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.Memory 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]
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].Non-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]
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).We 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]
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. 2019; [Epub ahead of print].Sepsis 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]
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]
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]
Gabriel M, Anzalone A, Gratton E, Estrada LC.
A tracking-based nanoimaging method for fast detection of surfaces' inhomogeneities using gold nanoparticles.
Microsc Res Tech. 2019; 82(11): 1835-1842.The localization of surfaces inhomogeneities is central to many areas of technology, chemistry and biology, ranging from surface defects in industry to the identification and screening of early bio-defects inside cells. The development of methods that enable direct, sensitive, and rapid detection of those inhomogeneities is both relevant and timely. To address this challenge, we developed a far-field nanoimaging method to detect the presence of surface's nanodefects that modify the signal emitted by gold nanoparticles (AuNPs) under laser irradiation. Our technique is based on the formation of hot spots due to the confinement of light in the proximity of the AuNP, whose positions depend on the polarization direction of the incident beam. An inhomogeneity is detected as an increase in the intensity collected from the hot spots when a laser beam is orbiting the nanoparticle and the incident polarization direction of the laser beam is changed periodically.
Murata MM, Kong X, Moncada E, Chen Y, Imamura H, Wang P, Berns MW, Yokomori K, Digman MA.
NAD+ consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival.
Mol Biol Cell. 2019; 30(20): 2584-2597. PMCID: PMC6740200DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose- and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, our understanding of a cell-wide consequence of its activation in damaged cells is still limited. Using the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound NADH fraction in response to nuclear DNA damage, which is triggered by PARP-dependent NAD+ depletion. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of oxphos, but not glycolysis, resulted in parthanatos due to rapid PARP-dependent ATP deprivation, indicating that oxphos becomes critical for damaged cell survival. The results reveal ... [truncated at 150 words]
Ma N, de Mochel NR, Pham PD, Yoo TY, Cho KWY, Digman MA.
Label-free assessment of pre-implantation embryo quality by the Fluorescence Lifetime Imaging Microscopy (FLIM)-phasor approach.
Sci Rep. 2019; 9(1), 13206. PMCID: PMC6744410Development of quantitative, safe and rapid techniques for assessing embryo quality provides significant advances in Assisted Reproductive Technologies (ART). Instead of assessing the embryo quality by the standard morphologic evaluation, we apply the phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) method to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify, under hypoxic and non-hypoxic conditions, the unique spectroscopic trajectories at different stages of mouse pre-implantation development, which is referred to as the developmental, or "D-trajectory", that consists of fluorescence lifetime from different stages of mouse pre-implantation embryos. The D-trajectory correlates with intrinsic fluorescent species from a distinctive energy metabolism and oxidized lipids, as seen with Third Harmonic Generation (THG) that changes over time. In addition, we have defined a non-morphological Embryo Viability Index (EVI) to distinguish pre-implantation embryo quality using the Distance Analysis (DA), a machine learning algorithm to process the fluorescence lifetime ... [truncated at 150 words]
Trivedi P, Palomba F, Niedzialkowska E, Digman MA, Gratton E, Stukenberg PT.
The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex.
Nat Cell Biol. 2019; 21(9): 1127-1137.The inner centromere is a region on every mitotic chromosome that enables specific biochemical reactions that underlie properties, such as the maintenance of cohesion, the regulation of kinetochores and the assembly of specialized chromatin, that can resist microtubule pulling forces. The chromosomal passenger complex (CPC) is abundantly localized to the inner centromeres and it is unclear whether it is involved in non-kinase activities that contribute to the generation of these unique chromatin properties. We find that the borealin subunit of the CPC drives phase separation of the CPC in vitro at concentrations that are below those found on the inner centromere. We also provide strong evidence that the CPC exists in a phase-separated state at the inner centromere. CPC phase separation is required for its inner-centromere localization and function during mitosis. We suggest that the CPC combines phase separation, kinase and histone code-reading activities to enable the formation of a ... [truncated at 150 words]
Dong Y, Sameni S, Digman MA, Brewer GJ.
Reversibility of age-related oxidized free NADH redox states in Alzheimer's disease neurons by imposed external Cys/CySS redox shifts.
Sci Rep. 2019; 9(1), 11274. PMCID: PMC6677822Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/NAD+) are critical for maintaining redox homeostasis. Aging as a major risk factor for Alzheimer's disease (AD) is associated with oxidative shifts, decreases in anti-oxidant protection and dysfunction of mitochondria. Here, we examined the flexibility of mitochondrial-specific free NADH in live neurons from non-transgenic (NTg) or triple transgenic AD-like mice (3xTg-AD) of different ages under an imposed extracellular Cys/CySS oxidative or reductive condition. We used phasor fluorescence lifetime imaging microscopy (FLIM) to distinguish free and bound NADH in mitochondria, nuclei and cytoplasm. Under an external oxidative stress, a lower capacity for maintaining mitochondrial free NADH levels was found in old compared to young neurons and a further decline with genetic load. Remarkably, an imposed Cys/CySS reductive state rejuvenated the mitochondrial free NADH levels of old NTg neurons by 71% and old 3xTg-AD neurons by ... [truncated at 150 words]
Levi M, Gratton E, Forster IC, Hernando N, Wagner CA, Biber J, Sorribas V, Murer H.
Mechanisms of phosphate transport.
Nat Rev Nephrol. 2019; 15(8): 482-500.Over the past 25 years, successive cloning of SLC34A1, SLC34A2 and SLC34A3, which encode the sodium-dependent inorganic phosphate (Pi) cotransport proteins 2a-2c, has facilitated the identification of molecular mechanisms that underlie the regulation of renal and intestinal Pi transport. Pi and various hormones, including parathyroid hormone and phosphatonins, such as fibroblast growth factor 23, regulate the activity of these Pi transporters through transcriptional, translational and post-translational mechanisms involving interactions with PDZ domain-containing proteins, lipid microdomains and acute trafficking of the transporters via endocytosis and exocytosis. In humans and rodents, mutations in any of the three transporters lead to dysregulation of epithelial Pi transport with effects on serum Pi levels and can cause cardiovascular and musculoskeletal damage, illustrating the importance of these transporters in the maintenance of local and systemic Pi homeostasis. Functional and structural studies have provided insights into the mechanism by which these proteins transport Pi, whereas in vivo ... [truncated at 150 words]
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The spectral phasor approach as a potential diagnostic tool for breast cancer.
Master in Biomedical Engineering, University of California, Irvine, 2019.
Advisor: Enrico Gratton
Breast cancer is a significant concern within the United States, causing over 200,000 new cases and 40,000 deaths each year. A new potential solution in the form of the spectral phasor approach to data analysis, paired with the noninvasive imaging approach of DOSI, presents itself as a safe, robust, and cost-effective, answering some of the concerns presented by current imaging techniques. By performing a Fourier transform to translate a stack of absorption coefficients at varying wavelengths to a single value or cluster of points on a spectral phasor plot, we are able to visualize the data in a clearly displayed format. In this paper, we explore the spectral phasor approach to analyze data gathered from both a single point on a patient, and multiple points in a grid on a patient as means to a diagnosis. Despite the potential that exists in the solution that this approach presents for quick ... [truncated at 150 words]
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Assessment of embryo health and circulating tumor cell metabolism using the phasor-FLIM approach.
PhD in Biomedical Engineering, University of California, Irvine, 2019.
Advisor: Michelle A Digman
Cellular functional and structural changes associated with metabolism are essential for understanding healthy tissue development and the progression of numerous diseases. Quantitatively monitoring of metabolic processes would spur medical research towards developing precise diagnostic tools, treatment methods, and preventive strategies for reducing the impact of the diseases. Unfortunately, established methods for this purpose are either destructive or require the use of exogenous agents. Recent work has highlighted the potential of endogenous two-photon excited fluorescence as a method to monitor subtle metabolic changes. In this thesis, we apply two-photon fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores for label-free metabolic imaging in pre-implantation embryos and other biological samples.
We exploited the intrinsically fluorescent coenzyme reduced nicotinamide adenine dinucleotide (NADH), an endogenous probe extensively used for metabolic imaging. We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. Using phasor-FLIM, we ... [truncated at 150 words]
Dvornikov A, Malacrida L, Gratton E.
The DIVER microscope for imaging in scattering media.
Methods Protoc. 2019; 2(2): 53. PMCID: PMC6632175We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive to low level light signals than conventional epi-detection used in two-photon fluorescence microscopes. The DIVER detector efficiently collects scattered emission photons from a wide area of turbid samples at almost any entrance angle in a 2π spherical angle. Using an epi-detection scheme only photons coming from a relatively small area of a sample and at narrow acceptance angle can be detected. The transmission geometry of the DIVER imaging system makes it exceptionally suitable for Second and Third Harmonic Generation (SHG, THG) signal detection. It also has in-depth fluorescence lifetime imaging (FLIM) capability. Using special optical filters with sin-cos spectral response, hyperspectral analysis of images acquired in-depth in ... [truncated at 150 words]
Polyzos AA, Lee DY, Datta R, Hauser M, Budworth H, Holt A, Mihalik S, Goldschmidt P, Frankel K, Trego K, Bennett MJ, Vockley J, Xu K, Gratton E, McMurray CT.
Metabolic reprogramming in astrocytes distinguishes region-specific neuronal susceptibility in Huntington mice.
Cell Metab. 2019; 29(6): 1258-1273. PMCID: PMC6583797The basis for region-specific neuronal toxicity in Huntington disease is unknown. Here, we show that region-specific neuronal vulnerability is a substrate-driven response in astrocytes. Glucose is low in HdhQ(150/150) animals, and astrocytes in each brain region adapt by metabolically reprogramming their mitochondria to use endogenous, non-glycolytic metabolites as an alternative fuel. Each region is characterized by distinct metabolic pools, and astrocytes adapt accordingly. The vulnerable striatum is enriched in fatty acids, and mitochondria reprogram by oxidizing them as an energy source but at the cost of escalating reactive oxygen species (ROS)-induced damage. The cerebellum is replete with amino acids, which are precursors for glucose regeneration through the pentose phosphate shunt or gluconeogenesis pathways. ROS is not elevated, and this region sustains little damage. While mhtt expression imposes disease stress throughout the brain, sensitivity or resistance arises from an adaptive stress response, which is inherently region specific. Metabolic reprogramming may have ... [truncated at 150 words]
Rodriguez-Agudo D, Malacrida L, Kakiyama G, Sparrer T, Fortes C, Maceyka M, Sublerh MA, Windle JJ, Gratton E, Pandak WM, Gil G.
StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis.
J Lipid Res. 2019; 60(6): 1087-1098. PMCID: PMC6547630How plasma membrane (PM) cholesterol is controlled is poorly understood. Ablation of the gene encoding the ER stress steroidogenic acute regulatory-related lipid transfer domain 5 (StarD5) leads to a decrease in PM cholesterol content, a decrease in cholesterol efflux, and an increase in intracellular neutral lipid accumulation in macrophages, the major cell type that expresses StarD5. ER stress increases StarD5 expression in mouse hepatocytes, which results in an increase in accessible PM cholesterol in wild type but not in StarD5-/- hepatocytes. StarD5-/- mice store higher levels of cholesterol and triglycerides and leads to altered expression of cholesterol-regulated genes. In vitro, a recombinant GST-StarD5 protein transfers cholesterol between synthetic liposomes. StarD5 overexpression leads to a marked increase in PM cholesterol. Phasor analysis of LAURDAN FLIM data revealed an increase in PM fluidity in StarD5-/- macrophages. Taken together, these studies show that StarD5 is a stress responsive protein that regulates PM cholesterol ... [truncated at 150 words]
Chen H, Ma N, Kagawa K, Kawahito S, Digman M, Gratton E.
Widefield multifrequency fluorescence lifetime imaging using a two-tap complementary metal-oxide semiconductor camera with lateral electric field charge modulators.
J Biophotonics. 2019; 12(5), e201800223.Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) measures the fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD-FLIM system based on a complementary metal-oxide semiconductor camera equipped with two-tap true correlated double sampling lock-in pixels and lateral electric field charge modulators. Owing to the fast intrinsic response and modulation of the camera, our system allows parallel multifrequency FLIM in one measurement via fast Fourier transform. We demonstrate that at a fundamental frequency of 20 MHz, 31-harmonics can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean-Venus that shows Förster Resonance Energy Transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. These results indicate that our widefield multifrequency FD-FLIM system is a ... [truncated at 150 words]
