Tropomyosin

Tropomyosins

Page updated 27/6/03

First discovered by Bailey (Bailey, 1948), the tropomyosins are a group of proteins that bind to the sides of actin filaments and (together with troponins) regulate the interaction of the filaments with myosin in response to Ca²⁺. They are parallel, a-helical, coiled coil dimers. Vertebrates typically express in the region of 20 different tropomyosins from alternative spicing from about four tropomyosin genes (Perry, 2001 ). Tropomyosins exist as both homo-dimers and hetero-dimers held together by a series of salt bridges and hydrophobic interactions. They may also interact through their N and C termini in solution (especially in low ionic strength buffers) and this can be monitored by viscosity. There are two major groups of tropomyosin the high and low molecular weight tropomyosins. The former type exist primarily in muscle tissues (cardiac, smooth and skeletal) whereas non-muscle tissues express both high and low mol.wt. tropomyosins.

Actin-Binding Properties.

Tropomyosins bind cooperatively to actin filaments (Wegner, 1979; Yang et al, 1979; Zot & Potter, 1987) probably because there is end-to-end binding of the tropomyosin molecules but possibly also because tropomyosin restricts the movement of the filament causing the filament repeat distance to be made more regular (Stokes & DeRosier et al, 1987). The binding of tropomyosin to actin is strongly Mg²⁺ dependent (Wegner, 1979). High mol.wt. tropomyosins are most often 284 amino-acids and bind seven actin monomers, the low mol.wt. tropomyosins consist of about 245-250 amino-acids and span six actins. This means that for every 14 actins in a filament there are two high mol.wt tropomyosins.
The carboxy-terminal region of tropomyosin is important for strong actin-binding. Unacetlyated striated muscle tropomyosin has a very low affinity for actin filaments (Hitchcock-DeGregori & Heald, 1987; Cho et al, 1990) and does not associate with other tropomyosin dimers in the usual head to tail manner. Unacetlyated smooth muscle tropomyosin however binds strongly to F-actin (Cho & Hitchcock-DeGregori, 1991). The requirement for N-terminus acetylation of striated muscle tropomyosin for F-actin binding can be overcome by the incorporation of the dipeptide Alanine-serine at the N-terminus (). For smooth muscle tropomyosin other important residues include the carboxyterminal Glu276 and Thr277 (Cho, 2000). Like striated muscle tropomyosin the gene product of the Saccharomyces Tpm1 gene requires acetylation to bind actin (Singer & Shaw, 2003). Saccharomyces tropomyosin Tpm1 requires the cooperation of two gene products, Nat3p, acetyl transferase and Mdm20p (Singer & Shaw, 2003).

Intracellular Localization

Tropomyosin was one of the first actin-binding proteins to be studied by indirect immunofluorescence (Jorgensen et al, 1975; Osborn & Weber, 1979). It is primarily localised (as one might anticipate) along the actin filaments but is excluded from the leading edge of moving cells (DesMarais et al, 2002; Cooper, 2002) in line with their anti- ADF/Cofilin (Bernstein & Bamburg, 1982) and Arp2/3 complex activities (Blanchoin et al, 2001).

Competition with other Actin-Binding Proteins.

In addition to controlling myosin interaction with actin filaments tropomyosins may alter binding of calponin (Childs & Watson, 1992), gelsolin (Ishikawa et al, 1989b) , ADF/cofilins (Bernstein & Bamburg, 1982; Nishida et al, 1985; Maciver et al, 1991), MAP2 (Okagaki & Asakura, 1987).

Other Tropomyosin-Binding Proteins

A variety of proteins have been reported to bind tropomyosins. These studies have been biochemically and/or yeast two hybrid based. Their physiological significance has not always been readily explicable.

Protein	Function	Regulation	Reference
Calponin			Childs & Watson, 1992
CEACAM1	Adhesion protein		Schumann et al, 2001
Endostatin			MacDonald et al, 2001
Enigma	LIM		Guy et al, 1999
Gelsolin (sub-domain 2)	To regulate gelsolin severing function?	Ca²⁺ and pH	Koepf & Burtnick 1992, 1993; Maciver et al, 2000

S100A2			Gimona, et al, 1997



Table 1. Tropomyosin binding proteins

Regulation of Muscle Contraction by Tropomyosins.

Contraction of muscle and its the influence of tropomyosin was inferred early (Ebashi, 1963; Ebashi & Endo, 1968).

Regulation of Tropomyosins

The existence of a tropomyosin kinase activity has been reported (Montgomery & Mak, 1984) but remains poorly characterised. Regulation by caldesmon through Ca²⁺/calmodulin.

Phylogenetics

Tropomyosins are expressed widely in animal tissues being present in probably all vertebrates. Their status in lower eukaryotes is less certain. There are genes in the yeast Saccharomyces cerevisiae and in . However, despite being studied extensively no tropomyosin-like proteins have been isolated or detected from either Acanthamoeba or Dictyostelium. No tropomyosin homolog seems present in the now complete Arabidopsis thaliana genome. A tropomyosin-like protein (DivIVA) has been identified in the gram positive bacteria (Edwards et al, 2000) and it is possible that this protein is the ancestral tropomyosin.

Tropomyosins and Human Disease

Oncogenic Transformation. Mutations (), deletions of tropomyosin genes and the altered expression of tropomyosin genes (Hendricks & Weintraub, 1981; Lin et al, 1984; Leavitt et al, 1986; Prasad et al, 1993; Miyado et al, 1996; Lin et al, 2002) have often been reported to be associated with transformation of vertebrate cells and further that over-expressed of normal tropomyosin can inhibit transformation (Prasad et al, 1993) yet it seems that in some cases at least this is due to the 3' untranslated region of the gene (Rastinejad et al, 1993). Janssen & Mier 1997). The oncogene Ras has been implicated in tropomyosin mediated cell transformation that has in turn been associated with actin cytoskeleton alteration. Forced expression of Tm1 suppresses neoplastic growth characteristics of ras transformed cells through an uncertain mechanism (Prasad et al, 1993). There are some suggestions that anchorage-dependence of growth may be involved (Boyd et al, 1996).

The role in allergy. Allergies such as asthma seem to be increasingly common problem for no obvious reason. Many other allergies are caused by reactivity against cytoskeletal proteins. An example of this is that many hay fever sufferers produce a response to profilin from the pollen of many plants (see profilin). Tropomyosins too are implicated

Hypertrophic Cardiomyopathy. Hypertrophic cardiomyopathy (HCM) is usually an inherited disorder caused by mutations in a number of cardiac cytoskeletal proteins including myosin II heavy chain, troponin T and tropomyosin (Van Driest et al, 2002a b).

Nemaline Myopathy.

Tropomyosin-Like proteins

Several proteins possess regions similar to tropomyosins (Table 2).

TM30nm			Miyado et al, 1994

Purification of Tropomyosins from cells and tissues.

Tropomyosins are soluble in neutral to low pH buffers. Purification of tropomyosin from platelets (Cote & Smillie, 1981). From fibroblasts (Lin et al, 1985; Matsumara & Yamashiro-Matsumura, 1985)

References:-

Aspenström, P., U. Lindberg, et al. (1992). Site-specific amino-terminal mutant of yeast-expressed b-actin. Characterization of the interaction with myosin and tropomyosin. FEBS Lett. 303: 59-63.

Bailey, K. (1948) Tropomyosin: a new asymmetric protein component of the muscle fibril. Biochemical J. 43, 271-287.

Bernstein, B. W. and J. R. Bamburg (1982). Tropomyosin binding to F-actin protects the F-actin from disassembly by brain actin depolymerizing factor (ADF). Cell Motility 2: 1-8.

Betcher-Lange, S. L. and S. S. Lehrer (1978). Pyrene excimer fluorescence in rabbit skeletal aa tropomyosin labeled with N-(1-pyrene)maleimide. J.Biol.Chem. 253(11): 3757-3760.

Bikle, D. D., S. Munson, et al. (1993). Zipper Protein, a Newly Described Tropomyosin-like Protein of the Intestinal Brush Border. J. Biol. Chem. 268(1): 620-626.

Blanchoin, L., Pollard, T. D. & Hitchcock-DeGregori, S. E. (2001) Inhibition of the Arp2/3 complex-nucleated actin polymerization and branch formation by tropomyosin. Curr. Biol. 11, 1300-1304.

Boyd, J., Risinger, J. L., Wiseman, R. W., Merrick, B. A., Selkirk, J. K. & Barrett, J. C. (1996) Regulation of microfilament organization and anchorage-independent growth by tropomyosin. PNAS. 92, 11534-11538.

Bryce, N. S., Schevzov, G., Ferguson, V., Percival, J. M., Lin, J. J.-C., Matsumura, F., Bamburg, J. R., Jeffrey, P. L., Hardeman, E. C., Gunning, P. & Weinberger, R. P. (2003) Specification of Actin Filament Function and Molecular Composition by Tropomyosin Isoforms. Mol. Biol. Cell. 14, 1002-1016.

Childs, T. J., M. H. Watson, et al. (1992). Calponin and tropomyosin interactions. BBA 1121: 41-46.

Cho, Y. J., Liu, J. & Hitchcock-DeGregori, S. E. (1990) J.Biol.Chem. 265, 538-545.

Cho, Y. J. & Hitchcock-DeGregori, S. E. (1991) Relationship between alternatively spliced exons and functional domains in tropomyosin. PNAS. 88, 10153-10157.

Cho, Y.-J. (2000) The carboxyl terminal amino acid residues glutamine 276-threonine277 are important for actin affinity of the unacetylated smooth a-tropomyosin. J.Biochem.Mol.Biol. 33, 531-536.

Cooper, J. A. (2002) Actin dynamics: Tropomyosin provides stability. Curr. Biol. 12, R523-R525.

Cote, G.P, & Smillie, L.B. (1981) Preparation and some propreties of equinine platelet tropomyosin. J.Biol.Chem. 256, 11004-11010.

Dabrowska, R., H. Hinssen, et al. (1996). Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin mg2+-atpase activity. Biochemical J. 315: 753-759.

DesMarais, V., Ichetovkin, I., Condeelis, J. & Hitchcock-DeGregori, S. E. (2002) Spatial regulation of actin dynamics: a tropomyosin-free, actin-rich compartment at the leading edge. J Cell Sci. 115, 4649-4660.

Ebashi, S. (1963) Nature. 200, 1010.

Ebashi, S. & Endo, M. (1968) Prog.Biophys.Mol.Biol. 18, 123-183.

Edwards, D.H., Thomaides, H.B. & Errington, J. Promiscuous targeting of Bacillus subtilis cell division protein DivIVA to division sites in Escherichia coli and fission yeast. EMBO J. 19, 2719-2727.

Fattoum, A., J. H. Hartwig, et al. (1983). Isolation and some structural and functional properties of macrophage tropomyosin. Biochemistry 22: 1187-1193.

Gimona, M., J. A. Kazzaz, et al. (1996). Forced expression of tropomyosin 2 or 3 in v-Ki-ras-transformed fibroblasts results in distinct phenotypic effects. Pro.Nat.Acad.Sci.USA 93: 9618-9623.

Gimona, M., Z. Lando, et al. (1997). "Ca²⁺-dependent interaction of S100A2 with muscle and non-muscle tropomyosins." J.Cell Sci. 110: 611-621.

Guy, P. M., D. A. Kenny, et al. (1999). The PDZ domain of the LIM protein enigma binds to b-tropomyosin. FEBS letters 10: 1973-1984.

Greenfield, N. J., Palm, T. & Hitchcock-DeGregori, S. E. (2002) Structure and interactions of the carboxyl terminus of striated muscle a-tropomyosin: It is important to be flexible. Biophysical J. 83, 2754-2766.

Greenfield, N. J., Swapna, G. V. T., Huang, Y., Palm, T., Graboski, S., Montelione, G. T. & Hitchcock-DeGregori, S. E. (2003) The structure of the carboxyl terminus of striated a-tropomyosin in solution reveals an unusual parallel arrangement of interacting a-helices. Biochemistry. 42, 614-619.

Hendrick, M. & Weintraub, H. (1981) Tropomyosin is decreased in transformed cells. PNAS. 78, 5633-5637.

Hitchcock-DeGregori, S. E. & Heald, R. W. (1987) J.Biol.Chem. 262, 9730-9735.

Hitchcock-DeGregori, S. E., Song, Y. & Greenfield, N. J. (2002) Functions of tropomyosin's periodic repeat. Biochemistry. 41, 15036-15044.

Honda, H., Y. Kitano, et al. (1996). Dual role of tropomyosin on chemically modified actin filaments from skeletal muscle. FEBS Lett. 383: 55-58.

Ishikawa, R., S. Yamashiro, et al. (1989a). Annealing of gelsolin-severed actin fragments by tropomyosin in the presence of calcium. J. Biol. Chem. 264: 16764-16770.

Ishikawa, R., S. Yamashiro, et al. (1989b). Differential modulation of actin-severing activity of gelsolin by multiple isoforms of cultured rat cell tropomyosin. J. Biol. Chem. 264: 7490-7497.

Janssen, R. A. J. and J. W. Mier (1997). Tropomysoin-2 cDNA lacking the 3' untranslated region riboregulator induces growth inhibition of v-Ki-ras-transformed fibroblasts. Mol.Biol.Cell 8: 897-908.

Jorgensen, A.O., Subrahmanyan, L. & Kalnins, V.I. (1975). Localization of tropomyosin in mouse embryo fibroblasts. Am.J.Anat. 142, 519-525.

Koepf, E. K. and L. D. Burtnick (1992). Interaction of plasma gelsolin with tropomyosin. FEBS 309(1): 56-58.

Koepf, E. K. and L. D. Burtnick (1993). Horse plasma gelsolin labelled with fluorescein isothiocyanate responds to calcium and actin. Eur. J. Biochem. 212: 713-718.

Landis, C., N. Back, et al. (1999). Effects of tropomyosin internal deletions on thin filament function. J.Biol.Chem. 274(44): 31279-31285.

Leavitt, J., Latter, G., Lutomski, L., Goldstein, D. & Burbeck, S. (1986) Tropomyosin isoform switching in tumorigenic human fibroblasts. Mol.Cell Biol. 6, 2721-2726.

Leszyk, J., D. Mornet, et al. (1989). Amino acid sequence of a 15 kilodalton actin-binding fragment of turkey gizard caldesmon; similarity with dystrophin, tropomyosin and the tropomyosin-binding region of troponin T. Biochem.Biophys.Res.Comm. 160(1): 210-216.

Lin, J.J.C., Helfman, D.M., Hughes, S.H. & Chou, C.S. (1985) Tropomyosin isoforms in chicken embryo fibroblasts: Purification, characterization and changes in Rous sarcoma virus-transformed cells. J.Cell Biol. 100, 692-703.

Lin, J. L., Geng, X., Bhattacharya, S. D., Yu, J. R., Reiter, R. S., Sastri, B., Glazier, K. D., Mirza, Z. K., Wang, K. K., Amenta, P. S., Das, K. M. & Lin, J. J. (2002) Isolation and sequencing of a novel tropomyosin isoform preferentially associated with colon cancer. Gastroenterology. 123, 152-162.

Liu, H. and A. Bretscher (1992). Characterization of TMP1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport. J.Cell Biol. 118: 285-299.

Mabuchi, K. (1996) Electron microscopic study of tropomyosin binding to actin filaments reveals tethering molecules representing weak binding., J. Struct.Biol. 116, 278-289.

MacDonald, N. J., Shivers, W. Y., Narum, D. L., Plum, S. M., Wingard, J. N., Fuhrmann, S. R., Liang, H., Holland-Linn, J., Chen, D. H. T. & Sim, B. K. L. (2001) Endostatin binds tropomyosin. J.Biol.Chem. 276, 25190-25196.

Maciver, S. K., Ternent, D., & McLaughlin, P.J. (2000). Domain 2 of gelsolin binds directly to tropomyosin. FEBS letters 473: 71-75.

Marston, S. B. and C. S. Redwood (1993). The essential role of tropomyosin in cooperative regulation of smooth muscle thin filament activity by caldesmon. J.Biol.Chem. 268(17): 12317-12320.

Matsumara, F. & Yamashiro-Matsumura, S. (1985) Purification and characterization of multiple isoforms of tropomyosin from rat cultured cells. J.Biol.Chem. 260, 13851-13859.

Michele, D. E., F. P. Albayya, et al. (1999). Thin filament protein dynamics in fully differentiated adult cardiac myocytes: Towards a model of sarcomere maintenance. J.Cell Biol. 145: 1483-1495.

Montgomery, K. and A. S. Mak (1984). In Vitro phosphorylation of tropomyosin by a kinase from chicken embryo. J.Biol.Chem. 259(9): 5555-5560.

Miyado, K., Katsuki, M. & Taniguchi, S. (1994) Isolation of a yeast tropomyosin-related cDNA clone that encodes a novel transmembranous protein having a C-terminal highly basic region. BBRC. 199, 1363-1371.

Miyado, K., Kimura, M. & Taniguchi, S. (1996) Decreased expression of a single tropomyosin isoform, TM5/TM30nm, results in reduction in motility of highly metastatic B16-F10 mouse melanoma cells. BBRC. 225, 427-435.

Nishida, E., S. Maekawa, et al. (1984). Cofilin, a Protein in Porcine Brain That Binds to Actin Filaments and Inhibits their Interactions with Myosin and Tropomyosin. Biochemistry 23: 5307-5313.

Nishida, E., E. Muneyuki, et al. (1985). An Actin-Depolymerizing Protein (Destrin) from Porcine Kidney. Its Action on F-Actin Containing or Lacking Tropomyosin. Biochemistry 24: 6624-6630.

Nowak, E., H. Hinssen, et al. (1993). Synergistic and antagonistic modulation of actomyosin ATPase by caldesmon, tropomyosin and gelsolin. J.Muscle Res.Cell Motil. 14: 244.

Okagaki, T. and S. Asakura (1987). Tropomyosin reverses cross-linking of F-actin with microtubule- associated protein-2. J.Biochem. 101: 189-197.

Ooi, T., Mihashi, K. & Kobayashi, H. (1962) On the polymerization of tropomyosin. Arch. Biochem.Biophys. 98, 1-11.

Osborn, M. & Weber, K. (1979) Microfilament-associated proteins in tissue culture cells viewed by stereo immunofluorescence. Eur. J. Cell Biology. 20, 28-36.

Pelham, R. J., J. J.-C. Lin, et al. (1996). A high molecular mass non-muscle tropomyosin isoform stimulates retrograde organelle transport. J.Cell Sci. 109: 981-989.

Perry, S.V. (2001). J.Muscle Res. Cell Motil. 22, 5-49.

Pittenger, M. F., J. A. Kazzaz, et al. (1994). Functional properties of non-muscle tropomyosin isoforms. Curr. Op.Cell Biol. 6: 96-104.

Prasad, G. L., Fuldner, R. A. & Cooper, H. L. (1993) Expression of transduced tropomyosin 1 cDNA suppresses neoplastic growth of cells transformed by the ras oncogene. PNAS. 90, 7039-7043.

Reid, S. W., E. K. Koepf, et al. (1993). Fluorescent responses of acrylodan-labeled plasma gelsolin. Archives Of Biochemistry And Biophysics 302: 31-36.

Sen, A. and J. M. Chalovich (1998). Caldesmon-Actin-Tropomyosin contains two types of binding sites for myosin S1. Biochemsitry 37: 7526-7531.

Shaw, T. J., V. Terry, et al. (1998). Alterations in distribution of actin binding proteins in uterine stromal cells during decidualization in the rat. Cell Biol.Internat. 22: 237-243.

Simon, V. R., S. L. Karmon, et al. (1997). Mitochondrial inheritance: Cell cycle and actin cable dependence of polarized mitochondrial movements in Saccharomyces cerevisiae. Cell Mot.Cytoskel. 37: 199-210.

Singer, J. M. & Shaw, J. M. (2003) Mdm20 protein functions with Nat3 protein to acetylate Tpm1 protein and regulate tropomyosin-actin interactions in budding yeast. PNAS. 100, 7644-7649.

Stokes, D. L. & D. J. DeRosier (1987). The variable twist of actin and its modulation by actin-binding proteins. J.Cell Biol. 104: 1005-1017.

Takahashi, K., M. Abe, et al. (1988). A novel troponinT-like protein (calponin) in vascular smooth muscle: interaction with tropomyosin paracrystals. J.Hypertension. 6(4): S40-S43.

Van Driest, S. L., Will, M. L., Atkins, D. L. et al. (2002a) A novel TPM1 mutation in a family with hypertrophic cardiomyopathy and sudden cardiac death in childhood. Am. J. Cardiol. 90, 1123-1127.

Van Driest, S. L., Ackerman, M. J., Ommen, S. R., Shakur, R., Will, M. L., Nishimura, R. A., Tajik, A. J. & Gersh, B. J. (2002b) Prevalence and severity of "benign" mutations in the b-myosin heavy chain, cardiac troponin T, and a-tropomyosin genes in hypertrophic cardiomyopathy. Circulation. 106, 3085-3090.

Warren, K. S., J. L.-C. Lin, et al. (1994). Overexpression of human fibroblast caldesmon fragment containing actin-, Ca⁺⁺/Calmodulin-, and tropomyosin-binding domains stabilizes endogenous tropomyosin and microfilaments. J.Cell Biol. 125(2): 359-368.

Watts, R. G. and T. H. Howard (1994). Role of tropomyosin, a-actinin, and actin binding protein 280 in stabilizing triton insoluble F-actin in basal and chemotactic factor activated neutrophils. Cell Mot.Cytoskel. 28: 155-164.

Wegner, A. (1979). Equilibrium of the actin-tropomyosin interaction. J.Mol.Biol. 131: 839-853.

Yang, Y.-Z., E. D. Korn, & Eisenberg, E. (1979). Cooperative binding of tropomyosin to muscle and Acanthamoeba actin. J.Biol.Chem. 254(15): 7137-7140.

Zot, A. S. & Potter, J. D. (1987) Structural aspects of troponin-tropomyosin regulation of skeletal muscle contraction. Ann.Rev.Biophys.Biophys.Chem. 16, 535-539.