RhoA/ROCK-mediated switching between Cdc42- and Rac1-dependent protrusion in MTLn3 carcinoma cells

Abstract

Rho GTPases are versatile regulators of cell shape that act on the actin cytoskeleton. Studies using Rho GTPase mutants have shown that, in some cells, Rac1 and Cdc42 regulate the formation of lamellipodia and filopodia, respectively at the leading edge, whereas RhoA mediates contraction at the rear of moving cells. However, recent reports have described a zone of RhoA/ROCK activation at the front of cells undergoing motility. In this study, we use a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated cells. Inhibition of Rho or ROCK enhanced protrusion, yet markedly inhibited cell motility; these changes correlated with a marked activation of Rac-1 at the cell edge. Surprisingly, whereas EGF-stimulated protrusion in control MTLn3 cells is Rac-independent and Cdc42-dependent, the opposite pattern is observed in MTLn3 cells after inhibition of ROCK. Thus, Rho and ROCK suppress Rac-1 activation at the leading edge, and inhibition of ROCK causes a switch between Cdc42 and Rac-1 as the dominant Rho GTPase driving protrusion in carcinoma cells. These data describe a novel role for Rho in coordinating signaling by Rac and Cdc42.

Multiple roles for RhoA during T cell transendothelial migration

“…14 However, studies examining where Rho GTPases are active in migrating cells demonstrated RhoA activity at the front as well as at the back of a variety of cell types migrating on rigid surfaces. [15][16][17][18] Our work shows for the first time that RhoA is active at the front of T cells under physiological conditions, migrating on and through the pliable EC surface.…”

“…For example, RhoA/ ROCK signaling suppresses Rac signaling at the leading edge of EGF-stimulated carcinoma cells, and inhibition of ROCK increased protrusion but reduced migration. 18 Additionally, ROCK/p-MLC is implicated in protrusive force generation at the leading edge of sarcoma cells. 24 ROCKs may drive retraction events to allow cells to reorient their direction of migration.…”

The regulation of rhoA by stard 13 in focal adhesions is essential for astrocytoma cell motility. (c2013)

“…This paper also shows that RhoA should be activated at the leading edge of astrocytoma cells, as previously established for breast cancer cells (El-Sibai et al, 2008).…”

“…Even though protrusion is unaffected, the absence of focal adhesions leads to an ineffective protrusion with not enough traction force on the ECM for the cells to move forward (Gupton, & Waterman-Storer, 2006). In this system and in other systems (El-Sibai et al, 2008), we have shown that defective adhesions due to inhibition of Rac or RhoA, though they do not affect protrusion, inhibit cell motility. Hence, as we describe a need to inhibit RhoA at the tail of cells, we also confirm the newly described role of RhoA at the leading edge of cancer cells undergoing movement, contrary to the canonical distribution of RhoA activation ( Figure 7).…”

“…The role of RhoA at the edge is to allow the maturation of Rac-mediated focal complexes into focal adhesions since the absence of RhoA leads to the absence of focal adhesions and the accumulation of focal complexes (El-Sibai et al, 2008). Even though protrusion is unaffected, the absence of focal adhesions leads to an ineffective protrusion with not enough traction force on the ECM for the cells to move forward (Gupton, & Waterman-Storer, 2006).…”