The Small GTPase Rac1 Increases Cell Surface Stiffness and Enhances 3D Migration Into Extracellular Matrices

Abstract

Membrane ruffling and lamellipodia formation promote the motility of adherent cells in two-dimensional motility assays by mechano-sensing of the microenvironment and initiation of focal adhesions towards their surroundings. Lamellipodium formation is stimulated by small Rho GTPases of the Rac subfamily, since genetic removal of these GTPases abolishes lamellipodium assembly. The relevance of lamellipodial or invadopodial structures for facilitating cellular mechanics and 3D cell motility is still unclear. Here, we hypothesized that Rac1 affects cell mechanics and facilitates 3D invasion. Thus, we explored whether fibroblasts that are genetically deficient for Rac1 (lacking Rac2 and Rac3) harbor altered mechanical properties, such as cellular deformability, intercellular adhesion forces and force exertion, and exhibit alterations in 3D motility. Rac1 knockout and control cells were analyzed for changes in deformability by applying an external force using an optical stretcher. Five Rac1 knockout cell lines were pronouncedly more deformable than Rac1 control cells upon stress application. Using AFM, we found that cell-cell adhesion forces are increased in Rac1 knockout compared to Rac1-expressing fibroblasts. Since mechanical deformability, cell-cell adhesion strength and 3D motility may be functionally connected, we investigated whether increased deformability of Rac1 knockout cells correlates with changes in 3D motility. All five Rac1 knockout clones displayed much lower 3D motility than Rac1-expressing controls. Moreover, force exertion was reduced in Rac1 knockout cells, as assessed by 3D fiber displacement analysis. Interference with cellular stiffness through blocking of actin polymerization by Latrunculin A could not further reduce invasion of Rac1 knockout cells. In contrast, Rac1-expressing controls treated with Latrunculin A were again more deformable and less invasive, suggesting actin polymerization is a major determinant of observed Rac1-dependent effects. Together, we propose that regulation of 3D motility by Rac1 partly involves cellular mechanics such as deformability and exertion of forces.

Dexamethasone and Glucocorticoid-Induced Matrix Temporally Modulate Key Integrins, Caveolins, Contractility, and Stiffness in Human Trabecular Meshwork Cells

“…Here, we show that DEX transiently upregulated RhoA in hTM cells, and this transient increase was time-dependent, consistent with prior studies that reported increased cell contractility, and/or fibrotic changes in hTM cells 49,51,65 and non-ocular tissues. 50 Although there was significant interaction between treatment and time for the expression of Rac 1/2/3 in hTM cells, no marked differences were observed between Veh and DEX. This finding may probably suggest DEX-induced fibrotic phenotypes in hTM cells is independent of Rac 1, 33 which may be dormant in hTM cells.…”

“…We recently documented crosslinked CDM stiffens hTM cells 81 and our group has previously reported DEX increases stiffness of hTM cells 7 ; herein, we document that GIM-mediated increased cell stiffening is consistent across multiple time points. Several studies have implicated substratum stiffness, 118 integrins, 73 caveolins, 119 and Rho GTPases 50,[120][121][122] in altered biomechanics in ocular and non-ocular cells / tissues. Therefore, the time-independent increase in stiffness of cells cultured on GIMs may be due in part to the integration of FN ED-A / FN ED-B overexpression, specific integrins, caveolins and Rho GTPases in hTM cells or aberrant TGFβ2 signaling.…”

“…On the other hand, DEX-induced ECM remodeling deemed responsible for increased resistance to aqueous outflow encompasses aberrant changes to its structural, matricellular, crosslinking, and turnover components typically resulting in increased ECM deposition and/or stiffness. 7,25,[36][37][38][39][40][41][42][43] Specific cell-matrix mechanoreceptors, like integrin subunits/integrin adhesomes 38,[44][45][46] and caveolins, 32,47 and key intracellular cytoskeletal-related proteins, [48][49][50][51][52][53] have been identified as critical mediators in these DEX-induced TM cell and/or ECM changes. For instance, β1, 52,54,55 β3, 30,40,54 and αVβ3 40,46,56 integrins, and integrin linked kinase 44 have all been implicated in aberrant remodeling of the actin cytoskeleton (such as CLANs formation) and/or ocular hypertension.…”

“…Similarly, membranebound cave-like structures, caveolae, have been implicated in fibrotic phenotypes, ECM remodeling, autophagy, and ocular hypertension. 32,47,[57][58][59] Moreover, cytoskeletal-related proteins, like RhoA, 49,51 Cdc42, 48 Rac 1/2/3, 50 and intermediate filament vimentin, 52,53,60 typically downstream of integrins and caveolae, 45,[61][62][63] have also been implicated in fibrotic phenotypes. 64,65 However, most of these studies either focused on only few crucial mechanoreceptors and cytoskeletal-related proteins at any given time or performed experiments at a single/limited time point(s).…”

Mechanical Cues Affect Migration and Invasion of Cells From Three Different Directions

“…Therefore, the hypothesis is that mechanical stimulation decreases the expression of integrin β3, which in turn decreases the activation of Rac1. In opposition to other investigations, in which Rac1 expression strengthened the migration and invasion of cells (Kunschmann et al, 2019). It is hypothesized that various types of force use distinct signal transduction pathways, in which the force is primarily modulated by the interaction of cells with various ligands.…”