Ranjit S, Henriksen K, Dvornikov A, Delsante M, Rosenberg A, Levi M, Gratton E.
Phasor approach to autofluorescence lifetime imaging FLIM can be a qantitative biomarker of chronic renal Parenchymal injury.
Kidney Int. 2020; Online ahead of print.Diabetic kidney disease continues to be the leading cause of chronic kidney disease, often advancing to end stage kidney disease. In addition to the well characterized glomerular alterations including mesangial expansion, podocyte injury, and glomerulosclerosis, tubulointerstitial fibrosis is also an important component of diabetic kidney injury. Similarly, tubulointerstitial fibrosis is a critical component of any chronic kidney injury. Therefore, sensitive and quantitative identification of tubulointerstitial fibrosis is critical for the assessment of long-term prognosis of kidney disease. Here, we employed phasor approach to fluorescence lifetime imaging, commonly known as FLIM, to understand tissue heterogeneity and calculate changes in the tissue autofluorescence lifetime signatures due to diabetic kidney disease. FLIM imaging was performed on cryostat sections of snap-frozen biopsy material of patients with diabetic nephropathy. There was an overall increase in phase lifetime (τphase) with increased disease severity. Multicomponent phasor analysis shows the distinctive differences between the different disease states. Thus, ... [truncated at 150 words]
Chen YC, Sood C, Marin M, Aaron J, Gratton E, Salaita K, Melikyan GB.
Super-resolution fluorescence imaging reveals that Serine incorporator protein 5 inhibits human immunodeficiency virus fusion by disrupting envelope glycoprotein clusters.
ACS Nano. 2020; Online ahead of print.Serine incorporator protein 5 (SERINC5) is the host anti-retroviral factor that reduces HIV-1 infectivity by incorporating into virions and inhibiting the envelope glycoprotein (Env) mediated virus fusion with target cells. We and others have shown that SERINC5 incorporation into virions alters the Env structure and sensitizes the virus to broadly neutralizing antibodies targeting cryptic Env epitopes. We have also found that SERINC5 accelerates the loss of Env function over time compared to control viruses. However, the exact mechanism by which SERINC5 inhibits HIV-1 fusion is not understood. Here, we utilized 2D and 3D super-resolution microscopy to examine the effect of SERINC5 on the distribution of Env glycoproteins on single HIV-1 particles. We find that, in agreement with a previous report, Env glycoproteins form clusters on the surface of mature virions. Importantly, incorporation of SERINC5, but not SERINC2, which lacks antiviral activity, disrupted Env clusters without affecting the overall Env content. ... [truncated at 150 words]
Cervantes M, Forné I, Ranjit S, Gratton E, Imhof A, Sassone-Corsi P.
BMAL1 associates with NOP58 in the nucleolus and contributes to pre-rRNA processing.
iScience. 2020; 23(6), 101151.The transcription factor BMAL1 is a core element of the circadian clock that contributes to cyclic control of genes transcribed by RNA polymerase II. By using biochemical cellular fractionation and immunofluorescence analyses we reveal a previously uncharacterized nucleolar localization for BMAL1. We used an unbiased approach to determine the BMAL1 interactome by mass spectrometry and identified NOP58 as a prominent nucleolar interactor. NOP58, a core component of the box C/D small nucleolar ribonucleoprotein complex, associates with Snord118 to control specific pre-ribosomal RNA (pre-rRNA) processing steps. These results suggest a non-canonical role of BMAL1 in ribosomal RNA regulation. Indeed, we show that BMAL1 controls NOP58-associated Snord118 nucleolar levels and cleavage of unique pre-rRNA intermediates. Our findings identify an unsuspected function of BMAL1 in the nucleolus that appears distinct from its canonical role in the circadian clock system.
Vallmitjana A, Dvornikov A, Torrado B, Jameson DM, Ranjit S, Gratton E.
Resolution of 4 components in the same pixel in FLIM images using the phasor approach.
Methods Appl Fluoresc. 2020; 8(3), 035001.In several cellular systems, the phasor FLIM approach has shown the existence of more than 2 components in the same pixel, a typical example being free and bound NADH. In order to properly quantify the concentrations and the spatial distributions of fluorescence components associated with different molecular species we developed a general method to resolve 3 and 4 components in the same pixel using the phasor approach. The method is based on the law of linear combination of components valid after transformation of the decay curves to phasors for each pixel in the image. In principle, the linear combination rule is valid for an arbitrary number of components. For 3 components we use only the phasor position for the first harmonic, which has a small error, while for 4 components we need the phasor location at higher harmonics that have intrinsically more noise. As a result of the noise in ... [truncated at 150 words]
Sameni S, Zhang R, Digman MA.
The phasor FLIM method reveals a link between a change in energy metabolism and mHtt protein spread in healthy mammalian cells when co-cultured with Huntington diseased cells.
Methods Appl Fluoresc. 2020; [Epub ahead of print].Huntington Disease (HD) is a late-onset autosomal neurodegenerative disease characterized by the aggregations of mutant Huntingtin proteins (mHTT). A glutamine stretch (PolyQ) at the N-terminal of the Huntingtin protein is generated by the abnormal expansion of CAG trinucleotide repeats in exon 1 of the HTT gene. While the resulting polyQ aggregates are the predominate feature of HD , the intercellular spread of the expanded protein and the effect upon this transfer inside healthy cells have not yet fully understood. Here, we have employed the phasor Fluorescence Lifetime Imaging Microscopy (FLIM) method to measure NADH fluorescence lifetime change after the internalization of the PolyQ protein. Based on our analysis, we have found a significant decrease in the fraction of bound NADH in both cytoplasmic and nucleus regions when cells are co-cultured or when healthy cells uptake the supernatant containing polyQ proteins and aggregates. Overall, our FLIM study combined with confocal fluorescence ... [truncated at 150 words]
Pascua SM, McGahey GE, Ma N, Wang JJ, Digman MA.
Caffeine and Cisplatin effectively targets the metabolism of a triple-negative breast cancer cell line assessed via Phasor-FLIM.
Int J Mol Sci. 2020; 21(7), E2443. PMCID: PMC7177700Triple-negative tumor cells, a malignant subtype of breast cancer, lack a biologically targeted therapy. Given its DNA repair inhibiting properties, caffeine has been shown to enhance the effectiveness of specific tumor chemotherapies. In this work, we have investigated the effects of caffeine, cisplatin, and a combination of the two as potential treatments in energy metabolism for three cell lines, triple-negative breast cancer (MDA-MB-231), estrogen-receptor lacking breast cancer (MCF7) and breast epithelial cells (MCF10A) using a sensitive label-free approach, phasor-fluorescence lifetime imaging microscopy (phasor-FLIM). We found that solely using caffeine to treat MDA-MB-231 shifts their metabolism towards respiratory-chain phosphorylation with a lower ratio of free to bound NADH, and a similar trend is seen in MCF7. However, MDA-MB-231 cells shifted to a higher ratio of free to bound NADH when cisplatin was added. The combination of cisplatin and caffeine together reduced the survival rate for MDA-MD231 and shifted their energy metabolism ... [truncated at 150 words]
Haensel D, Jin S, Sun P, Cinco R, Dragan M, Nguyen Q, Cang Z, Gong Y, Vu R, MacLean AL, Kessenbrock K, Gratton E, Nie Q, Dai X.
Defining epidermal basal cell states during skin homeostasis and wound healing using single-cell transcriptomics.
Cell Rep. 2020; 30(11): 3932-3947.e6. PMCID: PMC7218802Our knowledge of transcriptional heterogeneities in epithelial stem and progenitor cell compartments is limited. Epidermal basal cells sustain cutaneous tissue maintenance and drive wound healing. Previous studies have probed basal cell heterogeneity in stem and progenitor potential, but a comprehensive dissection of basal cell dynamics during differentiation is lacking. Using single-cell RNA sequencing coupled with RNAScope and fluorescence lifetime imaging, we identify three non-proliferative and one proliferative basal cell state in homeostatic skin that differ in metabolic preference and become spatially partitioned during wound re-epithelialization. Pseudotemporal trajectory and RNA velocity analyses predict a quasi-linear differentiation hierarchy where basal cells progress from Col17a1Hi/Trp63Hi state to early-response state, proliferate at the juncture of these two states, or become growth arrested before differentiating into spinous cells. Wound healing induces plasticity manifested by dynamic basal-spinous interconversions at multiple basal transcriptional states. Our study provides a systematic view of epidermal cellular dynamics, supporting a revised ... [truncated at 150 words]
Liang Z, Lou J, Scipioni L, Gratton E, Hinde E.
Quantifying nuclear wide chromatin compaction by phasor analysis of histone Förster resonance energy transfer (FRET) in frequency domain fluorescence lifetime imaging microscopy (FLIM) data.
Data Brief. 2020; 30, 105401. PMCID: PMC7152662The nanometer spacing between nucleosomes throughout global chromatin organisation modulates local DNA template access, and through continuous dynamic rearrangements, regulates genome function [1]. However, given that nucleosome packaging occurs on a spatial scale well below the diffraction limit, real time observation of chromatin structure in live cells by optical microscopy has proved technically difficult, despite recent advances in live cell super resolution imaging [2]. One alternative solution to quantify chromatin structure in a living cell at the level of nucleosome proximity is to measure and spatially map Förster resonance energy transfer (FRET) between fluorescently labelled histones - the core protein of a nucleosome [3]. In recent work we established that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) is a robust method for the detection of histone FRET which can quantify nuclear wide chromatin compaction in the presence of cellular autofluorescence [4]. Here we share FLIM data recording histone ... [truncated at 150 words]
Perinbam K, Kannan JVCA, Digman MA, Siryaporn A.
A shift in central metabolism accompanies virulence activation in Pseudomonas aeruginosa.
mBio. 2020; 11(2), e02730-18. PMCID: PMC7064766The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total ... [truncated at 150 words]
Davis RT, Blake K, Ma D, Gabra MBI, Hernandez GA, Phung AT, Yang Y, Maurer D, Lefebvre AEYT, Alshetaiwi H, Xiao Z, Liu J, Locasale JW, Digman MA, Mjolsness E, Kong M, Werb Z, Lawson DA.
Transcriptional diversity and bioenergetic shift in human breast cancer metastasis revealed by single-cell RNA sequencing.
Nat Cell Biol. 2020; 22(3): 310-320.Although metastasis remains the cause of most cancer-related mortality, mechanisms governing seeding in distal tissues are poorly understood. Here, we establish a robust method for the identification of global transcriptomic changes in rare metastatic cells during seeding using single-cell RNA sequencing and patient-derived-xenograft models of breast cancer. We find that both primary tumours and micrometastases display transcriptional heterogeneity but micrometastases harbour a distinct transcriptome program conserved across patient-derived-xenograft models that is highly predictive of poor survival of patients. Pathway analysis revealed mitochondrial oxidative phosphorylation as the top pathway upregulated in micrometastases, in contrast to higher levels of glycolytic enzymes in primary tumour cells, which we corroborated by flow cytometric and metabolomic analyses. Pharmacological inhibition of oxidative phosphorylation dramatically attenuated metastatic seeding in the lungs, which demonstrates the functional importance of oxidative phosphorylation in metastasis and highlights its potential as a therapeutic target to prevent metastatic spread in patients with breast ... [truncated at 150 words]
Titlow J, Robertson F, Järvelin A, Ish-Horowicz D, Smith C, Gratton E, Davis I.
Syncrip/hnRNP Q is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation.
J Cell Biol. 2020; 219(3), e201903135. PMCID: PMC7055005Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. Here, we show that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator ... [truncated at 150 words]
Tetreau G, Banneville AS, Andreeva EA, Brewster AS, Hunter MS, Sierra RG, Teulon JM, Young ID, Burke N, Grünewald TA, Beaudouin J, Snigireva I, Fernandez-Luna MT, Burt A, Park HW, Signor L, Bafna JA, Sadir R, Fenel D, Boeri-Erba E, Bacia M, Zala N, Laporte F, Després L, Weik M, Boutet S, Rosenthal M, Coquelle N, Burghammer M, Cascio D, Sawaya MR, Winterhalter M, Gratton E, Gutsche I, Federici B, Pellequer JL, Sauter NK, Colletier JP.
Serial femtosecond crystallography on in vivo-grown crystals drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade.
Nat Commun. 2020; 11(1): 1153. PMCID: PMC7052140Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.
Takahashi S, Luo Y, Ranjit S, Xie C, Libby AE, Orlicky DJ, Dvornikov A, Wang XX, Myakala K, Jones BA, Bhasin K, Wang D, McManaman JL, Krausz KW, Gratton E, Ir D, Robertson CE, Frank DN, Gonzalez FJ, Levi M.
Bile acid sequestration reverses liver injury and prevents progression of NASH in western diet-fed mice.
J Biol Chem. 2020; [Epub ahead of print]. PMCID: PMC7135973Non-alcoholic fatty liver disease (NAFLD) is a rapidly rising problem in the 21st century and is a leading cause of chronic liver disease that can lead to end-stage liver diseases, including cirrhosis and hepatocellular cancer. Despite this rising epidemic, no pharmacological treatment has yet been established to treat this disease. The rapidly increasing prevalence of NAFLD and its aggressive form, nonalcoholic steatohepatitis (NASH), requires novel therapeutic approaches to prevent disease progression. Alterations in microbiome dynamics and dysbiosis play an important role in liver disease, and these may represent targetable pathways to treat liver disorders. Improving microbiome properties or restoring normal bile acid metabolism may prevent or slow the progression of liver diseases such as NASH. Importantly, aberrant systemic circulation of bile acids can greatly disrupt metabolic homeostasis. Bile acid sequestrants (BAS) are orally administered polymers that bind bile acids in the intestine forming nonabsorbable complexes. BAS interrupts intestinal ... [truncated at 150 words]
Abram TJ, Cherukury H, Ou CY, Vu T, Toledano M, Li Y, Grunwald JT, Toosky MN, Tifrea DF, Slepenkin A, Chong J, Kong L, Pozo DVD, La KT, Labanieh L, Zimak J, Shen B, Huang SS, Gratton E, Peterson EM, Zhao W.
Rapid bacterial detection and antibiotic susceptibility testing in whole blood using one-step, high throughput blood digital PCR.
Lab Chip. 2020; 20(3): 477-489. PMCID: PMC7250044Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates ... [truncated at 150 words]
Palomba F, Scipioni L, Gratton E, Digman MA.
Water dynamics of a model protein phase separation via fluorescence lifetime and spectral analysis of ACDAN.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 308a, 1509-Pos.Cells can organize many of their biochemical reactions in membrane-less compartments. These can be achieved by self-assembly of proteins leading to protein phase separations (PPS). The mechanism of de-mixing can be physiological (inner centromere) and/or pathological (stress granules). PPS organize cellular regions via confinement and exclusion of the molecules affecting their dynamics within and surrounding the compartment. PPS, also called coacervates, can express different types of phase, from liquid- to solid-like. To characterize that, investigations commonly starts with the definition of the phase separation diagram of the purified protein that drive the water/protein liquid demixing. In our opinion, to better understand these complex phases, the characterization of the water dynamics is fundamental. In this contest, we proposed the study of the water dipolar relaxation measuring the emission spectra and fluorescence lifetime at optical resolution of ACDAN (6-acetyl-2-dimethylaminonaphthalene, solvofluorochromic dye) that colocalize on a model PPS. Indeed, we implement the method ... [truncated at 150 words]
Torrado B, Scipioni L, Gratton E, Badano JL, Malacrida LS, Irigoín F.
Primary cilium submicron organization and dynamics.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 437a, 2144-Pos.The aim of this work is to contribute to the characterization of the biophysical properties of the ciliary microenvironment in order to understand central processes of the organelle's biology such as protein import, movement and crowding organization. First, we analyzed the movement of a small soluble protein (EGFP) to establish its pattern of diffusion in different regions of the ciliary compartment (body and distal tip). Using advanced fluorescence fluctuation correlation spectroscopy (Comprehensive correlation analysis) with the Zeiss Airyscan Detector, we characterized the organization and dynamic fingerprint of these subcompartments. Preliminary results showed that both ciliary body and distal tip have permeable microdomains explaining why EGFP presents a pattern of diffusion compatible with dynamic partition rather than free diffusion. However, at the ciliary body diffusion is more confined than at the ciliary tip. Measurements on cytoplasm and nucleus were made to compare with cilia crowding organization. Moreover, to study ciliary structural ... [truncated at 150 words]
Hedde PN, Staaf E, Singh SB, Johansson S, Gratton E.
Pair correlation analysis reveals barriers to natural killer cell receptor motion at the synapse.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 246a, 1211-Pos.In living systems, barriers and obstacles to biomolecular dynamics, including receptor motion, critically regulate many mechanisms. Conventional methods including raster image correlation spectroscopy (RICS) and image mean square displacement (iMSD) analysis focus on measuring the average, isotropic motion of molecules in a larger region of interest [1]. Instead, the two-dimensional pair correlation function (2D-pCF) approach can visualize and quantify the paths taken by molecules with high spatial resolution to reveal spatial heterogeneities in motion as caused by barriers and obstacles that break the symmetry [2]. We used this method to study the anisotropy in the motion of natural killer (NK) cell receptors at the immune synapse. In cultured human HLA null 721.221 cells, we observed more barriers to motion for inhibitory receptor HLA-Cw4-YFP coexpressed with KIR3DL1 compared to inhibitory receptor HLA-B51-YFP coexpressed with KIR2DL1. Further, in a mouse model for NK cell education, we found the dynamic spatial organization of ... [truncated at 150 words]
Oneto M, Scipioni L, Sarmento M, Cainero I, Cerutti E, Pelicci S, Furia L, Pelicci PG, Dellino GI, Bianchini P, Faretta M, Gratton E, Diaspro A, Lanzano L.
Nanoscale distribution of nuclear sites analyzed by superresolution STED image cross-correlation spectroscopy.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 20a, 93-Plat.Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, superresolution microscopy has gained considerable interest as it can be used to probe the spatial organization of functional sites in intact single cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a pre-segmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet.
Hsu C, Gratton E, Anders R, Rosenberg A, Levi M, Ranjit S.
Image correlation microscopy approach to study collagen accumulation for distinguishing recurrence in liver cancer patients.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 309a-310a, 1514-Pos.Collagen alignment, also known as tumor associated collagen signature, has been used to predict worse prognosis in breast cancer. It has been shown in breast cancer studies that when collagen is aligned perpendicular to the tumor, it allows for cancer invasion. Cirrhosis is a risk factor for liver cancer development. Thus, we aim to test the hypothesis that there is a similar collagen signature in patients who recurred with liver cancer after liver transplant when compared to those who did not recur. In this study, we have used image correlation techniques to identify the changes in collagen structures around different cancer samples collected from liver cancer patients. Different properties of the image correlation function, including the anisotropic shape of the correlation function, the shape parameters of the ellipsoid describing the correlation function and orientation of the ellipses were used to create overlapping maps of image correlation properties with the SHG ... [truncated at 150 words]
Vallmitjana A, Vorontsova I, Torrado B, Schilling TF, Hall JE, Gratton E, Malacrida LS.
Measuring the spatial distribution of dipolar relaxation in live Zebrafish eye lenses during development.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 308a, 1508-Pos.This work presents image processing techniques we developed to analyze spectral images of in vivo zebrafish eye lenses using ACDAN (6-acetyl-2-dimethylaminonaphthalene) as a probe for intracellular dipolar relaxation (DR). Using these techniques we analyzed the development of regional DR in zebrafish lenses, as water homeostasis is crucial for normal development of structure and optical properties of the lens. We imaged lenses between two and nine days post-fertilization encompassing stages ranging from immature embryonic to functional emmetropic lenses at larval stages. ACDAN fluorescence was collected using a 32-channel spectral detector at anterior, equatorial and posterior planes of the ocular lens in anesthetized zebrafish incubated in ACDAN. We used the spectral phasor transformation to obtain a continuum of values within an arbitrarily-set interval in order to measure DR at each pixel of the images. From these images we interpolated radial DR profiles in all spatial directions from which a mean radial profile ... [truncated at 150 words]
Tedeschi G, Scipioni L, Abbott G, Digman MA.
Dynamic characterization of KCNQ1 and its regulatory subunits revealed by fluorescence fluctuation techniques.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 262a, 1285-Pos.KCNQ1 is the α subunit of a voltage-gated potassium channel expressed on the membrane of a variety of cells, including cardiac myocytes, where it has a key role in ventricular repolarization. KCNQ1 is known to interact with and be regulated by single-transmembrane domain ancillary subunits encoded by the family of KCNE genes, such as KCNE1 and KCNE2. Mutations in these genes are associated with life-threatening cardiac arrhythmias, including long QT syndrome.
Lefebvre AEYT, Adame FA, Liu M, Digman MA.
Non-destructively analyzing the metabolic dysregulation of Invasive cancer cells on an intracellular scale.
64th Annual Meeting of the Biophysical Society. San Diego, California. February 15-19, 2020.
Biophys J. 2020; 118(3, Suppl 1): 307a, 1505-Pos.Cancer has overtaken cardiovascular disease's top spot in non-communicable deaths. However, it is not cancer itself that looms as the largest killer, but rather cancer's spread to distant organs – a process known as metastasis – that finishes the job. Capturing metabolic alterations of migrating cells intracellularly, and in a non-destructive manner to both the surrounding environment and the cell is technically challenging. In this study, we use fluorescence lifetime imaging (FLIM) of the reduced form of nicotinamide adenine dinucleotide (NADH) and phasor analysis to examine metabolic changes on a single cell level. Additionally, we use phosphorescence lifetime imaging (PLIM) and phasor analysis of an oxygen sensitive probe ruthenium-tris(2,2’-bipyridyl) (Ru(BPY) 3 2+), whose lifetime is quenched in the presence of oxygen, in order to observe intracellular changes of oxygen concentration. Finally, we use spectral imaging and phasor analysis to quantify changes in intracellular lipid order using the fluorescent probe 6-dodecanoyl-N,N-dimethyl-2-naphthylamine ... [truncated at 150 words]
Sameni S, Zhang R, Digman MA.
Number and molecular brightness analysis reveals Htt25Q protein aggregation upon the uptake of Htt97Q aggregates.
Biochem Biophys Res Commun. 2020; 522(1): 133-137.Number and molecular Brightness (N&B) analysis is a powerful method used to monitor protein aggregation in living cells. Here, we used the N&B method to characterize the unexpanded HTT protein oligomerization after the internalization of the mutant HTT (mHTT) which contains a CAG repeat extensions encoding for long polyglutamine (polyQ) proteins resulting in misfolding and aggregation. HEK cells expressing Htt25Q-mCherry proteins were infected with Htt97Q-EGFP aggregates, by cell to cell uptake, in cultured conditions resulting in an increasing population of dimers and tetramers compared to our controls. This study shows for the first time the impact of protein aggregation in the unexpanded Htt25Q-mCherry expressing cells that occurs from cell to cell transfer of the expanded Htt97Q-EGFP. These results signify the sporadic behavior of the polyQ inclusion that gives insight into the mechanism of protein dynamics as a consequence of secreted mHTT aggregates.
Pedrote MM, Motta MF, Ferretti GDS, Norberto DR, Spohr TCLS, Lima FRS, Gratton E, Silva JL, de Oliveira GAP.
Oncogenic gain of function in Glioblastoma is linked to mutant p53 Amyloid oligomers.
iScience. 2020; 23(2), 100820. PMCID: PMC6976948Tumor-associated p53 mutations endow cells with malignant phenotypes, including chemoresistance. Amyloid-like oligomers of mutant p53 transform this tumor suppressor into an oncogene. However, the composition and distribution of mutant p53 oligomers are unknown and the mechanism involved in the conversion is sparse. Here, we report accumulation of a p53 mutant within amyloid-like p53 oligomers in glioblastoma-derived cells presenting a chemoresistant gain-of-function phenotype. Statistical analysis from fluorescence fluctuation spectroscopy, pressure-induced measurements, and thioflavin T kinetics demonstrates the distribution of oligomers larger than the active tetrameric form of p53 in the nuclei of living cells and the destabilization of native-drifted p53 species that become amyloid. Collectively, these results provide insights into the role of amyloid-like mutant p53 oligomers in the chemoresistance phenotype of malignant and invasive brain tumors and shed light on therapeutic options to avert cancer.
Castro-Castillo V, Gajardo J, Sandoval-Altamirano C, Gratton E, Sanchez S, Malacrida L, Gunther G.
CAPRYDAA, an anthracene dye analog to LAURDAN: a comparative study using cuvette and microscopy.
J Mater Chem B. 2020; 8(1). PMCID: PMC7029800We synthesized an anthracene derivative with solvatochromic properties to be used as a molecular probe for membrane dynamics and supramolecular organization. A nine carbon atom acyl chain and a dimethylamino substitution were introduced at positions 2 and 6 of the anthracene ring, respectively. This derivative, 2-nonanoyl-6-(dimethylamino)anthracene (termed CAPRYDAA), is a molecular probe designed to mimic the well-known membrane probe LAURDAN's location and response in the lipid membranes. Due to the larger distance between the electron donor and acceptor groups, its absorption and emission bands are red-shifted according to the polarity of the media. The photophysical behavior of CAPRYDAA was measured in homogeneous media, synthetic bilayer and cells, both in a cuvette and in a fluorescence microscope, using one and two-photon excitation. Our results show a comparable physicochemical behavior of CAPRYDAA with LAURDAN, but with the advantage of using visible light (488 nm) as an excitation source. CAPRYDAA was also excitable ... [truncated at 150 words]
Hedde PN, Staaf E, Singh SB, Johansson S, Gratton E.
Pair correlation analysis maps the dynamic two-dimensional organization of natural killer cell receptors at the synapse.
ACS Nano. 2019; 13(2): 14274-14282.In living systems, the contact between cells is the basis of recognition, differentiation, and orchestration of an immune response. Obstacles and barriers to biomolecular motion, especially for receptors at cellular synapses, critically control these functions by creating an anisotropic environment. Whereas conventional fluorescence fluctuation methods, such as fluorescence correlation spectroscopy or fluorescence recovery after photobleaching, can only measure the isotropic diffusion of molecules, the two-dimensional pair correlation function (2D-pCF) approach probes the anisotropic paths at different spatial locations within an image, allowing the creation of high-resolution maps that can visualize and quantify how molecules move in a living cell. In this work, we show how the 2D-pCF method maps the environment in cellular synapses as perceived by natural killer (NK) cell receptors. In cultured human HLA null 721.221 cells, 2D-pCF reveals the motion of inhibitory receptor HLA-Cw4-YFP coexpressed with KIR3DL1 to be highly directional around specific loci, while these restrictions ... [truncated at 150 words]
Planes N, Vanderheyden PPML, Gratton E, Caballero-George C.
Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells.
Microsc Res Tech. 2019; [Epub ahead of print].The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G-protein-coupled receptors namely Angiotensin II type 1 (AT1 ) and Endothelin 1 type A (ETA ) receptors and their corresponding receptor-ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT1 and ETA receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local ... [truncated at 150 words]
Sitiwin E, Madigan MC, Gratton E, Cherepanoff S, Conway RM, Whan R, Macmillan A.
Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques.
Sci Rep. 2019; 9(1), 18585. PMCID: PMC6901595Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive 'fit-free' approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and ... [truncated at 150 words]
Vorontsova I, Vallmitjana A, Nakazawa Y, Torrado B, Schilling TF, Hall JE, Gratton E, Malacrida L.
Aquaporin 0a is required for water homeostasis in the zebrafish lens in vivo.
6th International Conference on the Lens. Kailua Kona, Hawaii. December 8-13, 2019. 2019; ICL 2019 Meeting Program: 23.Purpose: Aquaporin 0 (AQP0) is essential for lens development and transparency. Mammalian AQP0 has multiple cellular functions including water transport and adhesion, which have been impossible to study individually. An ancestral teleost genome duplication gave rise to two AQP0 orthologs in zebrafish, aqp0a and aqp0b, which apparently have functionally diverged. In vitro, both Aqp0s permeate water. We have shown that CRISPR-Cas9 mediated null aqp0a-/- mutants form cataract, while aqp0b-/- lenses look like wild-type, consistent with distinct functions. In this study, we test adhesive properties, and the requirements of Aqp0a and Aqp0b for water homeostasis in zebrafish lenses in vivo.
Methods: Heterologous expression in adhesion-deficient mouse fibroblast L-cells was used to test adhesive properties of Aqp0s. To study lens water homeostasis in vivo, we have employed a permeable, non-toxic solvatochromic fluorescence probe, ACDAN (6-acetyl-2-dimethylaminonaphthalene). Spectral phasor analysis of the ACDAN signal allows mapping of dipolar relaxation (DR) onto lens images, which we ... [truncated at 150 words]
Oneto M, Scipioni L, Sarmento MJ, Cainero I, Pelicci S, Furia L, Pelicci PG, Dellino GI, Bianchini P, Faretta M, Gratton E, Diaspro A, Lanzanò L.
Nanoscale distribution of nuclear sites by super-resolved image cross-correlation spectroscopy.
Biophys J. 2019; 117(11): 2054-2065. PMCID: PMC6895719Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the ... [truncated at 150 words]
Ranjit S, Datta R, Dvornikov AS, Gratton E.
Multicomponent analysis of phasor plot in a single pixel to calculate changes of metabolic trajectory in biological systems.
J Phys Chem A. 2019; 123(45): 9865-9873.Phasor FLIM in cells undergoing oxidative stress and in mice liver sections have shown presence of a third autofluorescent component indicative of lipid droplets along with free and enzyme bound NADH with similar emission. This third component affects position and shape of the phasor distribution pushing it away from the metabolic trajectory. Phasor rule of addition is still valid and was exploited here to create a multicomponent analysis where the phasor distribution can be reassigned to the metabolic trajectory and changes in metabolism can be detected independently of the intensity of this third component. Calculation of multiple components from FLIM imaging data of biological systems is a difficult process, especially if different fluorescent species are present at a particular pixel. This paper describes the methodology that can be used to separate these multiple components when they are present in the phasor signature acquired in a single pixel of an image.
Ranjit S, Malacrida L, Stakic M, Gratton E.
Determination of the metabolic index using the fluorescence lifetime of free and bound NADH in the phasor approach.
J Biophotonics. 2019; 12(11), e201900156. PMCID: PMC6842045The fluorescence lifetime of nicotinamide adenine dinucleotide (NADH) is commonly used in conjunction with the phasor approach as a molecular biomarker to provide information on cellular metabolism of autofluorescence imaging of cells and tissue. However, in the phasor approach, the bound and free lifetime defining the phasor metabolic trajectory is a subject of debate. The fluorescence lifetime of NADH increases when bound to an enzyme, in contrast to the short multiexponential lifetime displayed by NADH in solution. The extent of fluorescence lifetime increase depends on the enzyme to which NADH is bound. With proper preparation of lactate dehydrogenase (LDH) using oxalic acid (OA) as an allosteric factor, bound NADH to LDH has a lifetime of 3.4 ns and is positioned on the universal semicircle of the phasor plot, inferring a monoexponential lifetime for this species. Surprisingly, measurements in the cellular environments with different metabolic states show a linear trajectory between ... [truncated at 150 words]
