Biology subjects

Genin, G. M.

Publications and source records attributed to Genin, G. M..

24 records · Page 2Linked to original sources

bioRxiv2021

3D Visualization of the Podocyte Actin Network using Integrated Membrane Extraction, Electron Microscopy, and Deep Learning

Although actin stress fibers are abundant in cultured cells, little is known about these structures in vivo. In podocytes of the kidney glomerulus, much evidence suggests that mechanobiological mechanisms underlie injury, with changes to actin stress fiber structures potentially responsible for pathological changes to cell morphology. However, this hypothesis is difficult to rigorously test in vivo due to challenges with visualization. We therefore developed the first visualization technique capable of resolving the three-dimensional (3D) podocyte actin network with unprecedented detail in healthy and injured podocytes, and applied this technique to reveal the changes in the actin network that occur upon podocyte injury. Using isolated glomeruli from healthy mice as well as from three different mouse injury models (Cd2ap-/-, Lamb2-/- and the Col4a3-/- model of Alport syndrome), we applied our novel imaging technique that integrates membrane-extraction, focused ion bean scanning electron microscopy (FIB-SEM), and deep learning image segmentation. In healthy glomeruli, we observed actin cables that link the interdigitating podocyte foot processes to newly described actin structures located at the periphery of the cell body. The actin cables within the foot processes formed a continuous, mesh-like, electron dense sheet that incorporated the slit diaphragms required for kidney filtration. After injury, the actin network was markedly different, having lost its organization and presenting instead as a disorganized assemblage of actin condensates juxtaposed to the glomerular basement membrane. The new visualization method enabled us, for the first time, to observe the detailed 3D organization of actin networks in both healthy and injured podocytes. Shared features of actin condensations across all three injury models further suggested common mechanobiological pathways that govern changes to podocyte morphology after injury.

cell biology↗

bioRxiv2020

Transient K+ current explains cross-species differences in the effects of myofibroblasts on myocytes

Electrical and paracrine couplings between cardiomyocytes (CMs) and myofibroblasts (MFBs) affect both physiology and pathophysiology of cardiac tissues in a range of animal models, but relating these observations to humans is a challenge because effects vary greatly across species. To address this challenge, we developed a mathematical model for mechanoelectrical interactions between CM and MFB, considering both electrical and paracrine couplings between CMs and MFBs, with the aim of identifying the sources of cross-species variation and extrapolating animal models to predicted effects in humans. Our results revealed substantial differences across species in how these couplings modulate excitation-contraction coupling and Ca2+ transients of CMs. Both classes of couplings prolong action potential and increase APD in rat CMs, but shorten action potential and decrease APD in human CMs. Electrical coupling attenuates Ca2+ transients and active tension generation in human CMs, but has no significant effect on rat CMs. Paracrine coupling reduces Ca2+ transients and active tension in both human and rat CM. The results suggest that the variance of functional interactions between CM and MFB in cross-species may be explained by differences in the transient outward K+ currents associated with the KCND2 gene, and thus suggest potential therapeutic pathways for fibrotic cardiomyopathy.

biophysics↗

bioRxiv2020

Negative durotaxis: cell movement toward softer environments

Durotaxis - the ability of cells to sense and migrate along stiffness gradients - is important for embryonic development and has been implicated in pathologies including fibrosis and cancer. Although cellular processes can sometimes turn toward softer environments, durotaxis at the level of cells has thus far been observed exclusively as migration from soft to stiff regions. The molecular basis of durotaxis, especially the factors that contribute to different durotactic behaviors in various cell types, are still inadequately understood. With the recent discovery of optimal stiffness, where cells generate maximal traction forces on substrates in an intermediate stiffness range, we hypothesized that some migratory cells may be capable of moving away from stiff environments and toward matrix on which they can generate more traction. Combining hydrogel-based stiffness gradients, live-cell imaging, genetic manipulations, and computational modeling, we found that cells move preferentially toward their stiffness optimum for maximal force transmission. Importantly, we directly observed biased migration toward softer environments, i.e. negative durotaxis, in human glioblastoma cells. This directional migration did not coincide with changes in FAK, ERK or YAP signaling, or with altered actomyosin contractility. Instead, integrin-mediated adhesion and motor-clutch dynamics alone are sufficient to generate asymmetric traction to drive both positive and negative durotaxis. We verified this mechanistically by applying a motor-clutch-based model to explain negative durotaxis in the glioblastoma cells and in neurites, and experimentally by switching breast cancer cells from positive to negative durotaxis via talin downregulation. Our results identify the likely molecular mechanisms of durotaxis, with a cells contractile and adhesive machinery dictating its capacity to exert traction on mechanically distinct substrates, directing cell migration.

biophysics↗

bioRxiv2019

Fluorescence correlation spectroscopy and photon count histograms in small domains. Part I: General theory

Analysis of fluctuations arising as fluorescent particles pass through a focused laser beam has enabled quantitative characterization of molecular kinetic processes. The mathematical frameworks of both fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis, which can measure these fluctuations, assume an infinite Gaussian beam, which prevents their application to particles within domains bounded at the nanoscale. We therefore derived general forms of FCS and PCH for bounded systems. The finite domain form of FCS differs from the classical form in its boundary and initial conditions and requires development of a new Fourier space solution for fitting data. Our finite-domain FCS predicts simulated data accurately and reduces to a previous model for the special case of molecules confined by two boundaries under Gaussian beams. Our approach enables estimation of the concentration of diffusing fluorophores within a finite domain for the first time. The method opens the possibility of quantification of kinetics in several systems for which this has never been possible, including in the one-dimensional lipid tubules discussed in Part 2 of this paper. Statement of SignificanceMethods based on fluorescence measurements of molecular concentration fluctuations, including Fluorescence Correlation Spectroscopy and Photon Count Histogram analysis, are widely used to determine rates of diffusion, chemical reaction and sizes of molecular aggregates. Typically, the range over which the molecules can diffuse is large compared to the size of the focused laser beam that excites the fluorescence. This work extends these measurements to systems that are comparable in size to the excitation laser beam. This extends the application of these methods to very small samples such as the interior of bacterial cells or the diffusion of molecules along individual macromolecules such as DNA.

biophysics↗

bioRxiv2018

Functional grading of pericellular matrix surrounding chondrocytes: potential roles in signaling and fluid transport

The extracellular matrix surrounding chondrocytes within cartilage and fibrocartilage has spatial gradients in mechanical properties. Although the function of these gradients is unknown, the potential exists for cells to tailor their mechanical microenvironment through these gradients. We hypothesized that these gradients enhance fluid transport around the cell during the slow loading cycles that occur over the course of a day, and that this enhancement changes the nature of the mechanical signals received at the surface of the cell. To test this hypothesis, we studied the effect of these gradients on the mechanical environment around a chondrocyte using a closed form, linearized model. Results demonstrated that functional grading of the character observed around chondrocytes in articular cartilage enhances fluid transport, and furthermore inverts compressive radial strains to provide tensile signals at the cell surface. The results point to several potentially important roles for functional grading of the pericellular matrix.

bioengineering↗

bioRxiv2018

Atomic force microscopy of phase separation on ruptured, giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) and supported lipid bilayers (SLBs) are synthetic model systems widely used in biophysical studies of lipid membranes. Phase separation behaviors of lipid species in these two model systems differ due to the lipid-substrate interactions that are present only for SLBs. Therefore, GUVs are believed to resemble natural cell membranes more closely, and a very large body of literature focuses on applying nano-characterization techniques to quantify phase separation on GUVs. However, one important technique, atomic force microscopy (AFM), has not yet been used successfully to study phase separation on GUVs. In the present study, we report that in binary systems, certain phase domains on GUVs retain their original shapes and patterns after the GUVs rupture on glass surfaces. This enabled AFM experiments on phase domains from binary GUVs containing 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) and either 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). These DLPC/DSPC and DLPC/DPPC GUVs both presented two different gel phases, one of which (bright phase) included a relatively high concentration of DiI-C20 but excluded Bodipy-HPC, and the other of which (dark phase) excluded both probes. The bright phases are of interest because they seem to stabilize dark phases against coalescence. Results suggested that the gel phases labeled by DiI-C20 in the DLPC/DSPC membrane, which surround the dark gel phase, is an extra layer of membrane, indicating a highly curved structure that might stabilize the interior dark domains. This phenomenon was not found in the DLPC/DPPC membrane. These results show the utility of AFM on collapsed GUVs, and suggest a possible mechanism for stabilization of lipid domains.

biochemistry↗