Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells

Abstract

Tropomyosin (Tm) is known to be an important gatekeeper of actin function. Tm isoforms are encoded by four genes, and each gene produces several variants by alternative splicing, which have been proposed to play roles in motility, proliferation, and apoptosis. Smooth muscle studies have focused on gizzard smooth muscle, where a heterodimer of Tm from the α-gene (Tmsm-α) and from the β-gene (Tmsm-β) is associated with contractile filaments. In this study we examined Tm in differentiated mammalian vascular smooth muscle (dVSM). Liquid chromatography-tandem mass spectrometry (LC MS/MS) analysis and Western blot screening with variant-specific antibodies revealed that at least five different Tm proteins are expressed in this tissue: Tm6 (Tmsm-α) and Tm2 from the α-gene, Tm1 (Tmsm-β) from the β-gene, Tm5NM1 from the γ-gene, and Tm4 from the δ-gene. Tm6 is by far most abundant in dVSM followed by Tm1, Tm2, Tm5NM1, and Tm4. Coimmunoprecipitation and coimmunofluorescence studies demonstrate that Tm1 and Tm6 coassociate with different actin isoforms and display different intracellular localizations. Using an antibody specific for cytoplasmic γ-actin, we report here the presence of a γ-actin cortical cytoskeleton in dVSM cells. Tm1 colocalizes with cortical cytoplasmic γ-actin and coprecipitates with γ-actin. Tm6, on the other hand, is located on contractile bundles. These data indicate that Tm1 and Tm6 do not form a classical heterodimer in dVSM but rather describe different functional cellular compartments

Localization and function of Xinα in mouse skeletal muscle

“…Other antibodies used were mouse monoclonal antibody (mAb) CG␤6 recognizing smooth muscle and nonmuscle tropomyosins (16,34) and anti-neurofilament 200 mAb N52 (Sigma, St Louis, MO). Alexa Fluor 488-conjugated ␣-bungarotoxin (B-13422) was purchased from Molecular Probes (Eugene, OR).…”

“…After preincubation with protein GSepharose beads (GE Healthcare, Piscataway, NJ) for 30 min at 4°C, the homogenate (150 g of total protein/immunoprecipitation) was cleared by centrifugation at 12,000 g for 15 min. The precleaned homogenate was incubated with anti-tropomyosin mAb LC24 (16,35,47,63), anti-filamin mAb FLMN01 (Abcam, Cambridge, MA), anti-vinculin mAb hVIN-1 (Sigma), anti-talin mAb 8D4 (Sigma), anti-␤-catenin mAb 15B8 (Sigma), or normal mouse serum control at 4°C overnight. Incubated again with protein G-Sepharose beads at 4°C for 1 h, the beads were collected by centrifugation and washed three times with immunoprecipitation buffer and one time with PBS.…”

Tropomyosin isoforms and reagents

“…These include LC1 and WSα/9c, 16 anti-sheep α/2a and CG1 72 and TM311. 73 In summary, one of the major limitations in evaluating the Tm isoform Tm4 recombinant proteins (Fig.…”

Cytoskeletal Reorganization Evoked by Rho-associated kinase- and Protein Kinase C-catalyzed Phosphorylation of Cofilin and Heat Shock Protein 27, Respectively, Contributes to Myogenic Constriction of Rat Cerebral Arteries

“…This is postulated to strengthen the cytoskeleton and enhance force transmission from the contractile apparatus to the cell membrane and extracellular matrix (24,25). Actin dynamics evoked by exposure to vasoconstrictor agonists involves a spatially restricted pool of nonmuscle ␥-and ␤-actin within the cell cortex and at focal adhesions and dense bodies, but not smooth muscle ␣-actin that interacts with tropomyosin and forms the contractile actin filaments within the core of VSMCs (40,41). Roles for additional cytoskeletal and membrane adhesion proteins in smooth muscle contraction have also been identified, including proteins such as talin that serve a structural role, and others that facilitate actin dynamics, such as the actin nucleation initiating factor, N-WASp (neuronal Wiskott-Aldrich Syndrome protein), cofilin, HSP20, HSP27, and VASP (vasodilator-stimulated phosphoprotein) (42)(43)(44)(45)(46)(47).…”