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Garner, R. M.

Publications and source records attributed to Garner, R. M..

4 recordsLinked to original sources

Tissue Fluidity: A Double-Edged Sword for Multicellular Patterning

The organization of cells into spatial patterns is a fundamental aspect of multicellularity. One major mechanism underlying tissue patterning is adhesion-based cell sorting, in which a heterogeneous mixture of cell types spontaneously separates into distinct domains based on differences in adhesion protein expression. Here, we identify tissue fluidity--the extent to which cells can move freely within a tissue--as a critical regulator of adhesion-based sorting. First, we describe a physically well-understood minimal tissue model that can integrate both tissue fluidity and adhesion-based sorting, and demonstrate that this model can quantitatively reproduce experimentally measured sorting dynamics in a fibroblast cell culture assay. We go on to show that altering tissue fluidity by any mechanism in the model leads to substantial changes in the rate or accuracy of sorting (or both). We further demonstrate that the balance between cell motility, which acts to fluidize the tissue, and homotypic cell-cell adhesion, which acts to solidify the tissue, sensitively tunes a fundamental trade-off between the rate and accuracy of sorting--such that sorting can only occur when motility and adhesion are tightly coupled. Intriguingly, best fits of the simulations to the experiments across a range of adhesion protein expression conditions suggest that cells may naturally scale their motility strength with their adhesion strength - thereby maintaining a permissive fluidity for sorting. Overall, our results indicate that tissue fluidity must be tightly regulated for sorting to occur, and that cells may have evolved a mechanism to naturally co-regulate their mechanical properties in order to sustain a patterning-competent fluidity. Statement of SignificanceTissue fluidity, or the ability of cells to freely rearrange within a tissue, is a universal property of multicellular organisms that plays central roles in development, cancer, and wound healing. Here, we identify tissue fluidity as a critical regulator of a major mechanism of multicellular patterning - adhesion-based cell sorting. The results of our combined experimental-computational investigation suggest that tissues can readily tune their fluidity in order to freeze, catalyze, or erase multicellular patterns - carrying significant implications for our understanding of how patterns are formed in development, lost in diseases affecting tissue organization (e.g., cancer), regained through the processes of wound healing and regeneration, and can be engineered in the creation of synthetic organoid and embryoid systems. One sentence summaryBiophysical modeling demonstrates how tissue fluidity is a key regulator of the rate and accuracy of adhesion-based sorting.

cell biology↗

A conserved pressure-driven mechanism for regulating cytosolic osmolarity

Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments like freshwater must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify basic principles governing contractile vacuole function, we here investigate the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages--the discoban Naegleria gruberi, and the amoebozoan slime mold Dictyostelium discoideum. Using quantitative cell biology we find that, although these species respond differently to osmotic challenges, they both use actin for osmoregulation, as well as vacuolar-type proton pumps for filling contractile vacuoles. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolate Paramecium multimicronucleatum. Because these three lineages diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.

cell biology↗

Vast heterogeneity in cytoplasmic diffusion rates revealed by nanorheology and Doppelgänger simulations

The cytoplasm is a complex, crowded, actively-driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem-cell fate. Little is known about the variance in these cytoplasmic properties. Here, we employed particle-tracking nanorheology on genetically encoded multimeric 40-nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of the fission yeast Schizosaccharomyces pombe. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the differences in average particle diffusivity among individual cells spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelganger Simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume a wide intra- and inter-cellular variation in cytoplasmic viscosity. The simulation combining intra- and inter-cellular variation in viscosity also predicted weak non-ergodicity in GEM diffusion, consistent with the experimental data. To probe the origin of this variation, we found that the variance in GEM diffusivity was largely independent of factors such as temperature, cytoskeletal effects, cell cycle stage and spatial locations, but was magnified by hyperosmotic shocks. Taken together, our results provide a striking demonstration that the cytoplasm is not "well-mixed" but represents a highly heterogeneous environment in which subcellular components at the 40-nm sizescale experience dramatically different effective viscosities within an individual cell, as well as in different cells in a genetically identical population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels. SignificanceBiophysical properties of the cytoplasm influence many cellular processes, from differentiation to cytoskeletal dynamics, yet little is known about how tightly cells control these properties. We developed a combined experimental and computational approach to analyze cytoplasmic heterogeneity through the lens of diffusion. We find that the apparent cytoplasmic viscosity varies tremendously - over 100-fold within any individual cell, and over 10-fold among individual cells when comparing averages of all particles measured for each cell. The variance was largely independent of temperature, the cytoskeleton, cell cycle stage, and localization, but was magnified under hyperosmotic shock. This suggests that cytoplasmic heterogeneity contributes substantially to biological variability within and between cells, and has significant implications for any cellular process that depends on diffusion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/491518v3_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@a6faeforg.highwire.dtl.DTLVardef@a49dc4org.highwire.dtl.DTLVardef@171d62borg.highwire.dtl.DTLVardef@a3c7bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

Leading edge stability in motile cells is an emergent property of branched actin network growth

Animal cell migration is predominantly driven by the coordinated, yet stochastic, polymerization of thousands of nanometer-scale actin filaments across micron-scale cell leading edges. It remains unclear how such inherently noisy processes generate robust cellular behavior. We employed high-speed imaging of migrating neutrophil-like HL-60 cells to explore the fine-scale shape fluctuations that emerge and relax throughout the process of leading edge maintenance. We then developed a minimal stochastic model of the leading edge that reproduces this stable relaxation behavior. Remarkably, we find lamellipodial stability naturally emerges from the interplay between branched actin network growth and leading edge shape - with no additional feedback required - based on a synergy between membrane-proximal branching and lateral spreading of filaments. These results thus demonstrate a novel biological noise-suppression mechanism based entirely on system geometry. Furthermore, our model suggests that the Arp2/3-mediated ~70-80{degrees} branching angle optimally smooths lamellipodial shape, addressing its long-mysterious conservation from protists to mammals. Significance StatementAll cellular functions are driven by the stochastic dynamics of macromolecules, and thus are subject to biological noise. Here, as a model system for noise-suppression in the context of cell migration, we investigate lamellipodial maintenance - where thousands of stochastically polymerizing filaments self-organize into a highly-stable, micron-scale leading edge. Combining experiment and computational modeling, we (1) establish lamellipodial stability is an emergent property of dendritically-branched actin network growth, (2) outline a noise-suppression mechanism based on the geometry of lamellipodial actin, and (3) determine the evolutionarily-conserved Arp2/3-mediated ~70-80{degrees} branching angle optimally suppresses stochastic fluctuations. Our results not only explain the essential role of Arp2/3-mediated branching in lamellipodial formation, but also address the decades-old question of why this specific geometry is so well-conserved.

cell biology↗