Abstract
The three canonical Rho GTPases RhoA, Rac1 and Cdc42 co-ordinate cytoskeletal dynamics. Recent studies indicate that all three Rho GTPases are activated at the leading edge of motile fibroblasts, where their activity fluctuates at subminute time and micrometer length scales. Here, we use a microfluidic chip to acutely manipulate fibroblast edge dynamics by applying pulses of platelet-derived growth factor (PDGF) or the Rho kinase inhibitor Y-27632 (which lowers contractility). This induces acute and robust membrane protrusion and retraction events, that exhibit stereotyped cytoskeletal dynamics, allowing us to fairly compare specific morphodynamic states across experiments. Using a novel Cdc42, as well as previously described, second generation RhoA and Rac1 biosensors, we observe distinct spatio-temporal signaling programs that involve all three Rho GTPases, during protrusion/retraction edge dynamics. Our results suggest that Rac1, Cdc42 and RhoA regulate different cytoskeletal and adhesion processes to fine tune the highly plastic edge protrusion/retraction dynamics that power cell motility.
Highlights
Rho GTPases regulate the actin and adhesion dynamics that power cell motility
We focused on two distinct stimuli to manipulate cell edge dynamics
These results indicate that specific F-actin and adhesion dynamics correlate with different edge motility states that are induced by platelet-derived growth factor (PDGF), Y-27632, or combined application of PDGF + Y-27632
Summary
Rho GTPases regulate the actin and adhesion dynamics that power cell motility. Initial models have proposed that Rac[1] controls membrane protrusion, Cdc[42] regulates filopodia and polarity, and RhoA promotes myosin contractility during tail retraction[1]. All three Rho GTPases have been observed to be active at the leading edge of motile fibroblasts[2,3,4,5,6]. A correct understanding of spatio-temporal Rho GTPase activation dynamics requires us to focus on context-dependent, specific subcellular morphodynamic processes, rather than on the entire cell[8,9]. While we start to unravel the diversity of spatio-temporal Rho GTPase signaling programs in different morphodynamic processes, their inherent complexity impedes a clear understanding of how they regulate cytoskeletal dynamics. This provides novel insight into how the three Rho GTPases co-operate to fine tune the cytoskeletal dynamics that power edge motility
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