Arp2/3 inhibition induces amoeboid-like protrusions in MCF10A epithelial cells by reduced cytoskeletal-membrane coupling and focal adhesion assembly.

Yvonne Beckham,Kareem Sayegh,Clement Campillo,Jonathan Stricker,Robert J Vasquez,Margaret L Gardel,Cara Gottardi

doi:10.1371/journal.pone.0100943

Yvonne Beckham, Kareem Sayegh + Show 5 more

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https://doi.org/10.1371/journal.pone.0100943

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Abstract

Here we demonstrate that Arp2/3 regulates a transition between mesenchymal and amoeboid protrusions in MCF10A epithelial cells. Using genetic and pharmacological means, we first show Arp2/3 inhibition impairs directed cell migration. Arp2/3 inhibition results in a dramatically impaired cell adhesion, causing deficient cell attachment and spreading to ECM as well as an 8-fold decrease in nascent adhesion assembly at the leading edge. While Arp2/3 does not play a significant role in myosin-dependent adhesion growth, mature focal adhesions undergo large scale movements against the ECM suggesting reduced coupling to the ECM. Cell edge protrusions occur at similar rates when Arp2/3 is inhibited but their morphology is dramatically altered. Persistent lamellipodia are abrogated and we observe a markedly increased incidence of blebbing and unstable pseuodopods. Micropipette-aspiration assays indicate that Arp2/3-inhibited cells have a weak coupling between the cell cortex and the plasma membrane, and suggest a potential mechanism for increased pseudopod and bleb formation. Pseudopods are not sensitive to reduced in formin or myosin II activity. Collectively, these results indicate that Arp2/3 is not necessary for rapid protrusion of the cell edge but plays a crucial role in assembling focal adhesions required for its stabilization.

Highlights

Cell migration is an essential physiological process in development, wound healing and immune response
In mesenchymal migration utilized by diverse cell types, including epithelial and fibroblast cells, coordination of protrusion and adhesion at the leading cell edge is facilitated by the lamellipodium
Human breast epithelial MCF10A cells plated on fibronectin-coated glass coverslips display a prominent lamellipodium, with approximately 30–40% of the cell periphery staining positive for cortactin (Fig. 1A)

Summary

Introduction

Cell migration is an essential physiological process in development, wound healing and immune response. In mesenchymal migration utilized by diverse cell types, including epithelial and fibroblast cells, coordination of protrusion and adhesion at the leading cell edge is facilitated by the lamellipodium. Nascent adhesions form within the lamellipodium in an actin-polymerization-dependent manner to couple the actin cytoskeleton to the ECM [3,9,10] and facilitate the effective transmission of forces, generated through actin polymerization, to advance the leading edge of the cell [11]. The coordination of adhesion assembly with actin polymerization is not completely understood but is thought to occur via interactions between Arp2/3 with vinculin (DeMali et al 2002). In the absence of lamellipodia, alternate actin-polymerization machinery within the lamella [13] or in filopodia [14,15,16,17] can facilitate cell edge protrusion and adhesion

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