Linkage of microtubules to special cortical regions is essential for cell polarization. CLIP-170 binds to the growing ends of microtubules and plays pivotal roles in orientation. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts with CLIP-170. In Vero fibroblasts, IQGAP1 localizes at the polarized leading edge. Expression of carboxy-terminal fragment of IQGAP1, which includes the CLIP-170 binding region, delocalizes GFP-CLIP-170 from the tips of microtubules and alters the microtubule array. Activated Rac1/Cdc42, IQGAP1, and CLIP-170 form a tripartite complex. Furthermore, expression of an IQGAP1 mutant defective in Rac1/Cdc42 binding induces multiple leading edges. These results indicate that Rac1/Cdc42 marks special cortical spots where the IQGAP1 and CLIP-170 complex is targeted, leading to a polarized microtubule array and cell polarization.
PIP2-binding proteins CAPS and Munc13 are required for Ca2+-triggered vesicle exocytosis. TIRF microscopy localized PIP2, DAG, CAPS, and Munc13. Exocytosis occurred at PIP2-rich sites. CAPS localized to vesicles but required PIP2, whereas Munc13 required stimulus-dependent recruitment to PIP2-rich domains, indicating distinct mechanisms.
We have previously proposed that IQGAP1, an effector of Rac1 and Cdc42, negatively regulates cadherinmediated cell-cell adhesion by interacting with -catenin and by causing the dissociation of ␣-catenin from cadherin--catenin-␣-catenin complexes and that activated Rac1 and Cdc42 positively regulate cadherin-mediated cell-cell adhesion by inhibiting the interaction of IQGAP1 with -catenin. However, it remains to be clarified in which physiological processes the Rac1-Cdc42-IQGAP1 system is involved. We here examined whether the Rac1-IQGAP1 system is involved in the cell-cell dissociation of Madin-Darby canine kidney II cells during 12-O-tetradecanoylphorbol-13-acetate (TPA)-or hepatocyte growth factor (HGF)-induced cell scattering. By using enhanced green fluorescent protein (EGFP)-tagged ␣-catenin, we found that EGFP-␣-catenin decreased prior to cell-cell dissociation during cell scattering. We also found that the Rac1-GTP level decreased after stimulation with TPA and that the Rac1-IQGAP1 complexes decreased, while the IQGAP1--catenin complexes increased during action of TPA. Constitutively active Rac1 and IQGAP1 carboxyl terminus, a putative dominant-negative mutant of IQGAP1, inhibited the disappearance of ␣-catenin from sites of cell-cell contact induced by TPA. Taken together, these results indicate that ␣-catenin is delocalized from cell-cell contact sites prior to cell-cell dissociation induced by TPA or HGF and suggest that the Rac1-IQGAP1 system is involved in cell-cell dissociation through ␣-catenin relocalization.Although cell-cell adhesion seems to be a static process for those who have not watched movies of cell-cell contacts, dynamic rearrangement of cell-cell adhesion occurs in a variety of cellular processes, including epithelial cell scattering, dispersal of cancer cells, cell division, and tissue rearrangement (for reviews, see references 2, 6, and 31). Cadherins, a family of cell-cell adhesion molecules, bind -catenin or plakoglobin (also known as ␥-catenin), which in turn is linked to the actin cytoskeleton via ␣-catenin (for a review, see reference 34). This linkage between cadherins and the actin-based cytoskeleton contributes to the development of a strong adhesive state. Indeed, ␣-catenin-deficient mouse teratocarcinoma F9 cells display a scattered-cell phenotype under conditions in which parental or ␣-catenin-reexpressing cells form compact colonies (16). In primary cultures of ␣-catenin-null keratinocytes, actin reorganization and polymerization at sites of cell-cell contact are prevented and adhesive contacts are not sealed (35). Loss of ␣-catenin expression has also been observed in lung carcinomas (36) and gastric carcinomas (22), and cells derived from these tumors show scattered-cell growth. Studies using optical tweezers and single-particle tracking have indicated that ϳ50% of the E-cadherin on the plasma membrane in epithelial cells is connected to the actin cytoskeleton, probably by ␣-catenin, but that the rest appears to be unattached (29).Taken together, these observations...
The actin cytoskeleton is dynamically remodeled during Fcγ receptor (FcγR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP2)-dependent manner. We investigated the role of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) γ and α isoforms, which synthesize PIP2, during phagocytosis. PIP5K-γ−/− bone marrow–derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcγR clustering. Delivery of exogenous PIP2 rescued these defects. PIP5K-γ knockout BMM also have more RhoA and less Rac1 activation, and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5K-γ by RNA interference inhibits particle attachment. In contrast, PIP5K-α knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein activation, and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5K-γ but not PIP5K-α is transiently activated by spleen tyrosine kinase–mediated phosphorylation. We propose that PIP5K-γ acts upstream of Rac/Rho and that the differential regulation of PIP5K-γ and -α allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.
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