Spatio-temporal co-ordination of RhoA, Rac1 and Cdc42 activation during prototypical edge protrusion and retraction dynamics

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.

Septins guide microtubule protrusions induced by actin-depolymerizing toxins like Clostridium difficile transferase (CDT)

“…We show that septins interact with microtubules via EB1, which is a multifunctional interaction partner at the microtubule tip (21)(22)(23)(24)(25). Pull-down experiments and surface plasmon resonance spectroscopy, exhibiting K d values in the nanomolar range, revealed that septins bind directly to EB1.…”

“…However, the structures involved are largely enigmatic. We asked whether the plus-end tracking protein-1 (EB1), which is a master regulator of protein interactions at the plus-ends of microtubules (21)(22)(23)(24)(25), plays a role in septin-microtubule communication. In fact, GST-pull-down experiments with EB1-loaded beads revealed a direct interaction of septins with EB1.…”

“…3D) (21). To analyze the spatiotemporal regulation of Cdc42, cells were transfected with a FRET sensor (24,25). After 24 h, transfected cells were treated with CDT and the activation of Cdc42 was monitored 1 h after toxin addition.…”

Periodic propagating waves coordinate RhoGTPase network dynamics at the leading and trailing edges during cell migration

“…2D). This periodic Rac1 activation drives actin polymerization at the leading edge pushing protrusion-retraction cycles (Machacek et al, 2009;Martin et al, 2016;Pertz, 2010;Tkachenko et al, 2011).…”

Rac1 switching at the right time and location is essential for Fcγ receptor-mediated phagosome formation

“…During FcγR-mediated phagocytosis, the processes of phagosome formation consisting of lamellipodial extension ( phagocytic cup formation) and phagocytic cup closure ( phagosome internalization) are highly dependent on actin assembly and remodelling (Aderem and Underhill, 1999;Araki et al, 1996Araki et al, , 2003Swanson, 2008;Swanson et al, 1999). The actin dynamics are known to be coordinated by both phosphoinositide metabolism and Rho family GTPases by which a variety of actin-binding proteins are recruited and/or activated (Beemiller et al, 2006(Beemiller et al, , 2010Caron and Hall, 1998;Chimini and Chavrier, 2000;Csépányi-Kömi et al, 2012;Martin et al, 2016;Niedergang et al, 2016). However, the precise mechanism underlying spatiotemporal regulation of phagosome formation remains to be elucidated.…”