Our research is aimed at understanding the molecular mechanisms of microfilament reorganization associated with the alterations of cell morphology seen during oncogenic transformation and during cell division. Morphological alterations are perhaps the most common of many phenotypic changes which occur upon oncogenic transformation. Microfilaments, which are at least in part responsible for the morphological alterations, are significantly re-organized in transformed cells. While "normal" cells with well-spread morphologies have numerous bundles of microfilaments and strongly adheres to the substrate, transformed cells typically display rounded morphologies with much less adhesiveness and show dispersed microfilament patterns.
Changes in the structure of actin cables, as well as in cell-substrate adhesion, are closely coupled with cell proliferation. At mitosis, normal cells become rounded and lose adhesiveness to the substrate, taking on characteristics similar to those of transformed cells. This resemblance of the morphology and adhesiveness of transformed cells and dividing normal cells accentuates the close relationship between oncogenic transformation and cell cycle control.
For several years, we have focused our studies on two microfilament-associated proteins, caldesmon and fascin. We previously found that caldesmon is disassociated from microfilaments as a result of its mitosis-specific phosphorylation by cdc2 kinase, and that expression of caldesmon is decreased upon cell transformation. Because caldesmon stimulates the actin binding activity of tropomyosin, and because tropomyosin stabilizes the structure of the microfilament, we hypothesized that the decrease in caldesmon expression in transformed cells, as well as the dissociation of caldesmon from microfilaments in mitotic cells, would destabilize microfilaments. Such destabilization likely contributes to the disorganization of stress fibers seen in many transformed cells and may be a prerequisite for the dynamic reorganization of microfilaments seen during mitosis. To examine the above possibility, we have generated a mutant of caldesmon in which all seven cdc2 phosphorylation sites were replaced with alanine, and examined its effects on microfilaments as well as on cell cycle progression. We have found that the expression of the mutant stabilizes the microfilament cytoskeleton during mitosis, delays entry into M-phase, and inhibits cell division. Interestingly, we also have found that the mutant stabilizes microfilaments during interphase and delays S-phase progression. These results suggest that there may be a mechanism by which the caldesmon-mediated regulation of microfilament organization affects cell cycle progression.
The functions of fascin in microfilament organization contrast with those of caldesmon: While caldesmon localizes mainly in stress fibers, fascin is present in filopodia, membrane ruffles and stress fibers. This localization is consistent with the actin bundling activity of fascin. Fascin and caldesmon show opposing regulatory effects on the actin binding activity of tropomyosin, and their expression is reciprocally altered upon cell transformation. We have found that ectopic expression of human fascin in normal epithelial cells causes major morphological alterations, including membrane protrusions and disorganization of cell-cell contacts, and that fascin-transfected epithelial cells show increased cell motility. We also have found that human fascin is phosphorylated in vivo and in vitro, resulting in the inhibition of its actin-binding and actin-bundling activities. These results suggest that fascin may function as an "activator" of cell motility, leading to an increase in the assembly of filopodia and membrane ruffles. By expressing mutant fascin lacking the phosphorylation site, we are studying the physiological significance of fascin phosphorylation in the formation of filopodia and membrane ruffles. Because small G-proteins including cdc42 and rac, as well as its effectors (IQGAP1 and N-WASP), have been shown to induce the formation of filopodia and lamellipodia, we also examine how these proteins are involved in the fascin-mediated cell motility and re-organization of the peripheral cytoskeleton.
