Homeostatic Actin Cytoskeleton Networks Are Regulated by Assembly Factor Competition for Monomers

Street, Chicago,IL 60637, USASummaryControlling the quantity and size of organelles throughcompetition for a limited supply of components is quicklyemerging as an important cellular regulatory mechanism[1]. Cells assemble diverse actin ﬁlament (F-actin) net-works for fundamental processes including division,motility, and polarization [2–4]. F-actin polymerization istightly regulated by activation of assembly factors suchas the Arp2/3 complex and formins at speciﬁc times andplaces. We directly tested an additional hypothesis thatdiverse F-actin networks are in homeostasis, wherebycompetition for actin monomers (G-actin) is critical forregulating F-actin network size. Here we show that inhibi-tion of Arp2/3 complex in the ﬁssion yeast Schizosacchar-omyces pombe not only depletes Arp2/3-complex-mediatedendocytic actin patches, but also induces a dramaticexcess of formin-assembled F-actin. Conversely, disrup-tion of formin increases the density of Arp2/3-complex-mediated patches. Furthermore, modiﬁcation of actin levelssigniﬁcantly perturbs the ﬁssion yeast actin cytoskeleton.Increasing actin favors Arp2/3-complex-mediated actin as-sembly, whereas decreasing actin favors formin-mediatedcontractile rings. Therefore, the speciﬁc actin concentra-tion in a cell is critical, and competition for G-actin helpsregulate the proper amount of F-actin assembly for diverseprocesses.Results and DiscussionTo control F-actin network density, actin polymerization istightly regulated through the activation of assembly (nucle-ation)factorsbyGTPasesignaling cascades, therateat whichF-actin barbed ends are capped, the rate at which assemblyfactors are turned off, and F-actin disassembly factors[2, 3, 5]. The supply of unassembled G-actin is not generallyconsidered to be limiting [6, 7]. Alternatively, it is possiblethat the actin cytoskeleton is homeostatic with a limitedconcentration of G-actin, which is competed for by assemblyfactors to help regulate its incorporation into diverse F-actinnetworks [3, 8–10]. However, this intriguing additional hypoth-esis has not been systematically tested.Fission yeast forms three F-actin network structures bythree different assembly factors [9]. The Arp2/3 complexassemblesshort-branched F-actinin endocytic actin patches,whereas the formins For3 and Cdc12 assemble long-straightF-actin in polarizing actin cables and the cytokinetic contrac-tile ring, respectively. The amount of actin and other compo-nents incorporated into actin patches and contractile rings isremarkably consistent, varying less than 50% for each struc-ture [11–13]. Although measuring the composition of actincables has been technically challenging, they may be similarlyconsistent. Of the w1 million actin molecules per cell, w35%to 50% are evenly distributed between 30 to 50 actin patches,w10% are incorporated into contractile rings, and perhaps asmuch as 15% are estimated to be consumed by actin cables[11–15].To directly test the hypothesis that assembly factors com-peteforG-actin,weinvestigatedtheconsequencesofsystem-atically disrupting individual assembly factors in ﬁssion yeastcells. Initially, we treated cells expressing the general F-actinmarker Lifeact-GFP with a range of concentrations of theArp2/3 complex inhibitor CK-666 [16], causing a dose-depen-dent decrease in the number of actin patches (Figures 1Aand 1B and Figure S1A available online), reduction in patchmotility, and increase in patch lifetime (Table S1). Strikingly,actin patch depletion coincides with the dramatic formationof new ectopic cable-like F-actin (Figures 1A and S1A), satu-rating at w100 mM CK-666 (Figure 1B). CK-666 treatmentfacilitates ectopic F-actin assembly in both minimal and richgrowth media, is visible with different general F-actin markersincluding rhodamine-phalloidin (Figures S1B–S1F), and isinhibited by the G-actin sequestering drug LatA (Figure S1G).Observation of cells in a microﬂuidic chamber revealedthat depletion of actin patches and the concomitant assemblyof ectopic F-actin occurs in w10–20 min after addition ofsaturating concentrations of CK-666 (Figure 1C and 1D andMovie S1). Ectopic F-actin rapidly disassembles upon washout of CK-666 with a corresponding reassembly of actinpatches in w10–40 min (Figures 1C and 1D). Actin patchproteins ArpC5-mCherry (Arp2/3 complex component) andAcp2-GFP (actin capping protein) are released into the cyto-plasm by CK-666 treatment, but do not incorporate into theectopic F-actin (Figures S1H–S1J).Genetic disruption of Arp2/3 complex also leads to ectopicF-actin assembly, albeit less prominently than with CK-666since actin patches are not depleted completely under theseconditions (Figures 1E–1H). Compared to wild-type (WT) cells,at the restrictive temperature of 19 C Arp2/3 complex cold-sensitive mutant arp3-C1 cells [17] have approximately halfthenumberofpatchesandacorrespondingstatisticallysignif-icant3-foldincreasein ectopicF-actin(p<0.0001)(Figures 1Eand 1F). Similarly, reduction of Arp2/3 complex expression byshutting off Arp3 (SO-arp3) for 46 hr also halves the number ofpatchespercellwhileincreasingtheamountofectopicF-actinby more than 3-fold (p < 0.0001) (Figures 1G and 1H).We next investigated whether the cable-like ectopic F-actinis spontaneously assembled, or is dependent upon remaining