The actin cytoskeleton undergoes extensive remodeling during cell morphogenesis and motility. The small guanosine triphosphatase Rho regulates such remodeling, but the underlying mechanisms of this regulation remain unclear. Cofilin exhibits actin-depolymerizing activity that is inhibited as a result of its phosphorylation by LIM-kinase. Cofilin was phosphorylated in N1E-115 neuroblastoma cells during lysophosphatidic acid-induced, Rho-mediated neurite retraction. This phosphorylation was sensitive to Y-27632, a specific inhibitor of the Rho-associated kinase ROCK. ROCK, which is a downstream effector of Rho, did not phosphorylate cofilin directly but phosphorylated LIM-kinase, which in turn was activated to phosphorylate cofilin. Overexpression of LIM-kinase in HeLa cells induced the formation of actin stress fibers in a Y-27632-sensitive manner. These results indicate that phosphorylation of LIM-kinase by ROCK and consequently increased phosphorylation of cofilin by LIM-kinase contribute to Rho-induced reorganization of the actin cytoskeleton.

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. . 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 replacingSer-39withAlaeliminatedthephosphorylation-dependent regulation of actin binding of fascin, indicating that phosphorylation at this site regulates the actin binding ability of fascin.

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Abstract. Two cDNAs, isolated from a Xenopus laevis embryonic library, encode proteins of 168 amino acids, both of which are 77% identical to chick cofilin and 66% identical to chick actin-depolymerizing factor (ADF), two structurally and functionally related proteins. These Xenopus ADF/cofilins (XACs) differ from each other in 12 residues spread throughout the sequence but do not differ in charge. Purified GST-fusion proteins have pH-dependent actin-depolymerizing and F-actin-binding activities similar to chick ADF and cofilin. Similarities in the developmental and tissue specific expression, embryonic localization, and in the cDNA sequences of the noncoding regions, suggest that the two XACs arise from allelic variants of the pseudotetraploid X. laevis.Immunofluorescence localization of XAC in oocyte sections with an XAC-specific monoclonal antibody shows it to be diffuse in the cortical cytoplasm. After fertilization, increased immunostaining is observed in two regions: along the membrane, particularly that of the vegetal hemisphere, and at the interface between the cortical and animal hemisphere cytoplasm. The cleavage furrow and the mid-body structure are stained at the end of first cleavage. Neuroectoderm derived tissues, notochord, somites, and epidermis stain heavily either continuously or transiently from stages 18-34. A phosphorylated form of XAC (pXAC) was identified by 2D Western blotting, and it is the only species found in oocytes. Dephosphorylation of >60% of the pXAC occurs within 30 min after fertilization. Injection of one blastomere at the 2 cell stage, either with constitutively active XAC or with an XAC inhibitory antibody, blocked cleavage of only the injected blastomere in a concentration-dependent manner without inhibiting nuclear division. The cleavage furrow of eggs injected with constitutively active XAC completely regressed. Blastomeres injected with neutralized antibody developed normally. These results suggest that XAC is necessary for cytokinesis and that its activity must be properly regulated for cleavage to occur.

Abstract. Radixin is a barbed end-capping actinmodulating protein which was first identified in isolated cell-to-cell adherens junctions from rat liver (Tsukita, Sa., Y. Hieda, and Sh. . J. Cell Biol. 108:2369-2382. In the present study, we have analyzed the distribution of radixin in dividing cells. For this purpose, an mAb specific for radixin was obtained using chicken gizzard radixin as an antigen. By immunofluorescence microscopy with this mAb and a polyclonal antibody obtained previously, it was clearly shown in rat fibroblastic cells (3Y1 cells) that radixin was highly concentrated at the cleavage furrow during cytokinesis. Radixin appeared to accumulate rapidly at the cleavage furrow at the onset of furrowing, continued to be concentrated at the furrow during anaphase and telophase, and was finally enriched at the midbody. This concentration of radixin at the cleavage furrow was detected in all other cultured cells we examined: bovine epithelial cells (MDBK cells), mouse myeloma cells (P3 cells), rat kangaroo Ptk2 cells, mouse teratocarcinoma cells, and chicken fibroblasts. Furthermore, it became clear that the epitope for the mAb was immunofluorescently masked in the cell-tocell adherens junctions. Together, these results lead as to conclude that radixin is present in the undercoat of the cell-to-cell adherens junctions and that of the cleavage furrow, although their respective molecular architectures are distinct. The possible roles of radixin at the cleavage furrow are discussed with special reference to the molecular mechanism of the actin filament-plasma membrane interaction at the furrow.TIN filaments are involved in many kinds of cell motility and are found in association with the plasma membrane in a variety of eukaryotic cells (18,28). An obvious function of the association of actin filaments with membranes may be that membranes serve as attachment sites for actin filaments in cell motility systems. In interphase eukaryotic nonmuscle cells, two types of well-developed actin filament bundles which can contract by themselves in cooperation with myosin molecules can be observed: stress fibers in cultured cells and circumferential bundles in epithelial cells (4, 31, 44). They are associated with plasma membranes at the ceU-to-substrate and cell-to-cell adherens junctions (AJ), t respectively (9, 13, 16, 37). To clarify the molecular organization of these actin filament attachment sites, intensive studies have been made mainly using immunohistochemical methods. So far many proteins have been identified as constituents of the undercoat of AJ. These AJ undercoat-constitutive proteins can he categorized into two groups: group A proteins such as ot-actinin (21) and filamin (14), which are localized not only at the AJ undercoat 1. Abbreviations used in this paper: AJ, adherens junction; HAT, hypoxanthine/aminopterine/thymidine; MDBK, Madin-Darby bovine kidney; pAb, polyclonal antibody; mAb, monoclonal antibody. but also along the actin bundles, and group B proteins such as vinculin (12, 15) and talin (2,...