Report Cellular Control of Cortical Actin Nucleation

Laboratoire d’Enzymologie et Biochimie Structurales, CNRS,91198 Gif-sur-Yvette, FranceSummaryThe contractile actin cortex is a thin layer of actin, myosin,and actin-binding proteins that subtends the membrane ofanimalcells.Thecortexisthemaindeterminantofcellshapeand plays a fundamental role in cell division [1–3], migration[4], and tissue morphogenesis [5]. For example, cortexcontractility plays a crucial role in amoeboid migration ofmetastatic cells[6]andduring division, whereits misregula-tion can lead to aneuploidy [7]. Despite its importance, ourknowledgeofthecortexispoor,andeventheproteinsnucle-ating it remain unknown, though a number of candidateshave been proposed based on indirect evidence [8–15].Here, we used two independent approaches to identifycortical actin nucleators: a proteomic analysis using cor-tex-rich isolated blebs, and a localization/small hairpinRNA (shRNA) screen searching for phenotypes with a weak-ened cortex or altered contractility. This unbiased study re-vealed that two proteins generated the majority of corticalactin:theforminmDia1andtheArp2/3complex.Eachnucle-atorcontributeda similaramountof F-actinto thecortexbuthad very different accumulation kinetics. Electron micro-scopy examination revealed that each nucleator affectedcortical network architecture differently. mDia1 depletionled to failure in division, but Arp2/3 depletion did not. Inter-estingly, despite not affecting division on its own, Arp2/3inhibitionpotentiatedtheeffectofmDia1depletion.Ourfind-ings indicate that the bulk of the actin cortex is nucleated bymDia1 and Arp2/3 and suggest a mechanism for rapid fine-tuning of cortex structure and mechanics by adjusting therelative contribution of each nucleator.Results and DiscussionHere, we took an unbiased approach to study cortical actinnucleation.Weusednaturalandinducedcellularblebsastools;expanding blebs are initially devoid of F-actin and progres-sively reassemble a contractile cortex prior to retraction [16],makingthemanidealmodeltostudydenovocortexassembly.Thus,wereasonedthattheproteinsnecessaryfortheregrowthof cortical actin should be present in blebs. Cortex assemblycouldoccurviaelongationofF-actinseedsormediatedbynu-cleators. We first examined several seed elongation corticalgrowth mechanisms and concluded that these were not sup-ported by experimental evidence (see Figure S1 available on-line). Therefore, we investigated the role of actin nucleators incortexassemblyusingtwoindependentunbiasedapproaches.First,weusedproteomicsonisolatedcorticestoidentifytheactin nucleators present in the cortex. To this aim, we sepa-rated blebs from constitutively blebbing M2 melanoma cellsby mechanical shearing as previously described [17], a proce-durethatallowsisolationofdynamicactincortices(Figure1A).We investigated the presence of actin nucleators in the actin-rich detergent-insoluble fraction of isolated blebs using massspectrometry analysis. We detected the presence of only twoactin nucleators: the formin mDia1 and the Arp2/3 complex(Figure1B),consistentwithsomereports[11,13]butincontra-diction with others [8–10, 12]. All seven subunits of the Arp2/3complex were detected along with the Arp2/3 nucleation-pro-moting factors cortactin and two subunits of the WAVE com-plex (SRA1 and NAP1).To verify these results using an independent approach, weundertook a screen based on mRNA expression profiles, pro-tein localization, and small hairpin RNA (shRNA) depletion.First, we determined which nucleators were expressed inbothM2andHeLacellsandlocalizedtotheircortex,reasoningthat basic mechanisms of cortex nucleation should beconserved across cell types. Quantitative PCR revealed thatten nucleators were expressed in both cell lines: the Arp2/3complex subunits ACTR2 and ACTR3; the formins DAAM1,DIAPH1, DIAPH3, FHOD1, FMNL1, INF1, and INF2; andSPIRE1 and SPIRE2 (Figure S2A). To distinguish potentialcortical actin nucleators, we examined the localization of theexpressed nucleators in M2 cells using imaging assays,reasoning that proteins clearly localized to the cell peripherywere good candidates for initiators of cortex growth, whereasnucleators strongly enriched in other locations were likelyfulfilling other functions. Of the expressed nucleators, onlyDaam1, Fhod1, mDia1, and the Arp2/3 complex were foundto localize to the cell periphery (Figures 1C–1F and S2B–S2I).Daam1 was present in the membrane of both expanding andretracting blebs (Figure S2B). Fhod1 accumulated at the actincortex late in bleb retraction (Figure S2C). Immunostaining for