Cells focus robust actin assembly at the interface between the plasma membrane and the cytoplasm and suppress assembly throughout the rest of the cytoplasm. Actin polymerization is focused at the plasma membrane: cytosol interface by the site-specific recruitment/activation at this interface of the major protein complexes that promote nucleation (i.e. Arp2/3, formins). Actin polymerization at this interface is also supported by the site-specific recruitment/activation of factors (e.g. VASP, formins) that promote filament elongation by antagonizing the action of the major cellular barbed end capper, Capping Protein (CP). Another leading edge elongation factor may be CARMIL, as it exhibits two robust anti-CP activities: weak barbed end capping by the CARMIL: CP complex and the ability to uncap CP-capped filaments. Finally, the cell may also use proteins that globally sequester CP in an inactive form to promote leading edge polymerization. One such sequestering protein may be V-1/myotrophin, an abundant (cellular concentration 8 M, versus 1 uM for CP) protein that binds CP 1:1 with an affinity of about 20 nM, resulting in a complex that has no affinity for the barbed end. Here we have explored at the biochemical level the mechanism of an exchange reaction that drives the transfer of CP from its sequestered state (CP: V-1) to its weak barbed end binding state (CP: CARMIL). These efforts yielded direct evidence of a CP: CARMIL: V-1 ternary complex, as well as an estimate of the relative contributions of this ternary complex pathway and a competitive binding pathway to the overall exchange reaction. Using both bulk actin polymerization assays and single filament imaging, we then provide evidence for the conversion of CP from its sequestered state to its weak barbed end binding state following the addition of CARMIL to assays containing the CP: V-1 complex. Finally, we show that CARMIL is recruited to the plasma membrane: cytosol interface in vivo and only during periods of active polymerization/edge advance. Together, these results suggest that a CARMIL-driven exchange reaction funnels inactive CP sequestered in freely-diffusing, CP: V-1 complexes into weak barbed end capping CP: CARMIL complexes specifically at advancing edges. This mechanism, together with VASP, which shields the barbed end from CP at advancing edges, may serve to enhance the growth of newly-nucleated actin filaments in a narrow zone at the plasma membrane: cytosol interface.
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