Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells. Here, we utilize mouse embryonic fibroblast CRISPR knock-in lines with labeled ARP2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that ARP2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of ARP2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility through cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity. ### Competing Interest Statement The authors have declared no competing interest. NIGMS, R35GM130312, R35GM158040, F32GM131578
Motile cells can sense and exert forces on the extracellular environment through dynamic actin networks. Increased stress against the polymerizing barbed ends of branched actin networks has been shown to lead to an increase in the density of these networks through a force feedback mechanism, though this phenomenon has not been explored through the examination of real-time responses of endogenous actin networks in cells. Here, we utilize mouse embryonic fibroblast CRISPR knock-in lines with labeled ARP2/3 complex to identify cellular and extracellular conditions that regulate branched actin density and enrichment at the leading edge of lamellipodial protrusions. A common theme shared among all branched actin density-increasing conditions is higher levels of interface stress between the plasma membrane and the barbed ends of the lamellipodial actin network. Among these conditions, we find that ARP2/3 is specifically required for robust spreading and protrusion in response to increased extracellular viscosity. Interestingly, time-lapse traction force microscopy of ARP2/3-dependent viscosity responses show significantly reduced changes in strain energy applied to the substrate when compared to spreading and motility through cell-matrix adhesion. In addition, we find that increased extracellular viscosity can bypass the need for extracellular matrix proteins to support lamellipodial protrusion driven by optogenetic Rac activation. Our studies provide strong support for in vitro models of branched actin force feedback responses and further characterize an essential role for branched actin in mediating dramatic cell shape changes in response to increased extracellular viscosity. ### Competing Interest Statement The authors have declared no competing interest. NIGMS, R35GM130312, R35GM158040, F32GM131578
Entamoeba histolytica is a parasitic amoeba and the cause amoebiasis, a common but understudied human diarrheal disease. E. histolytica trophozoites (“amoebae”) kill human cells through a process of cell-nibbling called trogocytosis ( trogo -: nibble) that contributes to tissue damage. Amoebae can also perform phagocytosis, in which entire human cells are ingested. Based on studies in which human cells were artificially stiffened, it was suggested that amoebae perform phagocytosis on stiffer cells, and trogocytosis on less stiff cells. A handful of recent studies of macrophages that used artificial targets or artificially stiffened target cells also suggested a similar relationship between target stiffness and trogocytosis/phagocytosis efficiencies. To better evaluate the impact of target cell stiffness on amoebic ingestion, instead of using artificial targets or artificial cell stiffening, we created human cell mutants in which individual Rho-pathway genes were knocked down. Strikingly, amoebae performed quantitatively reduced levels of trogocytosis on all knockdown mutants, regardless of cytoskeletal F-actin organization. In contrast, amoebic phagocytosis efficiency was inversely correlated with human cell cortical F-actin density. Thus, human cell F-actin organization differentially influences amoebic trogocytosis and phagocytosis. This is more complex than the conclusions of studies that used artificial targets or artificially stiffened cells. Our results emphasize that the dynamic nature of the cytoskeleton in living cells impacts trogocytosis. In addition to shedding light on the burgeoning field of eukaryotic trogocytosis, this work extends knowledge of amoebic ingestion processes that contribute to disease. ### Competing Interest Statement The authors have declared no competing interest. National Institute of Allergy and Infectious Diseases, R01AI146914, R21AI193905
Nucleus shape is a sensitive indicator of cell state, influenced by numerous biochemical and physiological factors. While prior work has cataloged how perturbations alter nucleus morphology, we address the inverse: inferring underlying molecular changes from nucleus shape alone. We previously developed a mechanical model yielding two nondimensional parameters: flatness index and scale factor, which are surrogate measures for cortical actin tension and nuclear envelope compliance respectively. In this study, we apply these parameters to investigate the dynamics in cellular mechanics during confined migration. We fabricated polydimethylsiloxane (PDMS) microchannels with widths of 3 μm (high confinement) and 10 μm (low confinement) and tracked cells migrating through them. We captured high-frequency 3D nucleus shapes via double fluorescence exclusion microscopy and custom image analysis. Fitting the model and estimating flatness index and scale factor to time-resolved shapes revealed dynamic regulation in 3 μm channels: actin tension decreased and nucleus compliance increased immediately before nucleus entry into the constriction, with rapid restoration to baseline upon exit. No such changes occurred in 10 μm channels, indicating active, confinement-dependent cytoskeletal adaptation. Immunostaining for YAP and lamin-A,C confirmed these model inferences. Our results uncover mechanostasis, active mechanical homeostasis, during confined migration and establish the combination of double fluorescence exclusion microscopy and nondimensional nucleus shape parameters as a powerful, non-invasive tool for single-cell mechanobiology studies. ### Competing Interest Statement The authors have declared no competing interest.
BackgroundInflammatory bowel disease (IBD), including Crohn’s disease and ulcerative colitis, is a chronic and relapsing condition with complex pathogenesis ...
Actin is a key component of the cytoskeleton and also plays diverse roles within the cell nucleus. While polymerized F-actin can be detected using a wide r
Rxiv cytoskeleton
Rxiv mechanobio
