Tropomyosin (TM) is widely distributed in all cell types associated with actin as a fibrous molecule composed of two α-helical chains arranged as a coiled-coil. It is localised, polymerised end to end, along each of the two grooves of the F-actin filament providing structural stability and modulating the filament function. To accommodate the wide range of functions associated with actin filaments that occur in eucaryote cells TM exists in a large number isoforms, over 20 of which have been identified. These isoforms which are expressed by alternative promoters and alternative RNA processing of four genes, TPM1, 2, 3 and 4, all conform to a general pattern of structure. Their amino acid sequences consist of an integral number, six or seven in vertebrates, of quasiequivalent regions of about 40 residues that are considered to represent the actin-binding regions of the molecule. In addition to the variable regions a large part of the polypeptide chains of the TM isoforms, mainly centrally located and expressed by five exons, is invariant. Many of the isoforms are tissue and filament specific in their distribution implying that the exons expressed in them and the regions of the molecule they represent are of significance for the function of the filament system with which they are associated. In the case of muscle there is clear evidence that the TM moves its position on the F-actin filament during contraction and it is therefore considered to play an important part in the regulation of the process. It is uncertain how the role of TM in muscle compares to that in non-muscle systems and if its function in the former tissue is unique to muscle.

Vertebrate tropomyosin: distribution, properties and function.

Tropomyosin isoforms: divining rods for actin cytoskeleton function.

Functional properties of non-muscle tropomyosin isoforms

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca(2+)-calmodulin-regulated manner Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca(2+)-calmodulin or Ca(2+)-S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation

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Caldesmon inhibits nonmuscle cell contractility and interferes with the formation of focal adhesions.

Towards a molecular understanding of cytokinesis.

At least eight tropomyosin isoforms (hTM1, hTM2, hTM3, hTM4, hTM5, hTM5a, hTM5b, and hTMsm alpha) are expressed from four distinct genes in human fibroblasts. In order to elucidate isoform properties, we have subcloned hTM3 and hTM5 full-length cDNAs, as well as their chimeric cDNAs into the bacterial expression pET8C system. Bacterially expressed tropomyosin isoforms (called PEThTM3, PEThTM5, PEThTM5/3, and PEThTM3/5) were purified and characterized. Under optimal binding conditions, the binding of PEThTM5 isoform to F-actin was stronger than the PEThTM3 isoform. However, analysis of actin-binding by the McGhee and von Hippel equation revealed that PEThTM3 exhibits higher cooperativity in binding than PEThTM5 does. Furthermore, the chimera PEThTM5/3 which possessed the N-terminal fragment of hTM5 fused to the C-terminal fragment of hTM3 had even stronger actin binding ability. The reverse chimera PEThTM3/5 which possessed the N-terminal fragment of hTM3 fused to the C-terminal fragment of hTM5 demonstrated greatly reduced affinity to actin filaments. In addition, both chimeras had different KCl requirements for optimal binding to F-actin than their parental tropomyosins. A bacterially made C-terminal fragment of human fibroblast caldesmon (PETCaD39) and native chicken gizzard caldesmon were both able to enhance the actin-binding of these bacterially expressed tropomyosins. However, PETCaD39's enhancement of binding to F-actin was greater for PEThTM5 than PEThTM3. Under 30 mM KCl and 4 mM MgCl2, the low M(r) isoform PEThTM4 appeared to be able to amplify the actin-activated HMM ATPase activity by 4.7 fold, while the high M(r) isoform PEThTM3 stimulated the activity only 1.5 fold. The higher enhancement of ATPase activity by PEThTM5 than by PEThTM3 suggested that the low M(r) isoform hTM5 may be more involved in modulating nonmuscle cell motility than hTM3. These results further suggested that different isoforms of tropomyosin might have finite differences in their specific functions (e.g., cytoskeletal vs. motile) inside the cell.

Li-Fei Liu

Papers

In vitro functional characterization of bacterially expressed human fibroblast tropomyosin isoforms and their chimeric mutants.

Expression of smooth muscle and nonmuscle tropomyosins in Escherichia coli and characterization of bacterially produced tropomyosins