The specific functions of greater than 40 vertebrate nonmuscle tropomyosins (Tms) are poorly understood. In this article we have tested the ability of two Tm isoforms, TmBr3 and the human homologue of Tm5 (hTM5 NM1 ), to regulate actin filament function. We found that these Tms can differentially alter actin filament organization, cell size, and shape. hTm5 NM1 was able to recruit myosin II into stress fibers, which resulted in decreased lamellipodia and cellular migration. In contrast, TmBr3 transfection induced lamellipodial formation, increased cellular migration, and reduced stress fibers. Based on coimmunoprecipitation and colocalization studies, TmBr3 appeared to be associated with actin-depolymerizing factor/cofilin (ADF)-bound actin filaments. Additionally, the Tms can specifically regulate the incorporation of other Tms into actin filaments, suggesting that selective dimerization may also be involved in the control of actin filament organization. We conclude that Tm isoforms can be used to specify the functional properties and molecular composition of actin filaments and that spatial segregation of isoforms may lead to localized specialization of actin filament function. INTRODUCTIONThe actin microfilament network is a primary cytoskeletal system involved in the development and maintenance of morphology within cells. The dynamic nature of the actinbased system and its organization is thought to regulate specific structural changes within different cellular regions (Gunning et al., 1998b). The function and form of the actin cytoskeleton is largely determined by actin-binding proteins that are associated with the polymeric structure. Tropomyosins (Tms), along with actin, are integral components of the microfilament cytoskeleton, although not all actin filaments have Tms bound to them (Bamburg, 1999). Tms bind largely by electrostatic charge to the helical groove of the actin filament and the Ͼ40 isoforms are obtained by alternative splicing from four genes, of which almost all are nonmuscle variants (Lees-Miller et al., 1990;Goodwin et al., 1991;Beisel and Kennedy, 1994;Dufour et al., 1998;Cooley and Bergtrom, 2001). Although a considerable amount of information exists as to the biochemical regulation of microfilament dynamics, little is known about the function of this large family of proteins in vertebrate nonmuscle cells.In vitro studies have shown that nonmuscle Tms are able to differentially protect actin from the severing action of gelsolin (Ishikawa et al., 1989) and can regulate the MgATPase activity of myosins to varying degrees (Fanning et al., 1994). The different binding strengths to actin are thought to impart a range of stability to the filaments (Matsumura and Yamashiro-Matsumura, 1985;Hitchcock-DeGregori et al., 1988;Pittenger et al., 1995). The impact of Tms on vertebrate cell morphology is poorly understood even though studies suggest the importance of Tm isoforms in regulating Article published online ahead of print. Mol. Biol. Cell 10.1091/ mbc.E02-04 -0244. Article and publication dat...

The intercalated disk protein Xin was originally discovered in chicken striated muscle and implicated in cardiac morphogenesis. In the mouse, there are two homologous genes, mXinα and mXinβ. The human homolog of mXinα, Cmya1, maps to chromosomal region 3p21.2-21.3, near a dilated cardiomyopathy with conduction defect-2 locus. Here we report that mXinα-null mouse hearts are hypertrophied and exhibit fibrosis, indicative of cardiomyopathy. A significant upregulation of mXinβ likely provides partial compensation and accounts for the viability of the mXinα-null mice. Ultrastructural studies of mXinα-null mouse hearts reveal intercalated disk disruption and myofilament disarray. In mXinα-null mice, there is a significant decrease in the expression level of p120-catenin, β-catenin, N-cadherin, and desmoplakin, which could compromise the integrity of the intercalated disks and functionally weaken adhesion, leading to cardiac defects. Additionally, altered localization and decreased expression of connexin 43 are observed in the mXinα-null mouse heart, which, together with previously observed abnormal electrophysiological properties of mXinα-deficient mouse ventricular myocytes, could potentially lead to conduction defects. Indeed, ECG recordings on isolated, perfused hearts (Langendorff preparations) show a significantly prolonged QT interval in mXinα-deficient hearts. Thus mXinα functions in regulating the hypertrophic response and maintaining the structural integrity of the intercalated disk in normal mice, likely through its association with adherens junctional components and actin cytoskeleton. The mXinα-knockout mouse line provides a novel model of cardiac hypertrophy and cardiomyopathy with conduction defects. KeywordsXin repeat proteins; N-cadherin; β-catenin; p120-catenin; connexin 43The intercalated disk contains adherens junctions, desmosomes, and gap junctions that maintain the integrity of the association between cardiomyocytes and enable the myocardium Address for reprint requests and other correspondence: J. J.-C. Lin, Dept. of Biological Sciences, Univ. of Iowa, 340 Biology Bldg. East, 210 E. Iowa Ave., Iowa City, IA 52242 (e-mail: jim-lin@uiowa.edu).. NIH Public Access Author ManuscriptAm J Physiol Heart Circ Physiol. Author manuscript; available in PMC 2008 November 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript to function in synchrony. The expression and distribution of many of these junctional components are often altered in many types of heart disease (5,8,13,35). However, direct evidence to support a role for these proteins in contributing to cardiomyopathies remains incomplete. The best-characterized example involves the effects of deletion of a key adherens junction component, N-cadherin, on the intercalated disk. N-cadherin functions to mediate Ca 2+ -dependent homophilic cell-cell adhesion. Conditional deletion of N-cadherin in the adult mouse heart leads to a complete dissolution of the intercalated disk structure and a significant decrease in the express...

Calponin is an extensively studied actin-binding protein, but its function is not well understood. Among three isoforms of calponin, h2-calponin is found in both smooth muscle and non-muscle cells. The present study demonstrates that epidermal keratinocytes and fibroblast cells express significant amounts of h2-calponin. The expression of h2-calponin is cell anchorage-dependent. The levels of h2-calponin decrease when cells are rounded up and remain low when cells are prevented from adherence to a culture dish. h2-calponin expression resumes after the floating cells are allowed to form a monolayer in plastic dish. Cell cultures on polyacrylamide gels of different stiffness demonstrated that h2-calponin expression is affected by the mechanical properties of the culture matrix. When cells are cultured on soft gel that applies less traction force to the cell and, therefore, lower mechanical tension in the cytoskeleton, the level of h2-calponin is significantly lower than that in cells cultured on hard gel or rigid plastic dish. Force-expression of h2-calponin enhanced the resistance of the actin filaments to cytochalasin B treatment. Keratinocyte differentiation is accompanied by a mechanical tension-related up-regulation of h2-calponin. Lowering the tension of actin cytoskeleton by inhibiting non-muscle myosin II ATPase decreased h2-calponin expression. In contrast to the mechanical tension regulation of endogenous h2-calponin, the expression of h2-calponin using a cytomegalovirus promotor was independent of the stiffness of culture matrix. The results suggest that h2-calponin represents a novel manifestation of mechanical tension responsive gene regulation that may modify cytoskeleton function.Calponin is a family of actin-associated proteins first found in smooth muscle cells (1). Three calponin isoforms (h1-, h2-, and acidic calponins) are encoded by three homologous genes. h1-calponin (2, 3) is specifically expressed in differentiated smooth muscle cells and has been extensively studied for its role in the regulation of smooth muscle contractility (for review, see Refs. 4 -6). The acidic calponin is found in nervous tissues and implicates in neuronal regeneration and growth (7-8).h2-calponin (3) is found in both smooth muscle and non-muscle cells such as human epidermal keratinocytes (9). h2-calponin mRNA has been also detected in endothelial cells (10) and fibroblasts (11). The gene regulation and function of h2-calponin are largely unknown. Previous studies suggested that h2-calponin may play a role in the organization of actin cytoskeleton (12) and in cytokinesis (13). This hypothesis is supported by the observation that h2-calponin is expressed at significant levels in growing and remodeling tissues (13). Forced expression of h2-calponin in cells lacking endogenous calponin results in an association with the actin stress fibers and a decrease in the rate of cell proliferation (13). These results suggest a microfilament-associated activity of h2-calponin, which may regulate the function of actin cytoskeleto...

No abstract

Eight mouse monoclonal antibodies, CH1, CH106, CH291, CL2, CG1, CG3, CG beta 2 and CG beta 6, against chicken tropomyosin isoforms have been prepared and characterized. The antigens recognized by these isoform-specific monoclonal antibodies were identified by both solid-phase radioimmunoassay and protein immunoblotting. To some extent, most antibodies showed isoform-specific, but one (CG3) recognized all isoforms of tropomyosin from chicken materials. The effects of monoclonal antibodies on the binding of cardiac tropomyosin to F-actin were investigated. Antibodies CH1, CH106, and CH291 had the ability to interfere with the binding of tropomyosin to F-actin, whereas others appeared to have no effect. Monoclonal antibody CL2 was able to distinguish the skeletal muscle tropomyosin-enriched microfilaments from the fibroblastic tropomyosin-enriched microfilaments of differentiating muscle cells. This antibody will be most useful for studying the compartmentalization of microfilaments and microfilament-associated proteins, particularly actin and tropomyosin isoforms during muscle differentiation. Immunofluorescence microscopy with CG1 antibody which recognized CEF tropomyosin isoforms 1 and 3 revealed the continuous staining of stress fibers in some populations of CEF cells. On the other hand, both periodic fluorescent staining and continuous staining of stress fibers were observed with CG3 antibody in all CEF cells.