Actin polymerization in cells occurs via filament elongation at the barbed end. Proteins that cap the barbed end terminate this elongation. Heterodimeric capping protein (CP) is an abundant and ubiquitous protein that caps the barbed end. We find that the mouse homolog of the adaptor protein CARMIL (mCARMIL) binds CP with high affinity and decreases its affinity for the barbed end. Addition of mCARMIL to cell extracts increases the rate and extent of Arp2/3 or spectrin-actin seed-induced polymerization. In cells, GFP-mCARMIL concentrates in lamellipodia and increases the fraction of cells with large lamellipodia. Decreasing mCARMIL levels by siRNA transfection lowers the F-actin level and slows cell migration through a mechanism that includes decreased lamellipodia protrusion. This phenotype is reversed by full-length mCARMIL but not mCARMIL lacking the domain that binds CP. Thus, mCARMIL is a key regulator of CP and has profound effects on cell behavior.
Formins, characterized by formin homology domains FH1 and FH2, are required to assemble certain F-actin structures including actin cables, stress fibers, and the contractile ring. FH1FH2 in a recombinant fragment from a yeast formin (Bni1p) nucleates actin filaments in vitro. It also binds to the filament barbed end where it appears to act as a "leaky" capper, slowing both polymerization and depolymerization by approximately 50%. We now find that FH1FH2 competes with tight capping proteins (including gelsolin and heterodimeric capping protein) for the barbed end. We also find that FH1FH2 forms a tetramer. The observation that this formin protects an end from capping but still allows elongation confirms that it is a leaky capper. This is significant because a nucleator that protects a new barbed end from tight cappers will increase the duration of elongation and thus the total amount of F-actin. The ability of FH1FH2 to dimerize probably allows the formin to walk processively with the barbed end as the filament elongates.
A fragment of the yeast formin Bni1 containing the FH1FH2 domains increases the rate of filament nucleation from pure G-actin [Pruyne et al. (2002) Science 297, 612-615]. To determine the mechanism of nucleation, we compared the G-actin dependence of Bni1FH1FH2-induced polymerization with theoretical models. The data best fit a model suggesting that Bni1FH1FH2 stabilizes an actin dimer. We also show that nucleation increases with the square root of the Bni1FH1FH2 concentration. We demonstrate that this relationship is expected for any such nucleator, independent of nucleus size. The proline-rich FH1 domain binds profilin, and deletion of this domain decreases the contribution of profilin-actin to the nucleation. A role for profilin binding to the FH1 domain in filament nucleation was supported by the inability of Bni1FH1FH2 to utilize a mutant profilin, H133S profilin, with defective binding to polyproline. Bni1FH1FH2 partially inhibits barbed-end elongation, and we find that the rate constants for both polymerization and depolymerization are decreased by approximately 50%. Bni1FH1FH2 has no effect on pointed-end kinetics or on the critical concentration. To investigate the domains of Bni1 required for these activities, the experiments were all duplicated with the FH2 domain alone. The FH2 domain is as effective as the FH1FH2 domains together in inhibiting barbed-end kinetics; it is less effective as a nucleator but the mechanism is again best fit by dimer stabilization.
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