Dynamin2 organizes lamellipodial actin networks to orchestrate lamellar actomyosin.

Manisha Menon,Dorothy A Schafer,Olga L Askinazi

doi:10.1371/journal.pone.0094330

Abstract

Actin networks in migrating cells exist as several interdependent structures: sheet-like networks of branched actin filaments in lamellipodia; arrays of bundled actin filaments co-assembled with myosin II in lamellae; and actin filaments that engage focal adhesions. How these dynamic networks are integrated and coordinated to maintain a coherent actin cytoskeleton in migrating cells is not known. We show that the large GTPase dynamin2 is enriched in the distal lamellipod where it regulates lamellipodial actin networks as they form and flow in U2-OS cells. Within lamellipodia, dynamin2 regulated the spatiotemporal distributions of α-actinin and cortactin, two actin-binding proteins that specify actin network architecture. Dynamin2's action on lamellipodial F-actin influenced the formation and retrograde flow of lamellar actomyosin via direct and indirect interactions with actin filaments and a finely tuned GTP hydrolysis activity. Expression in dynamin2-depleted cells of a mutant dynamin2 protein that restores endocytic activity, but not activities that remodel actin filaments, demonstrated that actin filament remodeling by dynamin2 did not depend of its functions in endocytosis. Thus, dynamin2 acts within lamellipodia to organize actin filaments and regulate assembly and flow of lamellar actomyosin. We hypothesize that through its actions on lamellipodial F-actin, dynamin2 generates F-actin structures that give rise to lamellar actomyosin and for efficient coupling of F-actin at focal adhesions. In this way, dynamin2 orchestrates the global actin cytoskeleton.

Highlights

Cells assemble a spectrum of dynamic actin networks comprised of branched, crosslinked and bundled filaments that, together, move cells and organelles, enable cell division and organize cells within tissues
Plasmids to express GFP-actin, GFPa-actinin, mCh-a-actinin and GFP-myosin light chain 2 (MLC2) were from Dr Rick Horwitz (University of Virginia, Charlottesville, VA), GFP-paxillin was from Dr Martin Schwartz (Yale School of Medicine, New Haven, CT), RFP-cortactin was from Dr James Casanova (University of Virginia, Charlottesville, VA) and mCh-dynamin1 was from Dr David Castle (University of Virginia, Charlottesville, VA)
To determine how dynamin2, which is enriched in lamellipodial actin networks [19,29,30,31], influences lamellar actomyosin, we observed the formation of nascent actomyosin in living U2-OS cells lacking up to,90% of endogenous dynamin2

Summary

Introduction

Cells assemble a spectrum of dynamic actin networks comprised of branched, crosslinked and bundled filaments that, together, move cells and organelles, enable cell division and organize cells within tissues. Well-characterized actin networks include the sheetlike, dendritic networks of lamellipodia that support protrusion at cell membranes and the contractile actomyosin networks of lamellae. Interactions between these distinct actin networks occur, in part, at focal adhesions. Lamellipodial, lamellar and focal adhesionassociated actin networks are interdependent. In part, from Arp2/3-dependent actin filaments and from remnants of lamellipodial F-actin-rich structures such as filopodia or F-actin of retracting protrusions [1,2,3,4,5,6]. How lamellipodial actin filaments transition from their characteristic dendritic organization to engage nascent focal adhesions or generate actomyosin of the lamellum is not known. The molecular mechanisms that regulate the interdependent spatial organization of actin filament networks near the leading edge are likely complex, we report here that the large GTPase dynamin is involved

Methods

Results

Conclusion