 150 words]
Guizy M, David M, Arias C, Zhang L, Cofán M, Ruiz-Gutiérrez V, Ros E, Lillo MP, Martens JR, Valenzuela C.
Modulation of the atrial specific Kv1.5 channel by the n-3 polyunsaturated fatty acid, α-linolenic acid.
J Mol Cell Cardiol. 2008; 44(2): 323-335.Epidemiological, clinical and experimental studies suggest that the cardioprotective effect of fish intake is mainly due to the antiarrhythmic properties of marine n-3 polyunsaturated fatty acids (PUFA), which modulate ion currents. Emerging evidences point to similar effects of α-linolenic acid (ALA), a vegetable n-3 PUFA, but much less is known about its effects on the specific cardiac ion channels. Using electrophysiology, protein biochemistry and fluorescence anisotropy measurements, we tested the effects of ALA on the atrial specific Kv1.5 channel. In stably transfected Ltk− cells, ALA blocked Kv1.5 channels in a time- and voltage-dependent manner with an IC50 value of 3.7 ± 0.3 μM. ALA at 2.5 μM inhibited the Kv1.5 current, shifted the midpoint of the activation curve by − 8.8 ± 4.3 mV (p < 0.05), accelerated the activation kinetics of Kv1.5 due to a negative shift in its voltage dependency and slowed its deactivation process. Marine n-3 PUFA ... [truncated at 150 words]
Montes LR, Alonso A, Goñi FM, Bagatolli LA.
Giant unilamellar vesicles electroformed form native membranes and organic lipid mixtures under physiological conditions.
Biophys J. 2007; 93(10): 3548-3554. PMCID: PMC2072068In recent years, giant unilamellar vesicles (GUVs) have become objects of intense scrutiny by chemists, biologists, and physicists who are interested in the many aspects of biological membranes. In particular, this "cell size" model system allows direct visualization of particular membrane-related phenomena at the level of single vesicles using fluorescence microscopy-related techniques. However, this model system lacks two relevant features with respect to biological membranes: 1), the conventional preparation of GUVs currently requires very low salt concentration, thus precluding experimentation under physiological conditions, and 2), the model system lacks membrane compositional asymmetry. Here we show for first time that GUVs can be prepared using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures at physiological ionic strength. Additionally, for the GUVs composed of native membranes, we show that membrane proteins and glycosphingolipids preserve their natural orientation after electroformation. We anticipate our result to ... [truncated at 150 words]
Plasencia I, Norlén L, Bagatolli LA.
Direct visualization of lipid domains in human skin stratum corneum's lipid membranes: effect of pH and temperature.
Biophys J. 2007; 93(9): 3142-3155. PMCID: PMC2025644The main function of skin is to serve as a physical barrier between the body and the environment. This barrier capacity is in turn a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix. This lipid matrix is essentially composed of very long chain saturated ceramides, cholesterol, and free fatty acids. Three unsolved key questions are i), whether the stratum corneum extracellular lipid matrix is constituted by a single gel phase or by coexisting crystalline (solid) domains; ii), whether a separate liquid crystalline phase is present; and iii), whether pH has a direct effect on the lipid matrix phase behavior. In this work the lateral structure of membranes composed of lipids extracted from human skin stratum corneum was studied in a broad temperature range (10 degrees C-90 degrees C) using different techniques such as differential scanning calorimetry, fluorescence spectroscopy, and two-photon excitation and laser ... [truncated at 150 words]
Coutinho A, García C, González-Rodríguez J, Lillo MP.
Conformational changes in human integrin αIIbβ3 after platelet activation, monitored by FRET.
Biophys Chem. 2007; 130(1-2): 76-87.Integrin αIIbβ3, an abundant heterodimeric receptor at the surface of blood platelets, binds adhesive proteins after platelet activation and plays a primary role in haemostasis. In solution, it has been observed mainly in two conformations: the bent and the extended forms. Based on X-ray crystallography, electron microscopy and immunochemical observations of full-length integrin ectodomains and intact integrins, it has been agreed that unactivated integrins are in the bent conformation, both isolated in solution and in living cells. However, consensus is yet to emerge on the bent or extended conformation of activated integrins and on their mechanism of activation (the switchblade, the deadbolt and the S–S reduction models), which require further experimental tests at the cell level to become established facts.
Here, we tested the proposed structural rearrangements undergone by integrin αIIbβ3 after cell activation, by using Förster-type fluorescence resonance energy transfer (FRET) and attached fluorescent labels to Fab fragments of monoclonal ... [truncated at 150 words]
Jose M, Nair DK, Reissner C, Hartig R, Zuschratter W.
Photophysics of Clomeleon by FLIM: discriminating excited state reactions along neuronal development.
Biophys J. 2007; 92(6): 2237-2254. PMCID: PMC1861805In this work, fluorescence lifetime imaging microscopy in the time domain was used to study the fluorescence dynamics of ECFP and of the ratiometric chloride sensor Clomeleon along neuronal development. The multiexponential analysis of fluorophores combined with the study of the contributions of the individual lifetimes (decay-associated spectra) was used to discriminate the presence of energy transfer from other excited state reactions. A characteristic change of sign of the pre-exponential factors of lifetimes from positive to negative near the acceptor emission maxima was observed in presence of energy transfer. By fluorescence lifetime imaging microscopy, we could show that the individual conformations of CFP display differential quenching properties depending on their microenvironment. Suitability of Clomeleon as an optical indicator to obtain a direct readout of the intracellular chloride concentrations in living cells was verified by steady-state and time-resolved spectroscopy. The simultaneous study of the photophysical properties of Clomeleon, the calcium indicator ... [truncated at 150 words]
