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

Abstract

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

V-1 regulates capping protein activity in vivo

“…In total, our data show that V-1 plays a major role in the regulation of CP activity in vivo, thereby influencing the organization, dynamics, and function of cortical actin structures that drive many fundamental cellular processes. Moreover, our results provide additional support for the novel model of CP regulation in vivo proposed recently by Fujiwara et al (26).…”

“…We recently proposed a model for CP regulation in vivo in which V-1 globally inhibits CP in the cytoplasm, and active, plasma membrane-associated CARMIL at protruding cell edges drives a complex exchange reaction converting sequestered CP (CP:V-1) into a version that binds the barbed end with ∼50 nM affinity (CP: CARMIL) (26). In this model, therefore, the barbed end capping activity required to drive Arp2/3-dependent branched actin network assembly is only generated downstream of the CARMILdriven complex exchange reaction that liberates CP from V-1.…”

“…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).…”

“…Like CARMIL, V-1 is expressed throughout most of the Eukaryotic kingdom. Recent work in mouse embryo fibroblasts has indicated that V-1, like CP, is freely diffusing in the cytoplasm and that it is present in molar excess over CP (26). These observations argue that the majority of cellular CP may in fact be sequestered by V-1 at steady state, barring regulation.…”

“…Recent work by Fujiwara et al seeking to define the functional interplay between CARMIL and V-1 has yielded a novel model for how barbed end capping may be regulated in cells (26). This model postulates that V-1 globally inhibits CP in the cytoplasm and that active, plasma membrane-associated CARMIL at protruding cell edges drives a complex exchange reaction converting sequestered CP (CP:V-1) into a version that binds the barbed end with ∼50 nM affinity (CP:CARMIL).…”