Identification of Residues within Tropomodulin-1 Responsible for Its Localization at the Pointed Ends of the Actin Filaments in Cardiac Myocytes

Abstract

Tropomodulin is a tropomyosin-dependent actin filament capping protein involved in the structural formation of thin filaments and in the regulation of their lengths through its localization at the pointed ends of actin filaments. The disordered N-terminal domain of tropomodulin contains three functional sites: two tropomyosin-binding and one tropomyosin-dependent actin-capping sites. The C-terminal half of tropomodulin consists of one compact domain containing a tropomyosin-independent actin-capping site. Here we determined the structural properties of tropomodulin-1 that affect its roles in cardiomyocytes. To explore the significance of individual tropomyosin-binding sites, GFP-tropomodulin-1 with single mutations that destroy each tropomyosin-binding site was expressed in cardiomyocytes. We demonstrated that both sites are necessary for the optimal localization of tropomodulin-1 at thin filament pointed ends, with site 2 acting as the major determinant. To investigate the functional properties of the tropomodulin C-terminal domain, truncated versions of GFP-tropomodulin-1 were expressed in cardiomyocytes. We discovered that the leucine-rich repeat (LRR) fold and the Cterminal helix are required for its proper targeting to the pointed ends. To investigate the structural significance of the LRR fold, we generated three mutations within the C-terminal domain (V232D, F263D, and L313D). Our results show that these mutations affect both tropomyosin-independent actincapping activity and pointed end localization, most likely by changing local conformations of either loops or side chains of the surfaces involved in the interactions of the LRR domain.Studying the influence of these mutations individually, we concluded that, in addition to the tropomyosin-independent actin-capping site, there appears to be another regulatory site within the tropomodulin C-terminal domain.

Tropomodulins and Leiomodins: Actin Pointed End Caps and Nucleators in Muscles

“…Fifth, this model is consistent with the azimuthal sliding of TM on the surface of the actin filament (53,54), whereby TM can explore three structural states (blocked, closed, and open) without generating steric clashes with Tmod at the pointed end, albeit likely influenced by Tmod-TM isoform specific interactions (see below). Sixth, this model is consistent with the results of numerous mutagenesis and capping studies (5,31,38,44,55,56). One interesting feature of the model is that because of the relative structural independence and weak individual binding affinities of ABS1 and ABS2, they can be expected to bind and detach from the pointed end semiindependently, consistent with the observation of actin subunit exchange at the pointed end of the thin filaments in muscle sarcomeres (57,58).…”

Leiomodin 3 and Tropomodulin 4 have overlapping functions during skeletal myofibrillogenesis

“…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).…”

Alteration of Tropomyosin-binding Properties of Tropomodulin-1 Affects Its Capping Ability and Localization in Skeletal Myocytes

“…When residues Leu-27 and Ile-131, which were shown to be crucial for TM binding were mutated, the mutation I131D in site 2 caused an ϳ90% decrease in assembly with more than half of Tmod1 observed to be diffusely localized, whereas mutation L27E caused an ϳ20% decrease in assembly (18). Introduction of both mutations caused a dramatic loss of consistent assembly, with most staining appearing diffuse (e.g.…”

“…Isolated cells were plated in 35-mm tissue culture dishes containing 12-mm round glass coverslips coated with Matrigel (Fisher Scientific, Pittsburgh, PA) for staining (ϳ1 ϫ 10 6 cells/dish). Transfection was performed using Effectene (Qiagen, Valencia, CA) as described (18).…”

“…NM_21883) was subcloned into pEGFP-C1 (18). To insert Q144K, D12N/Q144K, and R11K/ D12N/Q144K mutations in GFP-Tmod1, the following oligonucleotides were used, respectively: 5Ј-CAC ACT CAT GAG CAA CAA GCA GTA CTA CCA AGC C-3Ј (mTmod1 Q144K); 5Ј-GAG AGC TGG AGA AAT ATC GAA ACC TGG ATG AGG ATG-3Ј (mTmod1 D12N); and 5Ј-CAG ACG AGA GCT GGA GAA ATA TAA AAA CCT GGA TGA GGA TG-3Ј (mTmod1 R11K).…”