WASP Restricts Active Rac to Maintain Cells' Front-Rear Polarization.

Clelia Amato,Laura M Machesky,Peter A Thomason,Andrew J Davidson,Karthic Swaminathan,Shehab Ismail,Robert H Insall

doi:10.1016/j.cub.2019.10.036

Clelia Amato, Laura M Machesky + Show 5 more

Open Access

https://doi.org/10.1016/j.cub.2019.10.036

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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.

Highlights

Filamentous actin (F-actin) fulfils numerous functions in migrating cells
Loss of Wiskott-Aldrich syndrome protein (WASP) Causes Accumulation of SCAR/WAVE and Active Rac at the Rear Previous work shows that knockout mutants in the Dictyostelium gene encoding WASP, wasA, migrate slower than their wild-type counterparts, due to a defect in rear retraction [6]
One intriguing possibility was that WASP may be responsible for the spatial restriction of SCAR/WAVE’s activators, preventing actin polymerization in unwanted areas of the cell

Summary

Introduction

Filamentous actin (F-actin) fulfils numerous functions in migrating cells. One crucial role is the generation of protrusions, such as pseudopods and lamellipods. A second is maintenance of intracellular trafficking, by driving endocytosis and vesicle sorting. These diverse functions depend upon the highly conserved Arp2/3 complex, which drives branching and growth of the actin network [1]. Cells rely on the Wiskott-Aldrich syndrome protein (WASP) family of nucleation-promoting factors (NPFs) to control the Arp2/3 complex spatially and temporally. The founding member of the family, WASP [2], is specific to hematopoietic lineages. Vertebrates possess a ubiquitous WASP paralog, N-WASP, which was originally described as a neural-specific gene [3] expressed in most cell types [4]. Other eukaryotes, including Dictyostelium, yeasts, and Drosophila, express a single WASP, which is the ortholog of vertebrates’ ubiquitous isoform [5]. WASPs’ principal role is to facilitate clathrin-mediated endocytosis (CME) [6,7,8,9]

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