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

Burke, Thomas A.; Christensen, Jenna R; Barone, Elisabeth; Suarez, Cristian; Sirotkin, Vladimir; Kovar, David R.

doi:10.1016/j.cub.2014.01.072

Cited by 206 publications

(273 citation statements)

References 40 publications

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“…In this scheme, inhibition of formins or Arp2/3 shifts the turnover rate towards the Arp2/3-or formin-based kinetics, respectively (Burke et al, 2014;Fritzsche et al, 2013;Lomakin et al, 2015;Rotty et al, 2015;Suarez et al, 2015). To determine if such a balance was also at work in MCCs in vivo, we performed FRAP on apical actin after treatment with a specific Arp2/3 inhibitor, CK666.…”

Section: Rhoa Controls the Dynamics Of MCC Apical Emergencementioning

confidence: 99%

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells

Sedzinski¹,

Hannezo²,

Tu³

et al. 2017

Development

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Homeostatic replacement of epithelial cells from basal precursors is a multistep process involving progenitor cell specification, radial intercalation and, finally, apical surface emergence. Recent data demonstrate that actin-based pushing under the control of the formin protein Fmn1 drives apical emergence in nascent multiciliated epithelial cells (MCCs), but little else is known about this actin network or the control of Fmn1. Here, we explore the role of the small GTPase RhoA in MCC apical emergence. Disruption of RhoA function reduced the rate of apical surface expansion and decreased the final size of the apical domain. Analysis of cell shapes suggests that RhoA alters the balance of forces exerted on the MCC apical surface. Finally, quantitative time-lapse imaging and fluorescence recovery after photobleaching studies argue that RhoA works in concert with Fmn1 to control assembly of the specialized apical actin network in MCCs. These data provide new molecular insights into epithelial apical surface assembly and could also shed light on mechanisms of apical lumen formation.

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Section: Rhoa Controls the Dynamics Of MCC Apical Emergencementioning

confidence: 99%

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells

Sedzinski¹,

Hannezo²,

Tu³

et al. 2017

Development

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“…Given all this information, interventions that impair CP function, such as CP knockdown or V-1 overexpression, would be predicted to reduce the formation of cortical actin structures built by the Arp2/3 complex (lamellipodia and pseudopodia) and promote the formation of cortical actin structures built by formins and VASP (filopodia). Of note, the increase in filopodia number seen upon CP knockdown/V-1 overexpression is probably also due in significant part to an increase in the amount of monomer available for formin/VASP after the reduction in Arp2/3-dependent nucleation (15)(16)(17).…”

Section: Discussionmentioning

confidence: 99%

“…Although both proteins are fairly effective at physically shielding the barbed end from CP (10,13,14), it is likely that their robustness as filopodia generators in vivo would be increased by a reduction in CP levels. Given the recent work demonstrating that formins and the Arp2/3 complex compete for G-actin in vivo (15)(16)(17), the increase in filopodia number seen upon CP knockdown may also be due in part to an increase in the amount of monomer available for formin/VASP after the reduction in Arp2/ 3-dependent nucleation caused by CP knockdown.The studies discussed above suggest that cells could regulate their "actin phenotype" by regulating their level of active CP. Consistent with CP regulation in vivo, estimates of the half-life of CP on the barbed end near the plasma membrane in living cells are approximately three orders of magnitude shorter than CP's half-life on the barbed in vitro (i.e., ∼2-15 s in cells vs. ∼30 min for pure proteins) (8, 18).…”

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confidence: 99%

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V-1 regulates capping protein activity in vivo

Jung

Alexander

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Capping Protein (CP) plays a central role in the creation of the Arp2/ 3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."T he addition of Capping Protein (CP) to seed-initiated actin polymerization assays results in the rapid cessation of polymerization because CP binds with very high affinity to the fastgrowing barbed end of the actin filament to block further monomer addition (1). Direct extrapolation of this simple, potent biochemical property would suggest that the cell's content of F-actin should rise and fall as its content of CP is artificially forced to fall and rise, respectively. Indeed, this finding was reported many years ago in Dictyostelium amoeba (2). This simple view of CP's role in regulating actin assembly in vivo falls short of the whole story, however. The additional complexity arises from the critical relationship between CP and the Arp2/3 complex, the major actin nucleating machine that generates the branched actin networks comprising lamellipodia and pseudopodia (3). At the heart of this relationship is the fact that CP increases the rate of Arp2/3-dependent filament nucleation and promotes optimal branching by rapidly capping filaments (4). As a result, CP promotes actin-related proteins 2 and 3 (Arp2/3)-driven actin assembly and motility (4, 5). This effect was evident from early solution experiments focused on defining the function of the Arp2/3 complex (6), verified by in vitro reconstitution of the Arp2/3-dependent motility of Listeria (5), and explained mecha...

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“…One possible mechanism for modulating F-actin levels upon loss of CYK-1 or INFT2 is suggested by studies in fission yeast, where loss of one actin-polymerizing factor may promote the activity of another indirectly by increasing the pool of available cytoplasmic actin (Burke et al, 2014). However, in yeast this mechanism seems non-specific, as loss of one actin-polymerizing factor leads to increased activity of several others (Burke et al, 2014).…”

Section: Model For How the Exc-5−cdc-42 Pathway Regulates A Formin Nementioning

confidence: 98%

A network of conserved formins, regulated by the guanine exchange factor EXC-5 and the GTPase CDC-42, modulates tubulogenesis in vivo

Shaye

Greenwald

2016

Development

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The C. elegans excretory cell (EC) is a powerful model for tubulogenesis, a conserved process that requires precise cytoskeletal regulation. EXC-6, an ortholog of the diseaseassociated formin INF2, coordinates cell outgrowth and lumen formation during EC tubulogenesis by regulating F-actin at the tip of the growing canal and the dynamics of basolateral microtubules. EXC-6 functions in parallel with EXC-5/FGD, a predicted activator of the Rho GTPase Cdc42. Here, we identify the parallel pathway: EXC-5 functions through CDC-42 to regulate two other formins: INFT-2, another INF2 ortholog, and CYK-1, the sole ortholog of the mammalian diaphanous (mDia) family of formins. We show that INFT-2 promotes F-actin accumulation in the EC, and that CYK-1 inhibits INFT-2 to regulate F-actin levels and EXC-6-promoted outgrowth. As INF2 and mDia physically interact and cross-regulate in cultured cells, our work indicates that a conserved EXC-5−CDC-42 pathway modulates this regulatory interaction and that it is functionally important in vivo during tubulogenesis.

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

Cited by 206 publications

References 40 publications

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells

V-1 regulates capping protein activity in vivo

A network of conserved formins, regulated by the guanine exchange factor EXC-5 and the GTPase CDC-42, modulates tubulogenesis in vivo

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