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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.

SUBMITTER: Kunschmann T

PROVIDER: S-EPMC6531482 | biostudies-other | 2019 May

REPOSITORIES: biostudies-other

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

Kunschmann Tom T Puder Stefanie S Fischer Tony T Steffen Anika A Rottner Klemens K Mierke Claudia Tanja CT

Scientific reports 20190522 1

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 stil ...[more]

PMID: 31118438

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Project description:Cell migration performs a critical function in numerous physiological processes, including tissue homeostasis or wound healing after tissue injury, as well as pathological processes that include malignant progression of cancer. The efficiency of cell migration and invasion appears to be based on the mechano-phenotype of the cytoskeleton. The properties of the cytoskeleton depend on internal cytoskeletal and external environmental factors. A reason for this are connections between the cell and its local matrix microenvironment, which are established by cell-matrix adhesion receptors. Upon activation, focal adhesion proteins such as PINCH1 are recruited to sites where focal adhesions form. PINCH1 specifically couples through interactions with ILK, which binds to cell matrix receptors and the actomyosin cytoskeleton. However, the role of PINCH1 in cell mechanics regulating cellular motility in 3D collagen matrices is still unclear. PINCH1 is thought to facilitate 3D motility by regulating cellular mechanical properties, such as stiffness. In this study, PINCH1 wild-type and knock-out cells were examined for their ability to migrate in dense extracellular 3D matrices. Indeed, PINCH1 wild-type cells migrated more numerously and deeper in 3D matrices, compared to knock-out cells. Moreover, cellular deformability was determined, e.g., elastic modulus (stiffness). PINCH1 knock-out cells are more deformable (compliable) than PINCH1 wild-type cells. Migration of both PINCH1-/- cells and PINCH1fl/fl cells was decreased by Latrunculin A inhibition of actin polymerization, suggesting that actin cytoskeletal differences are not responsible for the discrepancy in invasiveness of the two cell types. However, the mechanical phenotype of PINCH1-/- cells may be reflected by Latrunculin A treatment of PINCH1fl/fl cells, as they exhibit resembling deformability to untreated PINCH1-/- cells. Moreover, an apparent mismatch exists between the elongation of the long axis and the contraction of the short axis between PINCH1fl/fl cells and PINCH1-/- cells following Latrunculin A treatment. There is evidence of this indicating a shift in the proxy values for Poisson's ratio in PINCH1-/- cells compared with PINCH1fl/fl cells. This is probably attributable to modifications in cytoskeletal architecture. The non-muscle myosin II inhibitor Blebbistatin also reduced the cell invasiveness in 3D extracellular matrices but instead caused a stiffening of the cells. Finally, PINCH1 is apparently essential for providing cellular mechanical stiffness through the actin cytoskeleton, which regulates 3D motility.

Dataset Information

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

Publications

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

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