Initiation of actin polymerization in cells requires nucleation factors. Here we describe an actinbinding protein, leiomodin, which 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 3-fold 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, suggesting a role for leiomodin in the nucleation of tropomyosindecorated filaments in muscles.Actin binding proteins suppress the spontaneous nucleation of actin monomers into filaments, so cells use nucleation factors to initiate actin polymerization. In non-muscle cells, the bestcharacterized filament nucleators are Arp2/3 complex and formins (1). Less is known about the initiation of actin filaments in striated and smooth muscle cells, where specialized proteins may be used to assemble and remodel the tropomyosin-decorated filaments.We identified leiomodin (Lmod) as a potential filament nucleator in muscle cells because sequence analysis suggested that it contained at least three actin-binding sites and could possibly recruit three actin monomers to form a polymerization nucleus. Thus, the first ~340 residues of Lmod are ~40% identical to tropomodulin (Tmod) ( fig. S1), a protein that caps actin filament pointed ends (2,3). The N-terminal portion of Tmod is unstructured, except for three helical segments involved in binding tropomyosin (residues 24-35 and 126-135) and actin (residues 65-75) (4). This region of Tmod, caps the pointed end of actin filaments in a tropomyosin-dependent manner (5). Tmod has a second, tropomyosin-independent, actinbinding and capping site within the C-terminal region (residues 160-359) (5), consisting almost †To whom correspondence should be addressed. droberto@mail.med.upenn.edu. 5 Present address: Elan Pharmaceuticals,

Cells sustain high rates of actin filament elongation by maintaining a large pool of actin monomers above the critical concentration for polymerization. Profilin-actin complexes constitute the largest fraction of polymerization-competent actin monomers. Filament elongation factors such as Ena/VASP and formin catalyze the transition of profilin-actin from the cellular pool onto the barbed end of growing filaments. The molecular bases of this process are poorly understood. Here we present structural and energetic evidence for two consecutive steps of the elongation mechanism: the recruitment of profilin-actin by the last poly-Pro segment of vasodilator-stimulated phosphoprotein (VASP) and the binding of profilin-actin simultaneously to this poly-Pro and to the G-actin-binding (GAB) domain of VASP. The actin monomer bound at the GAB domain is proposed to be in position to join the barbed end of the growing filament concurrently with the release of profilin.

SignificanceMore than 80% of human proteins are N-terminal (Nt)–acetylated during translation. In contrast, actin, the most abundant protein in the cytoplasm of animal cells, is Nt-acetylated posttranslationally and following a unique multistep mechanism that has remained poorly characterized. Here, we describe the discovery of actin’s N-terminal acetyltransferase (NAT), NAA80. We further demonstrate that actin Nt-acetylation plays essential roles in filament assembly, cytoskeleton organization, and cell motility, resulting in a net increase in the ratio of monomeric to filamentous actin and fewer lamellipodia and filopodia. These effects converge to reduce cell hypermotility. This work establishes the role of Nt-acetylation for the most abundant cytoskeletal protein in animals and reveals a NAT acting posttranslationally and on a single dedicated substrate.

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four ␤-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in ␤-trefoil domains 1 and 3. The site in ␤-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in ␤-trefoil-3 is related by pseudo-2-fold symmetry to that in ␤-trefoil-1. The two sites are ϳ5 nm apart, resulting in a distance between actin filaments in the bundle of ϳ8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.

Actin-related protein (Arp) 2/3 complex nucleates branched actin networks that drive cell motility. It consists of seven proteins, including two actin-related subunits (Arp2 and Arp3). Two nucleation-promoting factors (NPFs) bind Arp2/3 complex during activation, but the order, specific interactions, and contribution of each NPF to activation are unresolved. Here, we report the cryo–electron microscopy structure of recombinantly expressed human Arp2/3 complex with two WASP family NPFs bound and address the mechanism of activation. A cross-linking assay that captures the transition of the Arps into the activated filament-like conformation shows that actin binding to NPFs favors this transition. Actin-NPF binding to Arp2 precedes binding to Arp3 and is sufficient to promote the filament-like conformation but not activation. Structure-guided mutagenesis of the NPF-binding sites reveals their distinct roles in activation and shows that, contrary to budding yeast Arp2/3 complex, NPF-mediated delivery of actin at the barbed end of both Arps is required for activation of human Arp2/3 complex.

How proteins sharing a common fold have evolved different functions is a fundamental question in biology. Tropomodulins (Tmods) are prototypical actin filament pointed-end-capping proteins, whereas their homologs, Leiomodins (Lmods), are powerful filament nucleators. We show that Tmods and Lmods do not compete biochemically, and display similar but distinct localization in sarcomeres. Changes along the polypeptide chains of Tmods and Lmods exquisitely adapt their functions for capping vs. nucleation. Tmods have alternating tropomyosin (TM)- and actin-binding sites (TMBS1, ABS1, TMBS2, ABS2). Lmods additionally contain a C-terminal extension featuring an actin-binding WH2 domain. Unexpectedly, the different activities of Tmods and Lmods do not arise from the Lmod-specific extension. Instead, nucleation by Lmods depends on two major adaptations – the loss of pointed-end-capping elements present in Tmods and the specialization of the highly conserved ABS2 for recruitment of two or more actin subunits. The WH2 domain plays only an auxiliary role in nucleation.

Myosin V is a double-headed molecular motor involved in organelle transport. Two distinctive features of this motor, processivity and the ability to take extended linear steps of Ϸ36 nm along the actin helical track, depend on its unusually long light chainbinding domain (LCBD). The LCBD of myosin V consists of six tandem IQ motifs, which constitute the binding sites for calmodulin (CaM) and CaM-like light chains. Here, we report the 2-Å resolution crystal structure of myosin light chain 1 (Mlc1p) bound to the IQ2-IQ3 fragment of Myo2p, a myosin V from Saccharomyces cerevisiae. This structure, combined with FRET distance measurements between probes in various CaM-IQ complexes, comparative sequence analysis, and the previously determined structures calmodulin ͉ IQ motif ͉ x-ray crystallography ͉ FRET M yosin V is a molecular motor involved in a range of organelle-transporting functions, including the transport of melanosomes and synaptic vesicles in mammals and vacuoles and mRNA in yeast (1-4). Myosin V is composed of two identical heavy chains and 12 light chains. Each heavy chain consists of an N-terminal motor domain, containing the actinbinding and ATP catalytic sites, followed by the light chainbinding domain (LCBD), formed by six IQ motifs in tandem, and the tail domain, composed of regions of coiled-coil and a globular domain involved in cargo binding. The coiled-coil regions mediate the association of the heavy chains into dimers. The IQ motifs are Ϸ25-aa segments, centered around the consensus sequence IQxxxRGxxxR, and constitute the binding sites for the light chains, which can be either calmodulin (CaM) or CaM-related molecules (5, 6).A number of features distinguish myosin V from other myosin families. Myosin V has a high duty cycle, defined as the property to remain attached to actin for a large fraction of the mechanochemical cycle (7-9). The high duty cycle of myosin V is explained by a slow rate of ADP release, which becomes the rate-limiting step in the ATPase cycle (7). This kinetic adaptation allows myosin V to take multiple steps without dissociating from the actin filament, that is, myosin V is a processive motor (10-12). Linked with processivity is the ability of myosin V to take large steps of Ϸ36 nm (10), a distance equal to the helical repeat of the actin filament. Such a step size allows myosin V to walk in a straight line on the actin filament, in a hand-over-hand fashion (13-16). These characteristics seem to adapt myosin V for its cellular function, the transport of large cargoes atop the actin filament while avoiding collisions with cellular structures (3). Yet, central to this motor's uniqueness is its unusually long LCBD (4). A number of laboratories have recently established a direct connection between the length and structural integrity of the LCBD and the step size and processivity of myosin V (4,(17)(18)(19)(20). These studies focus on the role of the LCBD as a passive structural device whose function is to amplify small nucleotidedependent motions originating in the motor doma...

Previous structures of Arp2/3 complex, determined in the absence of a nucleation-promoting factor and actin, reveal its inactive conformation. The study of the activated structure has been hampered by uncontrollable polymerization. We have engineered a stable activated complex consisting of Arp2/3 complex, the WCA activator region of N-WASP, and one actin monomer, and studied its structure in solution by small angle X-ray scattering (SAXS). The scattering data support a model in which the first actin subunit binds at the barbed end of Arp2, and disqualify an alternative model that places the first actin subunit at the barbed end of Arp3. This location of the first actin and bound W motif constrains the binding site of the C motif to subunits Arp2 and ARPC1, from where the A motif can reach subunits Arp3 and ARPC3. The results support a model of activation that is consistent with most of the biochemical observations.