Molecular Basis for Barbed End Uncapping by CARMIL Homology Domain 3 of Mouse CARMIL-1

Zwolak, Adam; Uruno, Takehito; Piszczek, Grzegorz; Hammer, John A.; Tjandra, Nico

doi:10.1074/jbc.m110.134221

Cited by 31 publications

(52 citation statements)

References 56 publications

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“…A subsequent allosteric change in CP caused by ternary complex formation then drives V-1 dissociation, yielding the CP:CAH3 complex. Importantly, the binding sites on the surface of CP for CAH3 and V-1 are nonoverlapping, consistent with the possibility that these three proteins form a ternary complex (17)(18)(19)(20)(21).…”

mentioning

confidence: 57%

“…In the last step (step 4), 2 μM monomeric actin [containing 20% tetramethylrhodamine-5-maleimide (TMR)-labeled and 0.5% biotin-labeled actin], together with variable concentrations of CP (0-1 μM final) and a constant concentration of CAH3 (10 μM final), were added. Given CAH3's strong affinity for CP (1-3 nM) (17,19,25), this very high concentration of CAH3 should essentially preclude inhibition of actin polymerization by free CP. Fig.…”

Section: Cp:cah3 Complex Regulates the Rate Of Barbed-end Elongation mentioning

confidence: 99%

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Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges

Fujiwara¹,

Remmert²,

Piszczek

et al. 2014

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

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Although capping protein (CP) terminates actin filament elongation, it promotes Arp2/3-dependent actin network assembly and accelerates actin-based motility both in vitro and in vivo. In vitro, capping protein Arp2/3 myosin I linker (CARMIL) antagonizes CP by reducing its affinity for the barbed end and by uncapping CPcapped filaments, whereas the protein V-1/myotrophin sequesters CP in an inactive complex. Previous work showed that CARMIL can readily retrieve CP from the CP:V-1 complex, thereby converting inactive CP into a version with moderate affinity for the barbed end. Here we further clarify the mechanism of this exchange reaction, and we demonstrate that the CP:CARMIL complex created by complex exchange slows the rate of barbed-end elongation by rapidly associating with, and dissociating from, the barbed end. Importantly, the cellular concentrations of V-1 and CP determined here argue that most CP is sequestered by V-1 at steady state in vivo. Finally, we show that CARMIL is recruited to the plasma membrane and only at cell edges undergoing active protrusion. Assuming that CARMIL is active only at this location, our data argue that a large pool of freely diffusing, inactive CP (CP:V-1) feeds, via CARMIL-driven complex exchange, the formation of weakcapping complexes (CP:CARMIL) at the plasma membrane of protruding edges. In vivo, therefore, CARMIL should promote Arp2/3-dependent actin network assembly at the leading edge by promoting barbed-end capping there.cell migration | VASP

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confidence: 57%

Section: Cp:cah3 Complex Regulates the Rate Of Barbed-end Elongation mentioning

confidence: 99%

Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges

Fujiwara¹,

Remmert²,

Piszczek

et al. 2014

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

Self Cite

121

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“…However, substantial portions of the CARMIL fragment were not resolved in the structure, raising the possibility that these unresolved regions might be in contact with one of the several actin-binding surfaces of CP. In addition, a separate structural study, utilizing NMR, concluded that certain residues of CARMIL were in close contact with residues of CP known to contact actin (36).…”

Section: Discussionmentioning

confidence: 99%

Mechanism for CARMIL Protein Inhibition of Heterodimeric Actin-capping Protein

Kim

Ravilious

Sept

et al. 2012

Journal of Biological Chemistry

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“…conformation that binds the barbed end weakly (23)(24)(25). Recent work suggests that CARMIL proteins function in vivo at the plasma membrane:cytoplasm interface of protruding edges, where they appear to be recruited, unfolded, and activated to promote actin assembly (26).…”

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confidence: 99%

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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Molecular Basis for Barbed End Uncapping by CARMIL Homology Domain 3 of Mouse CARMIL-1

Cited by 31 publications

References 56 publications

Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges

Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges

Mechanism for CARMIL Protein Inhibition of Heterodimeric Actin-capping Protein

V-1 regulates capping protein activity in vivo

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