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Human fascin is an actin-bundling protein and is thought to play a role in the formation of microfilament bundles of microspikes and stress fibers in cultured cells. To explore the regulation of fascin-actin interaction, we have examined the effects of culture cell caldesmon and tropomyosin (TM) on actin binding activity of human fascin. Caldesmon alone or TM alone has little or no effect on the actin binding of fascin. However, caldesmon together with TM completely inhibits actin binding of human fascin. When calmodulin is added, the inhibition of fascin-actin interaction by caldesmon and TM becomes Ca2+ dependent because Ca2+/calmodulin blocks actin binding of caldesmon. Furthermore, as phosphorylation of caldesmon by cdc2 kinase inhibits actin binding of caldesmon, phosphorylation can also control actin binding of fascin in the presence of TM. As expected by the inhibition of fascin-actin binding, caldesmon coupled with TM also inhibits actin bundling activity of fascin. Whereas smooth muscle caldesmon alone or TM alone shows no effect, caldesmon together with TM completely inhibits actin bundling activity of fascin. This inhibition is again Ca2+ dependent when calmodulin is added to the system. These results suggest important roles for caldesmon and TM in the regulation of the function of human fascin.

Multiple isoforms of tropomyosin (TM) of rat cultured cells show differential effects on actin-severing activity of gelsolin. Flow birefringence measurements have revealed that tropomyosin isoforms with high Mrvalues (high MrTMs) partially protect actin filaments from fragmentation by gelsolin, while tropomyosins with low Mrvalues (low MrTMs) have no significant protection even when the actin filaments have been fully saturated with low MrTMs. We have also examined effect of nonmuscle caldesmon on the severing activity of gelsolin because 83-kDa nonmuscle caldesmon stimulates actin binding of rat cell TMs (Yamashiro-Matsumura, S., and Matsumura, F. (1988) J. Cell Biol.106, 1973–1983). While nonmuscle caldesmon alone or low MrTMs alone show no significant protection against fragmentation by gelsolin, the low MrTMs coupled with 83-kDa protein are able to protect actin filaments. Further, high MrTMs together with 83-kDa protein appear to block the severing activity completely. Electron microscopic analyses of length distribution of actin filaments have confirmed the results. The average length of control actin filaments is measured as 1.46 ± 1.0 µM, and gelsolin shortens the average length to 0.084 ± 0.039 µM. Similar short average lengths are obtained when gelsolin severs actin complexed with low MrTMs (0.080 ± 0.045 µM) or with nonmuscle caldesmon (0.11 ± 0.072 µM) while longer average length (0.22 ± 0.18 µM) is measured in the presence of high MrTMs. The simultaneous addition of nonmuscle caldesmon makes the average length considerably longer, i.e.0.61 ± 0.37 µMin the presence of low MrTMs and 1.57 ± 0.97 µMin the presence of high MrTMs. Furthermore, the actin binding of gelsolin is strongly inhibited by co-addition of high MrTMs and nonmuscle caldesmon. These results suggest that TM and gelsolin share the same binding site on actin molecules and that the differences in the actin affinities between TMs are related to their abilities of protection against gelsolin.

An F-actin-bundling protein with Mr of 55,000 has been purified from HeLa cells by a simple method using its affinity to F-actin. Briefly, muscle actin was mixed with supernatants of HeLa cell homogenates, and the resultant actin gel was precipitated by low speed centrifugation. The 55-kDa protein in the actin gel was dissociated by depolymerization of F-actin and purified sequentially by chromatography on DEAE-cellulose and hydroxylapatite. The Stokes radius and sedimentation coefficient of the 55-kDa protein were 32 A and 4.35 (S20,w), respectively. These results suggest that the 55-kDa protein is a monomeric globular protein with a native molecular weight of 57,000. The globular form of the protein was confirmed by electron microscopy of rotary shadowed specimens. The binding of the protein to actin was saturated at an approximate stoichiometry of 4 actin monomers to one 55-kDa molecule. The protein made F-actin aggregate side-by-side into bundles as has been reported for other F-actin-bundling proteins such as fimbrin (Mr = 68,000) and fascin (Mr = 58,000). The 55-kDa protein is a new actin-binding protein based on biochemical, morphological, and immunological characterization. Skeletal muscle tropomyosin inhibited the actin-bundling activity of 55-kDa protein by competitive binding to actin, suggesting that the 55-kDa protein binding site on F-actin is in the vicinity of the tropomyosin-binding site.

Using a newly developed method for microfilament isolation (Matsumura, F., Yamashiro-Matsumura, S. and Lin, J. J.-C. (1983) J. Biol. Chem. 258, 6636-6644), we have analyzed protein composition of microfilaments in “normal” and transformed rat tissue culture cells. They include REF-52 (an established rat embryo cell line) cells, REF-52 transformed by DNA viruses (SV40 or adenovirus type 5), normal rat kidney cells, and normal rat kidney cells transformed by RNA viruses (Kirsten or Rous sarcoma virus). Microfilaments from normal rat culture cells contain three major tropomyosins (apparent Mr = 40,000, 36,500, and 32,400) and two relatively minor tropomyosins (apparent Mr = 35,000 and 32,000). In transformed cells the levels of one or two of the major tropomyosins (Mr = 40,000 and 36,500) are decreased and the levels of one or both of the minor tropomyosins (Mr = 35,000 and 32,000) are increased. These changes in tropomyosin patterns were also observed in temperature shift experiments with rat-1 cells transformed with a Rous sarcoma virus mutant, temperature-sensitive for transformation. Cell-free translation of whole cell mRNA generated similar tropomyosin patterns on two-dimensional gels, suggesting that changes in the pattern of tropomyosin expression were largely effected at the level of RNA rather than by post-translational modification. Such changes in the tropomyosin composition of microfilaments were consistently found to accompany the various morphological alterations associated with transformation. We suggest that alterations in the pattern of tropomyosin expression are involved in, or cause, rearrangement of stress fibers and that this may be responsible (in part) for morphological transformation.

Previous results from our laboratory have shown that 1) cultured rat cells contain two classes of tropomyosin (TM), one (high MrTMs) with higher Mrvalues and greater affinity for actin than the other (low MrTMs); 2) presaturation of F-actin with high MrTMs, but not with low MrTMs, inhibits both actin-severing and actin binding activities of gelsolin; and 3) nonmuscle caldesmon not only enhances the inhibitory effects of high MrTMs but also makes low MrTMs capable of inhibiting the severing activity of gelsolin (Ishikawa, R., Yamashiro, S., and Matsumura, F. (1989) J. Biol. Chem. 264, 7490-7497). These results suggest that gelsolin has much lower affinity for F-actin-TM-caldesmon complexes than for pure F-actin. We have therefore examined whether addition of TM and/or caldesmon to gelsolin-severed actin filaments can make gelsolin dissociate from barbed ends of actin filaments, resulting in annealing of short actin filaments into long ones. Flow birefringence and electron microscopic studies have suggested that high MrTMs slowly and partially anneal gelsolin-severed actin fragments in 3 h, whereas low MrTMs have no effects. Nonmuscle caldesmon greatly potentiates the effects of high MrTMs and accelerates the process to 20 min, whereas nonmuscle caldesmon alone shows no effects. Furthermore, nonmuscle caldesmon makes low Mr TMs capable of reversing gelsolin-severing action. Actin binding assay has shown that gelsolin (or a gelsolin-actin complex) is dissociated from these annealed actin filaments. Smooth muscle TM and smooth muscle caldesmon also appear to anneal gelsolin-severed actin fragments as do high MrTMs and nonmuscle caldesmon. Calmodulin decreases the potentiation effects of caldesmon as calmodulin inhibits actin binding of caldesmon. These results suggest that tropomyosin and caldesmon may regulate both capping and severing activities of gelsolin.

We have shown previously that Rho plays a pivotal role in 1-oleoyl-lysophosphatidic acid (LPA)-dependent invasion of rat hepatoma cells (MM1). Herein we made stable transfectants of MM1 expressing active and Botulinum exoenzyme C3 (C3)-sensitive (Val14), or active and C3-insensitive (Val14/Ile41) forms of human RhoA. Both transfectants showed greatly promoted invasive ability in vitro in the absence of LPA as well as in vivo, adherence to the dish with scattered shape, and enhanced phosphorylation level of 20-kDa myosin light chain (MLC20). A specific MLC kinase inhibitor (KT5926) could inhibit their invasion and the phosphorylation level of MLC20. Stable active RhoA transfectants of W1 cells (low invasive counterpart of MM1) also demonstrated promoted invasive ability in vitro and in vivo, and enhanced phosphorylation level of MLC20. C3 treatment inhibited the invasiveness of the Val14 RhoA transfectant but not that of the Val14/Ile41 RhoA transfectant. LPA enhanced the invasiveness of both transfectants, and this enhancement was abolished by the C3 treatment. These results suggested that 1) the Rho signaling pathway and actomyosin system were linked in the transmigration of tumor cells, and 2) expressed active RhoA enhanced LPA-induced tumor cell invasion via the activation of endogenous RhoA pathway, indicating a positive feedback mechanism in the activation of RhoA.

Fascin is a 55-58-kDa actin-bundling protein, the actin binding of which is regulated by phosphorylation (Yamakita, Y., Ono, S., Matsumura, F., and Yamashiro, S. (1996) J. Biol. Chem. 271, 12632-12638). To understand the mechanism of fascin-actin interactions, we dissected the actin binding region and its regulatory site by phosphorylation of human fascin. First, we found that the C-terminal half constitutes an actin binding domain. Partial digestion of human recombinant fascin with trypsin yielded the C-terminal fragment with molecular masses of 32, 30, and 27 kDa. The 32- and 27-kDa fragments purified as a mixture formed a dimer and bound to F-actin at a saturation ratio of 1 dimer:11 actin molecules with an affinity of 1.4 x 10(6) M-1. Second, we identified the phosphorylation site of fascin as Ser-39 by sequencing a tryptic phosphopeptide purified by chelating column chromatography followed by C-18 reverse phase high performance liquid chromatography. Peptide map analyses revealed that the purified peptide represented the major phosphorylation site of in vivo as well as in vitro phosphorylated fascin. The mutation replacing Ser-39 with Ala eliminated the phosphorylation-dependent regulation of actin binding of fascin, indicating that phosphorylation at this site regulates the actin binding ability of fascin.

Human fascin is an actin-bundling protein that is thought to be involved in the assembly of actin filament bundles present in microspikes as well as in membrane ruffles and stress fibers. We have found that human fascin is phosphorylated in vivo upon treatment with 12-O-tetradecanoylphorbol-13-acetate, a tumor promoter. The in vivo phosphorylation is gradually increased from 0.13 to 0.30 mol/mol during 2 h of treatment, concomitant with disappearance of human fascin from stress fibers, membrane ruffles, and microspikes. Human fascin can also be phosphorylated in vitro as judged by phosphopeptide mapping. The extent of phosphorylation depends on pH: the stoichiometries are 0.05, 0.38, and 0.6 alone does not affect fascin-actin binding. With the incorporation of 0.25 mol of phosphate/mol of protein, the actin binding affinity is reduced from 6.7 x 10(6) to 1.5 x 10(6) m(-1). The actin bundling activity is also decreased. These results suggest that phosphorylation of fascin plays a role in actin reorganization after treatment with 12-O-tetradecanoylphorbol-13-acetate.

We have previously shown that rat cultured cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J. J.-C. (1983) J. Biol. Chem. 258, 6636-6644) and that these tropomyosins are differentially expressed upon cell transformation (Matsumura, F., Lin, J. J.-C., Yamashiro-Matsumura, S., Thomas, G. P., and Topp, W. C. (1983) J. Biol. Chem. 258, 13954-13964). To examine functions of tropomyosin in microfilament organization, we have purified and partially separated the multiple isoforms of tropomyosin by chromatography on hydroxylapatite. Analyses of cross-linked dimers produced by air oxidation have revealed that all isoforms except the tropomyosin isoform with apparent Mr of 35,000 form homodimers. Although these tropomyosins share many properties characteristic of tropomyosin, structural analyses at a peptide level and immunological analyses have shown that the five isoforms can be classified into two groups, i.e. tropomyosins with higher apparent Mr (Mr = 40,000, 36,500, and 35,000) and tropomyosins with lower apparent Mr (Mr = 32,400 and 32,000). The low Mr tropomyosins show less ability for head-to-tail polymerization and lower affinity to actin than the high Mr tropomyosins. We suggest that these differences in properties may be related to the changes in microfilament organization observed in transformed cells.

Phosphorylation of rat non-muscle caldesmon by cdc2 kinase causes reduction in most of caldesmon's properties, including caldesmon's binding to actin, myosin, and calmodulin, as well as its inhibition of actomyosin ATPase. We have generated and characterized the COOH terminus of caldesmon mutants lacking mitosis-specific phosphorylation sites, because the COOH-terminal half of caldesmon contains all 7 putative Ser or Thr sites for cdc2 kinase. Codons for the 7 putative Ser or Thr residues have been mutated to Ala, and resultant mutants were bacterially expressed. Analyses of the phosphopeptide maps of these mutants have identified 6 sites, including Ser-249, Ser-462, Thr-468, Ser-491, Ser-497, and Ser-527 as the mitosis-specific phosphorylation sites, whereas the phosphorylation of the remaining site, Thr-377, is not detected by this assay method. Actin binding experiments have suggested that 5 sites including Ser-249, Ser-462, Thr-468, Ser-491, and Ser-497 are important for the phosphorylation-dependent reduction in actin binding. Characterization of a mutant lacking all 7 Ser or Thr sites (7-fold mutant) has revealed that 7-fold mutation eliminates all phosphorylation sites by cdc2 kinase. While the in vitro properties of the 7-fold mutant, including actin, myosin, and calmodulin binding and inhibition of actomyosin ATPase, are very similar to those of nonmutated protein, such properties are not affected by the treatment with cdc2 kinase in contrast to nonmutated protein. This mutant should thus be useful to explore the functions of the mitosis-specific phosphorylation of caldesmon.

Cultured rat cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J.J.-C. (1983) J. Biol. Chem. 258, 6636-6644). To explore the roles of the multiple tropomyosin isoforms in the microfilament organization of cultured cells, we have examined effects of tropomyosins on the bundling activity of the 55-kDa protein recently purified from HeLa cells (Yamashiro-Matsumura, S., and Matsumura, F. (1985) J. Biol. Chem. 260, 5087-5097). Maximum bundling of F-actin was observed at a molar ratio of 55-kDa protein to actin higher than 1:8. None of the isoforms of cultured rat cell tropomyosin significantly altered the F-actin-bundling activity of 55-kDa protein at this ratio, whereas skeletal muscle tropomyosin inhibited the bundling activity to about 50%. Also, cultured cell tropomyosins did not inhibit binding of 55-kDa protein to actin, whereas skeletal muscle tropomyosin inhibited it by 50%. The effect of 55-kDa protein on the binding of tropomyosin to actin varied with the isoform type of tropomyosin. Most (80%) of the tropomyosins with low Mr values (Mr 32,400 or 32,000) were caused to dissociate from actin by 55-kDa protein, but only 20% of tropomyosins with high Mr values (Mr 40,000 or 36,500) was dissociated from actin in these conditions. Immunofluorescence has shown that, while tropomyosin was localized in stress fibers, 55-kDa protein was found in microspikes as well as stress fibers, both of which are known to contain bundles of microfilaments. Therefore, we suggest that 55-kDa protein together with the multiple tropomyosin isoforms may regulate the formation of two types of actin-filament bundles, bundles containing tropomyosin and those without tropomyosin.

We have developed a new method for the rapid isolation of tropomyosin-containing microfilaments from cultured cells using anti-tropomyosin monoclonal antibodies. Anti-tropomyosin monoclonal antibodies induce the bundle formation of microfilaments, which can be easily collected by low speed centrifugation. Electron microscopic studies of the isolated microfilaments show periodic localization of tropomyosin along the microfilaments of nonmuscle cells with a 33-34 nm repeat. Furthermore, the isolated microfilaments have the ability to activate the Mg2+-ATPase activity of skeletal muscle myosin to almost the same extent as skeletal muscle F-actin (filamentous actin). This microfilament isolation method is applicable to a variety of cell types, including REF-52 cells (an established rat embryo line), L6 myoblasts, 3T3 fibroblasts, Chinese hamster ovary cells, baby hamster kidney (BHK-21) cells, mouse neuroblastoma cells, gerbil fibroma cells, and chicken embryo fibroblasts. Sodium dodecyl sulfate-polyacrylamide gel analysis shows that, in addition to actin, microfilaments isolated from REF-52 cells contain five species of tropomyosin with apparent Mr = 40,000, 36,500, 35,000, 32,400, and 32,000, alpha-actinin, and as yet unknown proteins with apparent Mr = 83,000 and 37,000. The molar ratio of total tropomyosin (dimer) to actin in the isolated microfilaments is 1:8. The patterns of these multiple forms of tropomyosin were found to change when REF-52 cells were transformed with SV40 or adenovirus type 5.