Mammalian myotrophin/V-1 is a 13 kDa, ankyrin-repeat protein that binds Capping Protein (CP) in vitro with an affinity of 20 nM, creating a 1:1 complex (CP: V-1) that has no affinity for the barbed end. V-1 has the potential, therefore, to influence actin polymerization in vivo by reducing the extent of barbed end capping. Dictyostelium discoideum (D.d.) contains a single gene encoding a 13.2 kDa protein with high sequence similarity to mouse V-1. Like mouse V-1, Dd V-1 binds CP tightly, is present in cells at a three-fold molar excess over CP, and no longer binds CP when a function-blocking point mutation (FBM) is introduced into the first ankyrin loop (FBM Dd V-1). Consistent with V-1s ability to sequester CP, over-expression of Dd V-1 results in an elevation in total cellular F-actin content that scales positively with the degree of over-expression. Importantly, over-expression of FBM Dd V-1 does not alter cellular F-actin levels, arguing that the effect of V-1 over-expression is due to its ability to sequester CP. The over-expression of Dd V-1, but not FBM-Dd V-1, also induces the formation of actin-rich, filopodial-like structures that scales positively with the degree of over-expression. In contrast to over-expression, Dd V-1 null cells created by homologous recombination exhibit a large decrease in cellular F-actin content. Moreover, these cells exhibit significant decreases in growth rate, macropinocytosis rate, chemotactic streaming efficiency, polarity during migration, and random motility rate. Importantly, these defects are rescued by wild type V-1 but not by FBM Dd V-1. Together, these results argue that V-1 plays a major role in regulating actin assembly in cells. Moreover, the fact that FBM V-1 neither induces over expression phenotypes nor rescues null cell phenotypes argues that V-1 exerts its effects on the actin cytoskeleton by buffering cellular CP. We conclude, therefore, that reductions and elevations in cellular CP activity caused by the over-expression and knock out of V-1, respectively, result in changes in cellular actin content that are consistent with CPs barbed end capping activity. Moreover, the functional consequences of these changes, which involve alterations in numerous cellular activities that dependent on actin assembly, indicate that V-1 is physiologically important. While 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, CARMIL antagonizes CP by reducing its affinity for the barbed end and by uncapping CP-capped filaments, while 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 weak capping 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.

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U.S. National Heart Lung and Blood Inst
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Fujiwara, Ikuko; Remmert, Kirsten; Piszczek, Grzegorz et al. (2014) Capping protein regulatory cycle driven by CARMIL and V-1 may promote actin network assembly at protruding edges. Proc Natl Acad Sci U S A 111:E1970-9
Zwolak, Adam; Uruno, Takehito; Piszczek, Grzegorz et al. (2010) Molecular basis for barbed end uncapping by CARMIL homology domain 3 of mouse CARMIL-1. J Biol Chem 285:29014-26
Zwolak, Adam; Fujiwara, Ikuko; Hammer 3rd, John A et al. (2010) Structural basis for capping protein sequestration by myotrophin (V-1). J Biol Chem 285:25767-81
Fujiwara, Ikuko; Remmert, Kirsten; Hammer 3rd, John A (2010) Direct observation of the uncapping of capping protein-capped actin filaments by CARMIL homology domain 3. J Biol Chem 285:2707-20
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