Lee DH, Li X, Ma N, Digman MA, Lee AP.
Rapid and label-free identification of single leukemia cells from blood in a high-density microfluidic trapping array by fluorescence lifetime imaging microscopy.
Lab Chip. 2018; [Epub ahead of print].The rapid screening and isolation of single leukemia cells from blood has become critical for early leukemia detection and tumor heterogeneity interrogation. However, due to the size overlap between leukemia cells and the more abundant white blood cells (WBCs), the isolation and identification of leukemia cells individually from peripheral blood is extremely challenging and often requires immunolabeling or cytogenetic assays. Here we present a rapid and label-free single leukemia cell identification platform that combines: (1) high-throughput size-based separation of hemocytes via a single-cell trapping array, and (2) leukemia cell identification through phasor approach and fluorescence lifetime imaging microscopy (phasor-FLIM), to quantify changes between free/bound nicotinamide adenine dinucleotide (NADH) as an indirect measurement of metabolic alteration in living cells. The microfluidic trapping array designed with 1600 highly-packed addressable single-cell traps can simultaneously filter out red blood cells (RBCs) and trap WBCs/leukemia cells, and is compatible with low-magnification imaging and fast-speed fluorescence ... [truncated at 150 words]
Kim SM, Nguyen TT, Ravi A, Kubiniok P, Finicle BT, Jayashankar V, Malacrida L, Hou J, Robertson J, Gao D, Chernoff J, Digman MA, Potma EO, Tromberg BJ, Thibault P, Edinger AL.
PTEN deficiency and AMPK activation promote nutrient scavenging and anabolism in prostate cancer cells.
Cancer Discov. 2018; [Epub ahead of print].We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores suggesting that macropinocytosis can supply nutrients other than amino acids. Non-transformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically.
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Spectroscopic signatures of cells metabolism and extracellular species using phasor-FLIM (Conference Presentation).
BiOS 2018. Part of SPIE Photonics West. San Francisco, California, January 27 - February 1, 2018.
Multiphoton Microscopy in the Biomedical Sciences XVIII (Vol. 10498). 1049803, 2018. Fluorescence lifetime imaging has become a common technique in microscopy. The information contained in the fluorescence decay curve can be obtained with different methods. A major issue is that the fluorescence decay must be measured at many pixels either sequentially or in parallel, which generally results in relatively noisy curves. Another major issue is data analysis of the decay at each point of an image and the visualization of the results. Nonetheless, methods of data analysis and visualization have become relatively common and today we can analyze an image for different molecular species and molecular processes. In this talk I will discuss a method, called the phasor that was developed in my lab and that is now used in several commercial instruments. Using the phasor method applied to cell autofluorescence it has become possible to construct quantitative indices of cell metabolism and to identify molecular species characteristic to some diseases.
Kobylkevich BM, Sarkar A, Carlberg BR, Huang L, Ranjit S, Graham DM, Messerli MA.
Reversing the direction of galvanotaxis with controlled increases in boundary layer viscosity.
Phys Biol. 2018; 15(3): 036005.Weak external electric fields (EFs) polarize cellular structure and direct most migrating cells (galvanotaxis) toward the cathode, making it a useful tool during tissue engineering and for healing epidermal wounds. However, the biophysical mechanisms for sensing weak EFs remain elusive. We have reinvestigated the mechanism of cathode-directed water flow (electro-osmosis) in the boundary layer of cells, by reducing it with neutral, viscous polymers. We report that increasing viscosity with low molecular weight polymers decreases cathodal migration and promotes anodal migration in a concentration dependent manner. In contrast, increased viscosity with high molecular weight polymers does not affect directionality. We explain the contradictory results in terms of porosity and hydraulic permeability between the polymers rather than in terms of bulk viscosity. These results provide the first evidence for controlled reversal of galvanotaxis using viscous agents and position the field closer to identifying the putative electric field receptor, a fundamental, outside-in signaling ... [truncated at 150 words]
Malacrida L, Rao E, Gratton E.
Comparison between iMSD and 2D-pCF analysis for molecular motion studies on in vivo cells: the case of the epidermal growth factor receptor.
Methods. 2018; [Epub ahead of print].Image correlation analysis has evolved to become a valuable method of analysis of the diffusional motion of molecules in every points of a live cell. Here we compare the iMSD and the 2D-pCF approaches that provide complementary information. The iMSD method provides the law of diffusion and it requires spatial averaging over a small region of the cell. The 2D-pCF does not require spatial averaging and it gives information about obstacles for diffusion at pixel resolution. We show the analysis of the same set of data by the two methods to emphasize that both methods could be needed to have a comprehensive understanding of the molecular diffusional flow in a live cell.
Staaf E, Hedde PN, Singh SB, Piguet J, Gratton E, Johansson S.
Educated natural killer cells show dynamic movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A.
Sci Signal. 2018; 11(517), eaai9200.Educated natural killer (NK) cells have inhibitory receptors specific for self major histocompatibility complex (MHC) class I molecules and kill cancer cells more efficiently than do NK cells that do not have such receptors (hyporesponsive NK cells). The mechanism behind this functional empowerment through education has so far not been fully described. In addition, distinctive phenotypic markers of educated NK cells at the single-cell level are lacking. We developed a refined version of the image mean square displacement (iMSD) method (called iMSD carpet analysis) and used it in combination with single-particle tracking to characterize the dynamics of the activating receptor NKp46 and the inhibitory receptor Ly49A on resting educated versus hyporesponsive murine NK cells. Most of the NKp46 and Ly49A molecules were restricted to microdomains; however, individual NKp46 molecules resided in these domains for shorter periods and diffused faster on the surface of educated, compared to hyporesponsive, NK cells. In ... [truncated at 150 words]
Leemans SW, Dvornikov A, Gratton E.
Adaptive optics in deep tissue microscopy.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 533a, 2638-Pos.Multiphoton fluorescence microscopy is a powerful technique to visualize the form and function of the cells in tissue at sub-micron scales. To diminish fluorescence emission losses due to light scattering in the sample, our lab has developed a transmission fluorescence microscope to recover more of the emitted fluorescence from biological samples. Multiphoton fluorescence is sensitive to the peak power of the light, so the efficiency of the process improves when pulse duration is decreased and the focal volume is minimized. The DIVER (Deep Imaging Via Emission Recovery) microscope uses a pulse compressor that pre-compensates for dispersion in the excitation path to minimize pulse duration. Spatial variations in the index of refraction in tissue distort the focal volume, resulting in decreased resolution as imaging depth is increased. We compensate for spatial variations in index by conjugating the wavefront of the excitation light to the optical aberrations that are introduced by the ... [truncated at 150 words]
Scipioni L, Diaspro A, Lanzanò L, Gratton E.
AiryScan comprehensive superresolution correlation analysis.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 533a, 2641-Pos.AiryScan is a detector array developed by Zeiss that consists in 32 GaAs PMT detectors arranged in a hexagonal pattern. The development of single point detector arrays is recently increasing also thanks to the possibility of achieving super-resolution without signal loss by means of Image Scanning Microscopy (ISM, Sheppard et al. 2013). Furthermore, super-resolution techniques applied to fluorescence correlation techniques have shown to be effective in retrieving information that is not accessible with diffraction-limited methods, giving new valuable tools to biological research. Being essentially a high-speed nano-camera, AiryScan allows, in principle, for the implementation of most of the state-of-the-art correlation techniques at super-resolution in a single analysis. In this work we will present the parallel implementation of fluorescence correlation spectroscopy (FCS, Digman et al. 2011), pair-correlation function (pCF, Hinde et al. 2010) and connectivity analysis, image-derived mean square displacement (iMSD, Di Rienzo et al. 2014), number and brightness (N&B, Digman ... [truncated at 150 words]
Malacrida LS, Hedde PN, Ranjit S, Cardarelli F, Gratton E.
Elucidating invisible barriers and obstacles to molecular diffusion in live cells by the spatial pair-correlation function: a connectivity view of the cell.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 166a, 835-Pos.Despite recent advances in optical super-resolution, we lack of methods to visualize the path followed by diffusing molecules in the cell. Fluorescence correlation spectroscopy (FCS) provides molecular dynamics at the single molecule level; however, the classical single point FCS lacks spatial resolution. Methods based on image analysis like raster-scanning and spatiotemporal image correlation still needs spatial averaging over relatively large areas; hence the spatial resolution is compromised. Here, we develop a new method based on the spatial pair-cross-correlation in two dimensions (2D-pCF) to obtain relatively high-resolution images of molecular diffusion dynamics and transport in live cells. The 2D-pCF method measures the time for a particle to go from one location to another by cross-correlating the intensity fluctuations at specific points in an image. Hence, a visual map of connectivity for the average path followed by molecules is created. To test our approach we use a nanometer channel simulation and a ... [truncated at 150 words]
Torrado B, Malacrida L, Badano JL, Irigoín F, Gratton E.
Intracellular transport characterization of the transcription factor Gli2 by fluorescence correlation spectroscopy approaches.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 630a, 3123-Pos.Gli2 is the principal transcriptional activator of the Hedgehog signalingin mammals. Upon pathway activation, Gli2 moves into the cilium before reaching the nucleus. While two conserved classical nuclear localization signals (cNLS) mediate Gli2 nuclear localization via importin (Imp)-α/β1, these sequences are not required for Gli2 ciliary import (Torrado et al, 2016. PLoSONE ;11(8)). However, Importin-β2 is involved in Gli2 ciliary entry, and might also collaborate in Gli2 nuclear entry (Torrado et al, 2016. PLoSONE ;11(8)). Several questions about Gli2 intracellular traffic still remain opened. To analyze the nucleo-cytoplasmic transport of Gli2 we used fluorescence correlation spectroscopy methods, in particular pair correlation function and Number & Brightness approaches. We compared Gli2 nuclear transport when the pathway is on and off. Gli2 ciliary transport was analyzed by fluorescence correlation spectroscopy approaches combined with 3D-particle tracking so we could avoid the out of focus produced by the movement of the cilium. We applied ... [truncated at 150 words]
Ranjit S, Dvornikov A, Levi M, Gratton E.
Multicomponent analysis of phasor plot to decipher changes in metabolic trajectory of biological systems.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 167a, 843-Pos.Recent developments of phasor approach to Fluorescence Lifetime Imaging (FLIM) in cells undergoing oxidative stress or cultured in oleic acid enriched media and in mice liver sections having high fat diet, have shown presence of a third autofluorescent component indicative of lipid droplets besides the phasor components from free and enzyme bound NADH in the 400-450 nm emission wavelength. This third component changes the position and shape of the phasor distribution away from the metabolic trajectory, defined by the line joining the phasor position of free and enzyme bound NADH, and makes the study of changes in metabolism using the ratio of free to bound NADH difficult. However, the phasor rule of addition applies to any number of components and this was exploited to create a multicomponent analysis. A change in phasor distribution towards free or bound NADH can then be separated and analyzed independent of the third individual. Law ... [truncated at 150 words]
Digman MA, Palomba F, Kong X, Yokomori K, Gratton E.
Pair correlation analysis of Ku dynamics upon DNA damage.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 351a, 1740-Pos.We used the pair correlation function (pCF) analysis developed for fast frame acquisition to measure the changes in molecular connectivity of the Ku protein following double strand DNA damage. Ku protein binds to the ends of double-strand breaks and is required in DNA-repair for non-homologous end joining (NHEJ). Before damage, Ku diffuses freely with low anisotropy in the entire nucleus. After damage, we observe the formation of barriers of Ku at the damage site. In the rest of the nucleus the diffusion coefficient of Ku increases that persists four hours post damage. The diffusion anisotropy tensor of Ku in the nucleus was measured and an increase in anisotropy compared to the control cells without damage was observed. Given that DNA-PKcs play a critical role in NHEJ, loss of their activity can lead to a reduced repair rate and would alter Ku motility at the damage sites. To test this, we ... [truncated at 150 words]
Hedde PN, Malacrida L, Ahrar S, Siryaporn A, Gratton E.
SideSPIM - a flexible multipurpose platform for light sheet microscopy.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 187a, 945-Plat.We devised a flexible multipurpose platform for light sheet microscopy. Based on a regular inverted microscope, a custom designed chamber is used to allow selective plane illumination from the side (sideSPIM). This eliminates the need of dipping into the sample container, hence small sample volumes (~100 µl) can be realized and objective lenses can be interchanged quickly. We demonstrate that this flexible multipurpose platform is suitable for a large variety of applications involving samples of substantially different sizes and geometries. For example, we captured the microcirculation of erythrocytes in a zebrafish embryo at 5 stacks/s (40 planes), studied the growth of bacteria biofilms under flow using a fluidic device, and probed the diffusion of EGFP tagged proteins in mammalian cells by acquiring fast time series (200 fps) and subjecting them to fluorescence fluctuation based image mean square displacement (iMSD) and two-dimensional pair cross-correlation function (2D-pCF) analysis. We further evaluated the ... [truncated at 150 words]
Cinco R, Hedde PN, Digman MA, Gratton E.
Spatial characterization of NADH concentration and diffusion in cells and tissue.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 335a, 1662-Pos.Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH play a central role in bioenergetics as an enzymatic cofactor and reducing equivalent in cellular metabolic and redox reactions. The NADH/NAD+ ratio has significant impact on energy production, cell survival, proliferation, longevity and aging. Lifetime measurements of free/bound NADH autofluorescence has been demonstrated to approximate the NADH/NAD+ ratio and has been established as a reliable means for estimating the mode of metabolism used by cells and tissues. NADH microenvironment concentrations, diffusion speed, and potential sequestration areas may dictate NADH availability for biochemical reactions and thereby modulate cell metabolism and signaling. To approximate the spatial distribution and diffusion of NADH in cells, we excited NADH at 740 nm 2-Photon excitation and performed raster image correlation spectroscopy (RICS) and number and brightness (N&B). Application of RICS to determine NADH diffusion did not yield enough photons for a correlation to be detected, since the ... [truncated at 150 words]
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Spectral resolution in fluorescence microscopy in strongly scattering media.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 166a, 838-Pos.Spectral phasors were introduced as alternative to spectral demixing, for the determination of FRET and for the measurement of dipolar relaxation in membranes. The basic concept in the spectral phasor approach is that the entire spectrum is not needed for some analysis, but only some of the elements of the spectral distribution are sufficient. In the case in which the spectrum is dominated by few spectral bands, this approach is very efficient and provides fast and valid alternative to the analysis of the full spectrum. One example of a system that could benefit from the phasor analysis is in tissue spectroscopy in which the light to be collected has undergone multiple scattering so that it cannot be easily focused on the entrance slit of a dispersive element or transformed in a parallel beam for use with hyperspectral cameras. In a typical application of thick tissue spectroscopy, the light, after being ... [truncated at 150 words]
Murata MM, Kong X, Yokomori K, Digman MA.
Fluorescence lifetime of NADH reveals PARP-dependent increase of oxidative phosphorylation critical for cell survival.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 83a, 425-Pos.The DNA damage response (DDR) pathway triggers a chain of reactions that are critical for the maintenance of cell survival and genome integrity. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor that catabolizes nicotinamide adenine dinucleotide (NAD+) to form poly(ADP-ribose) on target proteins for initial chromatin organization and DNA repair pathway choice at damage sites. It has been reported that this depletion of NAD+ leads to a loss of a ATP and subsequent metabolic collapse ultimately leading to cellular death in DNA damaged cells. However, it is not clear whether or not regulating metabolism can overcome this PARP-dependent cell death pathway as a cell survival mechanism. We are investigating if inhibition of oxidative phosphorylation increases cellular damage sensitivity and if that correlates with cell survival. This may be dependent on PARP activity and the effect can last longer than the transient depletion of the total NADH level. Here we ... [truncated at 150 words]
Mah EJ, Yee AF, Digman MA.
Metabolism modulation of cancer cells on varying substrate stiffnesses.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 19a, 101-Plat.ancer cells sense their microenviroment and respond to biophysical cues that activate signaling networks through ion channels and integrin proteins. This allows cells to adapt by adjusting their cellular shape and tension that remodels the extracellular matrix (ECM). New evidence shows that these ECM signaling cascades may increase glucose uptake leading to altered metabolic states. The hallmark of metabolic alteration of increase glycolysis, i.e. Warburg effect, in cancer cells together with atypical ECM structure may be responsible for tumor cell aggressiveness and drug resistance. While it is known that tumor cells stiffen the ECM as the tumor progression occurs, a direct relationship between ECM stiffness and altered metabolism has not been explicitly measured. Here we apply the phasor approach to fluorescence lifetime imaging microscopy (FLIM) as a method to measure metabolic as a function of ECM mechanics. We imaged two breast cancer cell lines of high and low aggressiveness (MDA-MB231 ... [truncated at 150 words]
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Laurdan imaging and spectral phasor analysis reveals increased membrane fluidity in Huntington disease.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 177a, 891-Pos.Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeats in exon 1 of the gene encoding polyglutamine (polyQ). This is an incurable neurodegenerative disorder with complex pathogenies including protein aggregation and dysregulation of lipid homeostasis. Lipids are vital for brain function. Altered lipid metabolism has been linked to dysfunction in Huntington disease. In particular, cholesterol metabolism appears to be disturbed in patients with HD and in animal models. Here we used a biophysical approach to examine the effect of HD in the cell plasma membrane fluidity influenced by the pathogenic protein (97QmRuby) compared to non-pathogenic protein (25QmRuby) and non-labeled cells. To characterize membrane fluidity and hydration in HD, we used the fluorescent membrane probe LAURDAN that is sensitive to physical states of phospholipids. Using LAURDAN emission we characterized membrane fluidity. We further validated our results using secondary fluorescent membrane probe Nile ... [truncated at 150 words]
Kong L, Murata MM, Digman MA.
Absence of REV3L promotes p53-regulated cancer cell metabolism in cisplatin-treated lung carcinoma cells.
Biochem Biophys Res Commun. 2018; 496(1): 199-204.Lung cancer is one of the deadliest cancers in the world because of chemo-resistance to the commonly used cisplatin-based treatments. The use of low fidelity DNA polymerases in the translesional synthesis (TLS) DNA damage response pathway that repairs lesions caused by cisplatin also presents a mutational carcinogenic burden on cells that needs to be regulated by the tumor suppressor protein p53. However, there is much debate over the roles of the reversionless 3-like (REV3L) protein responsible for TLS and p53 in regulating cancer cell metabolism. In this study, the fluorescence lifetime of the metabolic coenzyme NADH reveals that the absence of REV3L can promote the p53-mediated upregulation of oxidative phosphorylation in cisplatin-treated H1299 lung carcinoma cells and increases cancer cell sensitivity to this platinum-based chemotherapy. These results demonstrate a previously unrecognized relationship between p53 and REV3L in cancer cell metabolism and may lead to improvements in chemotherapy treatment plans that ... [truncated at 150 words]
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Apparatus and method for an inclined single plane imaging microscope box (iSPIM box).
Patent US9874736B2, 23 Jan 2018.An apparatus for inclined single plane Illumination microscopy of a sample includes a laser for launching excitation light beams at a plurality of wavelengths, a laser beam expander, an injection arm optically coupled to the laser beam expander, a conventional back-to-back microscope system, a universal dichroic mirror optically coupled to the injection arm to direct the excitation light beams into the conventional back-to-back microscope onto a sample plane in an imaging plane, and to receive fluorescence light from the sample, a universal optical adaptor optically coupled to the universal dichroic mirror, a re-imaging component optically coupled to the universal optical adaptor; and a camera output connector optically coupled to the re-imaging component, where the laser beam expander, injection arm, universal optical adapter, re-imaging component, and camera are combined in a modular unit which is arranged and configured to be coupled to the conventional back-to-back microscope.
Mäntylä E, Chacko JV, Aho V, Parrish CR, Shahin V, Kann M, Digman MA, Gratton E, Vihinen-Ranta M.
Viral highway to nucleus exposed by image correlation analyses.
Sci Rep. 2018; 8(1): 1152. PMCID: PMC5773500Parvoviral genome translocation from the plasma membrane into the nucleus is a coordinated multistep process mediated by capsid proteins. We used fast confocal microscopy line scan imaging combined with image correlation methods including auto-, pair- and cross-correlation, and number and brightness analysis, to study the parvovirus entry pathway at the single-particle level in living cells. Our results show that the endosome-associated movement of virus particles fluctuates from fast to slow. Fast transit of single cytoplasmic capsids to the nuclear envelope is followed by slow movement of capsids and fast diffusion of capsid fragments in the nucleoplasm. The unique combination of image analyses allowed us to follow the fate of intracellular single virus particles and their interactions with importin β revealing previously unknown dynamics of the entry pathway.
Sameni S, Malacrida L, Tan Z, Digman MA.
Alteration in fluidity of cell plasma membrane in Huntington disease revealed by spectral phasor Analysis.
Sci Rep. 2018; 8(1): 734. PMCID: PMC5768877Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here, we investigated how membrane fluidity in the living cells (differentiated PC12 and HEK293 cell lines) are affected using a hyperspectral imaging of widely used probes, LAURDAN. Using phasor approach, we characterized the fluorescence of LAURDAN that is sensitive to the polarity of the immediate environment. LAURDAN is affected by the physical order of phospholipids (lipid order) and reports the membrane fluidity. We also validated our results using a different fluorescent membrane probe, Nile Red (NR). The plasma membrane ... [truncated at 150 words]
Wang XX, Wang D, Luo Y, Myakala K, Dobrinskikh E, Rosenberg AZ, Levi J, Kopp JB, Field A, Hill A, Lucia S, Qiu L, Jiang T, Peng Y, Orlicky D, Garcia G, Herman-Edelstein M, D’Agati V, Henriksen K, Adorini L, Pruzanski M, Xie C, Krausz KW, Gonzalez FJ, Ranjit S, Dvornikov A, Gratton E, Levi M.
FXR/TGR5 dual agonist prevents progression of nephropathy in diabetes and obesity.
J Am Soc Nephrol. 2018; 29(1). PMCID: PMC5748904Bile acids are ligands for the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5. We have shown that FXR and TGR5 have renoprotective roles in diabetes- and obesity-related kidney disease. Here, we determined whether these effects are mediated through differential or synergistic signaling pathways. We administered the FXR/TGR5 dual agonist INT-767 to DBA/2J mice with streptozotocin-induced diabetes, db/db mice with type 2 diabetes, and C57BL/6J mice with high-fat diet-induced obesity. We also examined the individual effects of the selective FXR agonist obeticholic acid (OCA) and the TGR5 agonist INT-777 in diabetic mice. The FXR agonist OCA and the TGR5 agonist INT-777 modulated distinct renal signaling pathways involved in the pathogenesis and treatment of diabetic nephropathy. Treatment of diabetic DBA/2J and db/db mice with the dual FXR/TGR5 agonist INT-767 improved proteinuria and prevented podocyte injury, mesangial expansion, and tubulointerstitial fibrosis. INT-767 exerted coordinated effects on multiple ... [truncated at 150 words]
Malacrida L, Hedde PN, Ranjit S, Cardarelli F, Gratton E.
Visualization of barriers and obstacles to molecular diffusion in live cells by spatial pair-cross-correlation in two dimensions.
Biomed Opt Express. 2018; 9(1): 303-321. PMCID: PMC5772584Despite recent advances in optical super-resolution, we lack a method that can visualize the path followed by diffusing molecules in the cytoplasm or in the nucleus of cells. Fluorescence correlation spectroscopy (FCS) provides molecular dynamics at the single molecule level by averaging the behavior of many molecules over time at a single spot, thus achieving very good statistics but at only one point in the cell. Earlier image-based methods including raster-scan and spatiotemporal image correlation need spatial averaging over relatively large areas, thus compromising spatial resolution. Here, we use spatial pair-cross-correlation in two dimensions (2D-pCF) to obtain relatively high resolution images of molecular diffusion dynamics and transport in live cells. The 2D-pCF method measures the time for a particle to go from one location to another by cross-correlating the intensity fluctuations at specific points in an image. Hence, a visual map of the average path followed by molecules is created.
Gach JS, Bouzin M, Wong MP, Chromikova V, Gorlani A, Yu KT, Sharma B, Gratton E, Forthal DN.
Human Immunodeficiency Virus type-1 (HIV-1) evades antibody-dependent phagocytosis.
PLoS Pathog. 2017; 13(12), e1006793. PMCID: PMC5760106Fc gamma receptor (FcyR)-mediated antibody functions play a crucial role in preventing HIV infection. One such function, antibody-dependent phagocytosis (ADP), is thought to be involved in controlling other viral infections, but its role in HIV infection is unknown. We measured the ability of HIV-specific polyclonal and monoclonal antibodies (mAbs) to mediate the internalization of HIV-1 virions and HIV-1-decorated cells by phagocytes. To measure ADP of virions, we primarily used a green-fluorescent protein-expressing molecular clone of HIV-1JRFL, an R5, clinical isolate, in combination with polyclonal HIVIG or mAbs known to capture and/or neutralize HIV-1. THP-1 and U937 cells, as well as freshly isolated primary monocytes from healthy individuals, were used as phagocytic effector cells, and uptake of virions was measured by cytometry. We surprisingly found minimal or no ADP of virions with any of the antibodies. However, after coating virions with gp41 or with gp41-derived peptides, gp41- (but not gp120-) specific ... [truncated at 150 words]
Davey RJ, Digman MA, Gratton E, Moens PDJ.
Quantitative image mean squared displacement (iMSD) analysis of the dynamics of Profilin 1 at the membrane of live cells.
Methods. 2017; [Epub ahead of print].Image mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A). Using total internal reflection fluorescence microscopy images, we obtained data on isotropic and confined diffusion coefficients, the proportion of cell areas where isotropic diffusion is the major diffusion mode compared to the confined diffusion mode, the size of ... [truncated at 150 words]
Trinh AL, Chen H, Chen Y, Hu Y, Li Z, Siegel ER, Linskey ME, Wang PH, Digman MA, Zhou YH.
Tracking functional tumor cell subpopulations of malignant glioma by phasor fluorescence lifetime imaging microscopy of NADH.
Cancers (Basel). 2017; 9(12), E168. PMCID: PMC5742816Intra-tumoral heterogeneity is associated with therapeutic resistance of cancer and there exists a need to non-invasively identify functional tumor subpopulations responsible for tumor recurrence. Reduced nicotinamide adenine dinucleotide (NADH) is a metabolic coenzyme essential in cellular respiration. Fluorescence lifetime imaging microscopy (FLIM) of NADH has been demonstrated to be a powerful label-free indicator for inferring metabolic states of living cells. Using FLIM, we identified a significant shift towards longer NADH fluorescence lifetimes, suggesting an increase in the fraction of protein-bound NADH, in the invasive stem-like tumor-initiating cell (STIC) subpopulation relative to the tumor mass-forming cell (TMC) subpopulation of malignant gliomas. By applying our previously studied model to transition glioma from a majority of STIC to a majority of TMC in serum-adherent culture conditions following serial passages, we compared changes in NADH states, cellular respirations (oxidative phosphorylation and glycolysis), EGFR expression, and cell-growth speed over passages. We identified a significant positive ... [truncated at 150 words]
Bohórquez-Hernández A, Gratton E, Pacheco J, Asanov A, Vaca L.
Cholesterol modulates the cellular localization of Orai1 channels and its disposition among membrane domains.
Biochim Biophys Acta. 2017; 1862(12): 1481-1490. PMCID: PMC5902182Store Operated Calcium Entry (SOCE) is one of the most important mechanisms for calcium mobilization in to the cell. Two main proteins sustain SOCE: STIM1 that acts as the calcium sensor in the endoplasmic reticulum (ER) and Orai1 responsible for calcium influx upon depletion of ER. There are many studies indicating that SOCE is modulated by the cholesterol content of the plasma membrane (PM). However, a myriad of questions remain unanswered concerning the precise molecular mechanism by which cholesterol modulates SOCE.
In the present study we found that reducing PM cholesterol results in the internalization of Orai1 channels, which can be prevented by overexpressing caveolin 1 (Cav1). Furthermore, Cav1 and Orai1 associate upon SOCE activation as revealed by FRET and coimmunoprecipitation assays. The effects of reducing cholesterol were not limited to an increased rate of Orai1 internalization, but also, affects the lateral movement of Orai1, inducing movement in a linear pattern ... [truncated at 150 words]
Digiacomo L, Cardarelli F, Pozzi D, Palchetti S, Digman MA, Gratton E, Capriotti AL, Mahmoudi M, Caracciolo G.
An apolipoprotein-enriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles.
Nanoscale. 2017; 9(44): 17254-17262. PMCID: PMC5700750Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in ... [truncated at 150 words]
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System and method for digital parallel frequency fluorometry.
Patent EP2529152B1, 15 Nov 2017.
Malacrida L, Jameson DM, Gratton E.
A multidimensional phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes.
Sci Rep. 2017; 7(1): 9215. PMCID: PMC5569084Mammalian cell membranes have different phospholipid composition and cholesterol content, displaying a profile of fluidity that depends on their intracellular location. Among the dyes used in membrane studies, LAURDAN has the advantage to be sensitive to the lipid composition as well as to membrane fluidity. The LAURDAN spectrum is sensitive to the lipid composition and dipolar relaxation arising from water penetration, but disentangling lipid composition from membrane fluidity can be obtained if time resolved spectra could be measured at each cell location. Here we describe a method in which spectral and lifetime information obtained in different measurements at the same plane in a cell are used in the phasor plot providing a solution to analyze multiple lifetime or spectral data through a common visualization approach. We exploit a property of phasor plots based on the reciprocal role of the phasor plot and the image. In the phasor analysis each pixel ... [truncated at 150 words]
Hedde PN, Malacrida L, Ahrar S, Siryaporn A, Gratton E.
sideSPIM - selective plane illumination based on a conventional inverted microscope.
Biomed Opt Express. 2017; 8(9): 3918-3937. PMCID: PMC5611913Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa. Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to ... [truncated at 150 words]
