Initiation of actin polymerization in cells requires nucleation factors. Here we describe an actin-binding protein, leiomodin, that acted as a strong filament nucleator in muscle cells. Leiomodin shared two actin-binding sites with the filament pointed end–capping protein tropomodulin: a flexible N-terminal region and a leucine-rich repeat domain. Leiomodin also contained a C-terminal extension of 150 residues. The smallest fragment with strong nucleation activity included the leucine-rich repeat and C-terminal extension. The N-terminal region enhanced the nucleation activity threefold and recruited tropomyosin, which weakly stimulated nucleation and mediated localization of leiomodin to the middle of muscle sarcomeres. Knocking down leiomodin severely compromised sarcomere assembly in cultured muscle cells, which suggests a role for leiomodin in the nucleation of tropomyosin-decorated filaments in muscles.

We used fluorescence microscopy to determine how polymerization of Mg-ADP-actin depends on the concentration of phosphate. From the dependence of the elongation rate on the actin concentration and direct observations of depolymerizing filaments, we measured the polymerization rate constants of ADP-actin and ADP-P _i -actin. Saturating phosphate reduces the critical concentration for polymerization of Mg-ADP-actin from 1.8 to 0.06 μM almost entirely by reducing the dissociation rate constants at both ends. Saturating phosphate increases the barbed end association rate constant of Mg-ADP-actin 15%, but this value is still threefold less than that of ATP-actin. Thus, ATP hydrolysis without phosphate dissociation must change the conformation of polymerized actin. Analysis of depolymerization experiments in the presence of phosphate suggests that phosphate dissociation near the terminal subunits is much faster than in the interior. Remarkably, 10 times more phosphate is required to slow the depolymerization of the pointed end than the barbed end, suggesting a weak affinity of phosphate near the pointed end. Our observations of single actin filaments provide clues about the origins of the difference in the critical concentration at the two ends of actin filaments in the presence of ATP.

Significance: Assembly of actin filaments near the plasma membrane drives extension of the cell edge as it migrates. Cells recruit a protein machine called the Arp2/3 complex to the leading edge to initiate actin filament branches that grow and push on the membrane. Equally important, capping protein (CP) binds to and terminates polymerization of the fast-growing end of the filament. Like other aspects of actin assembly, CP is subject to regulation. Specifically, the protein V-1/myotrophin inactivates CP, while capping protein Arp2/3 myosin I linker (CARMIL) proteins moderately inhibit CP. We describe how CARMIL and V-1 cooperate in a cycle of reactions to regulate CP and promote assembly of the actin network by the Arp2/3 complex.