γ-Tubulin localizes at actin-based membrane protrusions and inhibits formation of stress-fibers

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Abstract

γ-Tubulin serves as a template in the γ-TuRC machinery to nucleate microtubules. Curiously, γ-tubulin also interacts with Arp2/3, a complex that nucleates actin filaments, and with the Arp2/3 activator WASH. We previously reported that γ-tubulin and Arp2/3 colocalize at the centrosome, where WASH localizes. Here, we report that γ-tubulin localizes at actin-based membrane protrusions, where Arp2/3 operates. This was confirmed by the presence of tagged γ-tubulin at membrane protrusions in stimulated cells and by downregulation of γ-tubulin expression. Surprisingly, expression of tagged γ-tubulin dramatically inhibited the formation of stress-fibers, while having no effect on microtubules. This phenotype is similar to the disruption of stress-fibers by the overexpression of the WCA domain of WASH and other Wiskott–Aldrich syndrome (WAS) family members. We hypothesize that γ-tubulin regulates Arp2/3 and actin nucleation promoting factors such as WASH, explaining the similar effect of γ-tubulin expression and WCA domain expression on stress-fibers. The data presented here indicate that γ-tubulin has a profound relationship with actin filament dynamics.

Highlights

► γ-Tubulin localizes at actin-based membrane protrusions. ► The localization was supported by knock-down and overexpression of γ-tubulin. ► Expression of tagged γ-tubulin dramatically inhibited the formation of stress-fibers. ► This phenotype is similar to that obtained by overexpression of WCA domains.

Introduction

γ-Tubulin is part of the γ-tubulin ring complex (γ-TuRC), a molecular machine that nucleates microtubules at the centrosome, the mitotic spindle and the kinetochores [1], [2], [3], [4]. γ-Tubulin functions as a template for α- and β-tubulin dimer recruitment in microtubule nucleation [5]. γ-TuRC also regulates the minus-end and the plus-end of microtubules [6], [7]. Microtubules are required for various essential physiological events such as cell polarization, intracellular transport, organelle positioning, primary cilium formation and cell division.

In contrast to microtubules, which rely on only one molecular complex for nucleation, actin filaments are nucleated by a large number of protein complexes, including Arp2/3 [8]. Arp2/3 is activated by a large set of nucleation-promoting factors (NPFs), including Class I NPFs WASP and WASH [9]. All these Class I NPFs have a WCA domain in common which activates Arp2/3 [9], [10]. Intriguingly, expression of the WCA domain of WASH results in disruption of stress-fibers and this process is Arp2/3-dependent [11]. Furthermore, Arp2/3 and WASH localize at the centrosome and interact with γ-tubulin [11], [12], [13].

The cell relies on the coordinate action of three types of cytoskeletons. Intermediate filaments such as keratin provide mechanical rigidity and structure [14]. Microtubules are specialized in cellular transport processes, with cargo ranging from vesicles and organelles to sister chromosomes in anaphase. Actin filaments, with the help of the motor protein myosin, provide force required for processes such as cell mobility. Although typical functions can be assigned to each type of cytoskeleton, their functions nonetheless overlap and are tightly entwined [15], [16], [17], [18]. This study complements these connections and specifically describes the newly uncovered role of γ-tubulin in actin filament dynamics.

Section snippets

Reagents

Epidermal growth factor (EGF), l-α-lysophosphatidic acid (LPA) and 4,6-diamidino-2-phenylindole (DAPI) were obtained from Sigma–Aldrich (#E9644, #L7260 and #D8417). Alexa Fluor 488 Phalloidin was obtained from Molecular Probes (#A12379).

Antibodies

The following antibodies were used: rabbit anti-Arp3 was a kind gift from Dr. C. Ampe (VIB, Ghent University, Belgium). Rabbit anti-V5 was obtained from Sigma–Aldrich (#V8137). Mouse anti-V5 antibody was obtained from Invitrogen (#R96025). Alexa Fluor

Results and discussion

We previously examined the localization of several proteins at the centrosome, including CapG, actin, Exo70 and Arp2/3 and used γ-tubulin as a marker for the localization of the centrosome [13]. In the course of these studies, we consistently observed γ-tubulin at membrane protrusions, independently of cell line or fixation procedure (shown for MDA-MB-231 cells in Fig. 1A). Therefore, we undertook the study of γ-tubulin at membrane protrusions. To confirm the data obtained with

Acknowledgments

This work was supported by the Concerted Actions Program of Ghent University (GOA), the Interuniversity Attraction Poles (IUAP) and the Fund for Scientific Research-Flanders (FWO-Vlaanderen).

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