Morphological changes in the dendritic spines have been postulated to participate in the expression of synaptic plasticity. The cytoskeleton is likely to play a key role in regulating spine structure. Here we examine the molecular mechanisms responsible for the changes in spine morphology, focusing on drebrin, an actin-binding protein that is known to change the properties of actin filaments. We found that adult-type drebrin is localized in the dendritic spines of rat forebrain neurons, where it binds to the cytoskeleton. To identify the cytoskeletal proteins that associated with drebrin, we isolated drebrin-containing cytoskeletons using immunoprecipitation with a drebrin antibody. Drebrin, actin, myosin, and gelsolin were co-precipitated. We next examined the effect of drebrin on actomyosin interaction. In vitro, drebrin reduced the sliding velocity of actin filaments on immobilized myosin and inhibited the actin-activated ATPase activity of myosin. These results suggest that drebrin may modulate the actomyosin interaction within spines and may play a role in the structure-based plasticity of synapses.

Fascin-1 is a putative bundling factor of actin filaments in the filopodia of neuronal growth cones. Here, we examined the structure of the actin bundle formed by human fascin-1 (actin/ fascin bundle), and its mode of interaction with myosin in vitro. The distance between cross-linked filaments in the actin/ bundle was 8-9 nm, and the bundle showed the transverse periodicity of 36 nm perpendicular to the bundle axis, which was confirmed by electron microscopy. Decoration of the actin/fascin bundle with heavy meromyosin revealed that the arrowheads of filaments in the bundle pointed in the same direction, indicating that the bundle has polarity. This result suggested that fascin-1 plays an essential role in polarity of actin bundles in filopodia. In the in vitro motility assay, actin/ fascin bundles slid as fast as single actin filaments on myosin II and myosin V. When myosin was attached to the surface at high density, the actin/fascin bundle disassembled to single filaments at the pointed end of the bundle during sliding. These results suggest that myosins may drive filopodial actin bundles backward by interacting with actin filaments on the surface, and may induce disassembly of the bundle at the basal region of filopodia.

Cell transformations accompany alterations in cell morphology and microfilament patterns. Calvasculin encodes mRNA termed pEL‐98, 18A2, 42A, p9Ka, or mtsl, found to be elevated in several metastatic cell lines. We report the elevation of calvasculin expression in SR‐3Y1 cells, which show disappearance of ordered microfilaments, compared to that in 3Y1 cells and that the similar distribution of calvasculin to that of actin filaments. Interestingly, calvasculin co‐sediments with F‐actin and bundles actin filaments in a Ca2+‐dependent manner. This activity, along with the elevation of calvasculin following transformation, suggests that the disorganization of filaments in SR‐3Y1 cell is due to the cross‐linking activity of calvasculin.

The purification of drebrin, an actin‐binding protein that is specifically expressed in embryonic rat brain, was described previously. During the purification of drebrin, we found that an actin‐binding protein of 54 kDa was also expressed at high levels in embryonic brain, and this protein was identified by immunoblotting as fascin. To explore the roles of fascin in brain development, we purified fascin from brains of infant rats and characterized it. We found that the actin‐binding activity of fascin was strongly inhibited by drebrin. Fascin caused formation of actin bundles, a process that was inhibited in the presence of drebrin, as confirmed by electron microscopy and a low‐speed centrifugation assay. In PC12 cells, fascin was localized in the filopodia of growth cones, whereas drebrin was localized in the basal region of growth cones. Our results suggest that fascin might play an important role in the organization of actin in filopodia and that this organization might be regulated by drebrin.

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 Ca 2؉ dependent because Ca 2؉ /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 Ca 2؉ 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.Fascins belong to a unique family of actin-bundling proteins (1, 2), which include sea urchin fascin (3-8), HeLa 55-kDa actin-bundling protein (9, 10), and the gene products of Drosophila singed (11-13). All of these proteins make F-actin aggregate side-by-side into bundles (4, 8 -10) and are localized in the structures containing actin bundles including filopodia and stress fibers of cultured cells (1, 9, 10, 14), bristles of Drosophila, actin bundles of Drosophila nurse cells (11, 13), and microspikes and microvilli of sea urchin eggs and coelomocytes (6, 7). Some of these structures such as filopodia and microspikes are known to be dynamic structures responding to various biological signals. For example, the fertilization of sea urchin eggs induces the formation of fascin-containing microvilli on their surfaces (7). Filopodia contain fascin-actin bundles and are actively extending and retracting during cell movement of fibroblasts. Fascin should thus be involved in the assembly and disassembly of actin bundles in such structures. However, the mechanisms for the regulation of actin binding of fascin are not well understood.One way to regulate fascin-actin interaction is phosphorylation. We have shown that human fascin is phosphorylated at Ser-39 in vivo in human neuroblastoma cells upon treatment with 12-O-tetradecanoylphorbal-13-acetate, a tumor promoter (16). The same site is phosphorylated by protein kinase C, which results in the inhibition of actin binding of fascin (16,17). However, the stoichiometry of fascin phosphorylation by protein kinase C is low, suggesting that kinases other than protein kinase C may be involved. Furthermore, the stoichiometry of in vivo phosphorylation is also low in the a...