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1. Spatiotemporal Coordination of Rac1 and Cdc42 at the Whole Cell Level during Cell Ruffling

   https://doi.org/10.3390/cells12121638  

   Hladyshau, Siarhei ; Stoop, Jorik P. ; Kamada, Kosei ; Nie, Shuyi ; Tsygankov, Denis ( June 2023 , Cells)

   Rho-GTPases are central regulators within a complex signaling network that controls cytoskeletal organization and cell movement. The network includes multiple GTPases, such as the most studied Rac1, Cdc42, and RhoA, along with their numerous effectors that provide mutual regulation through feedback loops. Here we investigate the temporal and spatial relationship between Rac1 and Cdc42 during membrane ruffling, using a simulation model that couples GTPase signaling with cell morphodynamics and captures the GTPase behavior observed with FRET-based biosensors. We show that membrane velocity is regulated by the kinetic rate of GTPase activation rather than the concentration of active GTPase. Our model captures both uniform and polarized ruffling. We also show that cell-type specific time delays between Rac1 and Cdc42 activation can be reproduced with a single signaling motif, in which the delay is controlled by feedback from Cdc42 to Rac1. The resolution of our simulation output matches those of time-lapsed recordings of cell dynamics and GTPase activity. Our data-driven modeling approach allows us to validate simulation results with quantitative precision using the same pipeline for the analysis of simulated and experimental data.

    

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2. Chemo-mechanical diffusion waves explain collective dynamics of immune cell podosomes

   https://doi.org/10.1038/s41467-023-38598-z  

   Gong, Ze ; van den Dries, Koen ; Migueles-Ramírez, Rodrigo A. ; Wiseman, Paul W. ; Cambi, Alessandra ; Shenoy, Vivek B. ( December 2023 , Nature Communications)

   Immune cells, such as macrophages and dendritic cells, can utilize podosomes, mechanosensitive actin-rich protrusions, to generate forces, migrate, and patrol for foreign antigens. Individual podosomes probe their microenvironment through periodic protrusion and retraction cycles (height oscillations), while oscillations of multiple podosomes in a cluster are coordinated in a wave-like fashion. However, the mechanisms governing both the individual oscillations and the collective wave-like dynamics remain unclear. Here, by integrating actin polymerization, myosin contractility, actin diffusion, and mechanosensitive signaling, we develop a chemo-mechanical model for podosome dynamics in clusters. Our model reveals that podosomes show oscillatory growth when actin polymerization-driven protrusion and signaling-associated myosin contraction occur at similar rates, while the diffusion of actin monomers drives wave-like coordination of podosome oscillations. Our theoretical predictions are validated by different pharmacological treatments and the impact of microenvironment stiffness on chemo-mechanical waves. Our proposed framework can shed light on the role of podosomes in immune cell mechanosensing within the context of wound healing and cancer immunotherapy.

    

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3. Optogenetic Rac1 engineered from membrane lipid-binding RGS-LOV for inducible lamellipodia formation

   https://doi.org/10.1039/C9PP00434C  

   Berlew, Erin E. ; Kuznetsov, Ivan A. ; Yamada, Keisuke ; Bugaj, Lukasz J. ; Chow, Brian Y. ( March 2020 , Photochemical & Photobiological Sciences)

   We report the construction of a single-component optogenetic Rac1 (opto-Rac1) to control actin polymerization by dynamic membrane recruitment. Opto-Rac1 is a fusion of wildtype human Rac1 small GTPase to the C-terminal region of BcLOV4, a LOV (light-oxygen-voltage) photoreceptor that rapidly binds the plasma membrane upon blue-light activation via a direct electrostatic interaction with anionic membrane phospholipids. Translocation of the fused wildtype Rac1 effector permits its activation by GEFs (guanine nucleotide exchange factors) and consequent actin polymerization and lamellipodia formation, unlike in existing single-chain systems that operate by allosteric photo-switching of constitutively active Rac1 or the heterodimerization-based ( i.e. two-component) membrane recruitment of a Rac1-activating GEF. Opto-Rac1 induction of lamellipodia formation was spatially restricted to the patterned illumination field and was efficient, requiring sparse stimulation duty ratios of ∼1–2% (at the sensitivity threshold for flavin photocycling) to cause significant changes in cell morphology. This work exemplifies how the discovery of LOV proteins of distinct signal transmission modes can beget new classes of optogenetic tools for controlling cellular function. 

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4. Variable fluid flow regimes alter human brain microvascular endothelial c ell–cell junctions and cytoskeletal structure

   https://doi.org/10.1002/cm.21687  

   Ranadewa, Dilshan ; Wu, Jingwen ; Subramanianbalachandar, Vignesh A. ; Steward, Jr, Robert L. ( September 2021 , Cytoskeleton)

   The human brain microvasculature is constantly exposed to variable fluid flow regimes and their influence on the endothelium depends in part on the synchronous cooperative behavior between cell–cell junctions and the cytoskeleton. In this study, we exposed human cerebral microvascular endothelial cells to a low laminar flow (1 dyne⋅cm⁻²), high laminar flow (10 dyne⋅cm⁻²), low oscillatory flow (±1 dyne⋅cm⁻²), or high oscillatory flow (±10 dyne⋅cm⁻²) for 24 hr. After this time, endothelial cell–cell junction and cytoskeletal structural response was characterized through observation of zonula occludens‐1 (ZO‐1), claudin‐5, junctional adhesion molecule‐A (JAM‐A), vascular endothelial cadherin (VE‐Cad), and F‐actin. In addition, we also characterized cell morphology through measurement of cell area and cell eccentricity. Our results revealed the greatest change in junctional structure reorganization for ZO‐1 and JAM‐A to be observed under low laminar flow conditions while claudin‐5 exhibited the greatest change in structural reorganization under both low and high laminar flow conditions. However, VE‐Cad displayed the greatest structural response under a high laminar flow, reflecting the unique responses each cell–cell junction protein had to each fluid flow regime. In addition, cell area and cell eccentricity displayed most significant changes under the high laminar flow and low oscillatory flow, respectively. We believe this study will be useful to the field of cell mechanics and mechanobiology.

    

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5. Non-junctional role of Cadherin3 in cell migration and contact inhibition of locomotion via domain-dependent, opposing regulation of Rac1

   https://doi.org/10.1038/s41598-020-73862-y  

   Ichikawa, Takehiko ; Stuckenholz, Carsten ; Davidson, Lance A. ( October 2020 , Scientific Reports)

   Classical cadherins are well-known adhesion molecules responsible for physically connecting neighboring cells and signaling this cell–cell contact. Recent studies have suggested novel signaling roles for “non-junctional” cadherins (NJCads); however, the function of cadherin signaling independent of cell–cell contacts remains unknown. In this study, mesendodermal cells and tissues from gastrula stageXenopus laevisembryos demonstrate that deletion of extracellular domains of Cadherin3 (Cdh3; formerly C-cadherin inXenopus) disrupts contact inhibition of locomotion. In both bulk Rac1 activity assays and spatio-temporal FRET image analysis, the extracellular and cytoplasmic Cdh3 domains disrupt NJCad signaling and regulate Rac1 activity in opposing directions. Stabilization of the cytoskeleton counteracted this regulation in single cell migration assays. Our study provides novel insights into adhesion-independent signaling by Cadherin3 and its role in regulating single and collective cell migration.

    

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