Tropomodulin Binds Two Tropomyosins:  A Novel Model for Actin Filament Capping

Abstract

Tropomodulin, a tropomyosin-binding protein, caps the slow-growing (pointed) end of the actin filament regulating its dynamics. Tropomodulin, therefore, is important for determining cell morphology, cell movement, and muscle contraction. For the first time we show that one tropomodulin molecule simultaneously binds two tropomyosin molecules in a cooperative manner. On the basis of the tropomodulin solution structure and predicted secondary structure, we introduced a series of point mutations in regions important for tropomyosin-binding and actin-capping. Capping activity of these mutants was assayed by measuring actin polymerization using pyrene fluorescence. Using direct methods (circular dichroism and native gel-electrophoresis) for detecting tropomodulin/ tropomyosin binding, we localized the second tropomyosin-binding site to residues 109-144. Despite previous reports that the second binding site is for erythrocyte tropomyosin only, we found that both short non-muscle and long muscle α-tropomyosins bind there as well, though with different affinities. We propose a model for actin capping where one tropomodulin molecule can bind to two tropomyosin molecules at the pointed end. Keywordstropomodulin; tropomyosin; actin filament; pointed end Actin is a major component of the cell cytoskeleton and the sarcomeres of striated muscle, and plays a critical role in cell morphology, cell movement, and muscle contraction (1,2). It is known that actin filaments form vastly different structures in different cell types and in different locations within the cell. Regulation of the dynamics at actin's ends is of central importance in the assembly of these various structures. An actin filament has two distinct ends: a fastgrowing barbed end and a slow-growing pointed end (3). The exclusive properties of these ends are exploited by over 160 distinct actin binding proteins, including those that cap, sever, or cross-link filaments (4). Of particular interest is tropomodulin (Tmod), a tropomyosinbinding protein, which regulates actin filament dynamics by capping the pointed end.Tropomyosin (TM) is a two-chained coiled coil that binds head-to-tail (N to C terminus) along the length of both sides of actin filaments, spanning 6-7 actin monomers per TM molecule, depending on the isoform (5). There are a variety of TM isoforms encoded by different genes. The N-terminus of TM, which points towards the pointed end of the actin filament (6), is required for interaction with Tmod (7-9). Best known for its role in muscle contraction (10, 11), TM also regulates the stiffness of actin (12) and influences polymerization at the pointed To whom correspondence should be addressed: Alla S. Kostyukova, end (13,14). Tmod in combination with TM forms a cap at the pointed end, preventing addition or loss of G-actin (15-17).Tropomodulin, originally discovered as a TM-binding protein in erythrocyte membranes (18), is the only known pointed end specific capping protein (15). Presently, there are four known Tmod isoforms (16,19,20). These isofor...

Tropomodulin isoforms regulate thin filament pointed-end capping and skeletal muscle physiology

“…Our blot overlay data appear to contradict previous blot overlay and other binding experiments showing that residues 1-130 of chicken Tmod4 bind more strongly to N-terminal  fast -TM peptides than do residues 1-130 of chicken Tmod1 (Greenfield and Fowler, 2002). However, the truncated Tmods may be missing a portion of an additional TM-binding site (Kostyukova et al, 2006), thereby affecting the overall avidity of the Tmod-TM interaction. This confounding variable prevents side by side comparison of the results in these two studies.…”

“…Based on studies in cardiac muscle, changes in overall levels of Tmods via relative isoform expression could affect the extent of pointed-end capping, thus regulating thin filament lengths (for review see Littlefield and Fowler, 2008). Furthermore, at least Tmod1 and -4 have differential binding to TMs (Greenfield and Fowler, 2002;Kostyukova et al, 2006Kostyukova et al, , 2007. Therefore, we hypothesized that a combination of Tmod isoform expression and avidity for TMs regulates thin filament length in skeletal muscles.…”

“…Thin filaments are extremely uniform in length but dynamic at their ends, where new actin monomer can be incorporated by subunit exchange (Littlefield et al, 2001). Thin filament pointed ends are capped by tropomodulins (Tmods), which interact with tropomyosins (TMs) and cap TM-decorated actin ends more tightly than bare actin ends (Weber et al, 1994(Weber et al, , 1999Kostyukova et al, 2005Kostyukova et al, , 2006. Tmod-mediated pointed-end dynamics are a key regulator of thin filament length in mammalian cardiac muscle and invertebrate striated muscles (for review see Littlefield and Fowler, 2008), but Tmod's roles in thin filament length regulation in skeletal muscle and how thin filament length regulation influences in vivo skeletal muscle physiology remain unknown.…”

Leiomodin 3 and Tropomodulin 4 have overlapping functions during skeletal myofibrillogenesis

“…Although our observations suggest that Tmod4 and Lmod3 localize simultaneously at the actin thin filament pointed ends, it is still not clear whether they localize to the same or different thin filaments. According to a model proposed by Kostyukova and colleagues, Tmod caps thin filament ends by interacting with two tropomyosin molecules through its two tropomyosin binding sites (Kostyukova et al, 2006). To date, no reports have been published on the association of Lmod with actin filament pointed ends, although it has been reported that Lmod-tropomyosin and Tmod-tropomyosin interactions are isoform specific (Kostyukova et al, 2006;Kostyukova, 2007;Kostyukova, 2008).…”

“…In striated muscle, in cooperation with tropomyosin, Tmod1 binds to and limits the exchange of actin monomers at the pointed end of the actin thin filaments (Weber et al, 1994). The localization and capping functions of Tmod1 appear to be dependent on two actin-binding and two tropomyosin-binding domains, given that mutations in any of these regions affect these functions Kostyukova et al, 2005;Kostyukova et al, 2006;Greenfield et al, 2005;Kong and Kedes, 2006;Tsukada et al, 2011). Overexpression of Tmod1 in primary myocyte cultures results in a shortening of actin filaments (Sussman et al, 1999;Littlefield et al, 2001), whereas depletion of Tmod1 leads to abnormally long thin filaments (Gregorio et al, 1995;Sussman et al, 1998;Mudry et al, 2003).…”