The dynamic behaviour of myosin V molecules translocating along actin filaments has been mainly studied by optical microscopy. The processive hand-over-hand movement coupled with ATP hydrolysis was thereby demonstrated. However, the protein molecules themselves are invisible in the observations and have therefore been visualised by electron microscopy in the stationary states. Namely, the concomitant assessment of structure and dynamics has been unfeasible, a situation prevailing throughout biological research. Here, using high-speed atomic force microscopy, we directly visualise myosin V molecules walking along actin tracks. themselves. The structure of proteins has been studied by electron microscopy, x-ray crystallography, or NMR but the obtained structures are substantially static. To overcome this long-standing dilemma and enable to simultaneously record the structure and dynamics of functioning biomolecules, high-speed atomic force microscopy (HS-AFM) has been developed [2][3][4][5] . The recent significant improvement in its

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.

At mitosis in eukaryotic cells there are profound changes of shape and structure whose causes are almost entirely obscure. What is known is that there are changes in the organization of microfilaments, including the disassembly of microfilament bundles during prophases and the accompanying rounding-up of cultured cells; the formation of transient contractile rings during cytokinesis; and, subsequently, the reassembly of microfilament bundles and the respreading of the two daughter cells. As an initial step towards the biochemical understanding of these events, in which the disassembly and reassembly of microfilaments appear to play an important part, we searched for alterations of the molecular constitution of microfilaments during mitosis. We found that non-muscle caldesmon, a protein with a relative molecular mass (Mr) of 83,000 (83K) which binds to actin and calmodulin, is dissociated from microfilaments during mitosis, apparently as a consequence of phosphorylation. This process may contribute to the changes of shape and structure of cells in mitosis, as caldesmon inhibits actomyosin ATPase.

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.