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

Gallant C, Appel S, Graceffa P, Leavis P, Lin JJ, Gunning PW, Schevzov G, Chaponnier C, DeGnore J, Lehman W, Morgan KG. Tropomyosin variants describe distinct functional subcellular domains in differentiated vascular smooth muscle cells. Am J Physiol Cell Physiol 300: C1356-C1365, 2011. First published February 2, 2011; doi: 10.1152/ajpcell.00450.2010.-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 alpha-gene (Tmsm-alpha) and from the beta-gene (Tmsm-beta) 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-alpha) and Tm2 from the alpha-gene, Tm1 (Tmsm-beta) from the beta-gene, Tm5NM1 from the gamma-gene, and Tm4 from the delta-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 gamma-actin, we report here the presence of a gamma-actin cortical cytoskeleton in dVSM cells. Tm1 colocalizes with cortical cytoplasmic gamma-actin and coprecipitates with gamma-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.