Leiomodin Is an Actin Filament Nucleator in Muscle Cells

Abstract

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,

ArabidopsisFormin3 Directs the Formation of Actin Cables and Polarized Growth in Pollen Tubes

“…Within the cell, actin nucleation factors are responsible for the generation of actin nuclei, providing a mechanism for the cell to regulate when and where to assemble actin filaments. Several actin-nucleating factors are known to date (Welch et al, 1997;Pruyne et al, 2002;Quinlan et al, 2005;Chereau et al, 2008), among which the Arp2/3 complex and formins are two well-studied actin nucleators. The Arp2/3 complex initiates actin assembly from preexisting actin filaments to generate a branched network of actin filaments (Mullins et al, 1998), whereas formins induce polymerization of parallel actin filaments for the formation of actin bundles or cables.…”

Mechanisms of leiomodin 2-mediated regulation of actin filament in muscle cells

“…Recent studies have uncovered actin nucleation activities of the nebulin-N-WASP complex (9) and of formin proteins FHOD3 (10)(11)(12), mDia2, DAAM, FMNL1, and FMNL2 (13,14) in sarcomeres. In particular, leiomodin (Lmod) has been identified as a class of potent tandem-G-actin-binding nucleators in muscle cells (15,16); Lmod1 is found in smooth muscle of many human tissues, and Lmod2 and Lmod3 are found in cardiac and skeletal muscle (17). Lmod2 knockdown severely compromises sarcomere organization and assembly in muscle cells (15), whereas mutations, deletions (18), or instability (19) in Lmod3 underlies severe, often lethal, human nemaline myopathy.…”

X-ray scattering study of actin polymerization nuclei assembled by tandem W domains

“…Cells use filament nucleators to stabilize actin polymerization nuclei, whose formation is rate-limiting during actin assembly (1). In addition to Arp2/3 complex and formins (2), a number of filament nucleators have been recently discovered: Spire (3), Cobl (4), VopL (5), VopF (6), and Lmod (7). With the exception of formins, all filament nucleators use the W domain for interaction with actin.…”