WASP Restricts Active Rac to Maintain Cells’ Front-Rear Polarization

Abstract

SummaryEfficient motility requires polarized cells, with pseudopods at the front and a retracting rear. Polarization is maintained by restricting the pseudopod catalyst, active Rac, to the front. Here, we show that the actin nucleation-promoting factor Wiskott-Aldrich syndrome protein (WASP) contributes to maintenance of front-rear polarity by controlling localization and cellular levels of active Rac. Dictyostelium cells lacking WASP inappropriately activate Rac at the rear, which affects their polarity and speed. WASP’s Cdc42 and Rac interacting binding (“CRIB”) motif has been thought to be essential for its activation. However, we show that the CRIB motif’s biological role is unexpectedly complex. WASP CRIB mutants are no longer able to restrict Rac activity to the front, and cannot generate new pseudopods when SCAR/WAVE is absent. Overall levels of Rac activity also increase when WASP is unable to bind to Rac. However, WASP without a functional CRIB domain localizes normally at clathrin pits during endocytosis, and activates Arp2/3 complex. Similarly, chemical inhibition of Rac does not affect WASP localization or activation at sites of endocytosis. Thus, the interaction between small GTPases and WASP is more complex than previously thought—Rac regulates a subset of WASP functions, but WASP reciprocally restricts active Rac through its CRIB motif.

The WAVE Regulatory Complex Is Required to Balance Protrusion and Adhesion in Migration

“…tested here, N-WASP cannot compensate for the loss of the WRC, in contrast to what occurs in Dictyostelium [42].…”

“…As it was recently shown, in the absence of the Scar/WAVE complex, WASP requires active Rac1 to drive pseudopod extensions in Dictyostelium [42]. However, the mammalian counterpart to Dictyostelium WASP, N-WASP is activated by both Rac1 and Cdc42 [52], and Dictyostelium does not express Cdc42.…”

“…Our study confirmed previous findings that NCKAP1, (one of the two largest subunits of the WRC) is essential for the WRC's function in driving Arp2/3 mediated actin polymerisation at the leading edge [11,31]. However, other family members similar to WAVE, such as the ubiquitous neural-WASP (N-WASP) can compensate for the loss of the WRC and promote 3D migration and invasion of cancer cells [11] and also can substitute for WRC to rescue Dictyostelium discoideum migration [42,50]. When both Scar and WASP are deleted in Dictyostelium, they become immobile but the cells generate numerous filopodia due to excess activity of the formin dDia2 [51].…”

“…This one difference between Dictyostelium and mammalian cells may be why there is no compensation by N-WASP in 2D migration. Indeed there is plentiful active Rac1 in WRC depleted cells, especially after Rac1 activation or microinjection of active Rac1 that would have the ability to drive pseudopodia formation as seen in [42]. Therefore, something else is preventing this process in mammalian cells, possibly the high adhesiveness of mammalian cells compared to Dictyostelium [53] keeping the cells anchored to the substrate.…”

Cell–substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime

“…We therefore determined the effect of Rac1 binding on Scar/WAVE phosphorylation. First, we inhibited the Rac1 activity in both Dictyostelium and B16F1 cells by EHT1864, an effective inhibitor of RacGEF activity [29,30], and assessed the band shifts of Scar/WAVE. EHT1864 treatment resulted in complete loss of Pak-CRIB-mRFPmars2 from cell periphery and depolarized Dictyostelium cells (Fig 2A, S1 Video), confirming that the inhibitor greatly reduced active Rac levels.…”