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A network of protein interactions that centre around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells is identified. Rich1 and Amot maintain tight junctions (TJ) integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking.

Rho GTPases regulate reorganization of actin and microtubule cytoskeletal structures during both interphase and mitosis. The timing and subcellular compartment in which Rho GTPases are activated is controlled by the large family of Rho GTP exchange factors (RhoGEFs). Here, we show that the microtubule-associated RhoGEF Lfc is required for the formation of the mitotic spindle during prophase/prometaphase. The inability of cells to assemble a functioning spindle after Lfc inhibition resulted in a delay in mitosis and an accumulation of prometaphase cells. Inhibition of Lfc's primary target Rho GTPase during prophase/prometaphase, or expression of a catalytically inactive mutant of Lfc, also prevented normal spindle assembly and resulted in delays in mitotic progression. Coinjection of constitutively active Rho GTPase rescued the spindle defects caused by Lfc inhibition, suggesting the requirement of RhoGTP in regulating spindle assembly. Lastly, we implicate mDial as an important effector of Lfc signaling. These findings demonstrate a role for Lfc, Rho, and mDial during mitosis.

Research was conducted to characterize the unc-73 gene product from Caenorhabditis elegans as it relates to guided cell and growth cone migrations. Results showed that unc-73 affected several cell migrations in C. elegans and influenced pioneer and non-pioneer growth cone and cell migrations. They also indicated that the unc-73 peptide was capable of activating Rac and inducing actin reorganization characteristic of membrane ruffling.

Chimeric mice in which lymphocytes are deficient in the Syk tyrosine kinase have been created. Compared with Syk-positive controls, mice with Syk -/- lymphocytes display substantial depletion of intraepithelial [Gamma][Delta] T cells in the skin and gut, with developmental arrest occurring after antigen receptor gene rearrangement. In this dependence on Syk, subsets of intraepithelial [Gamma][Delta] T cells are similar to B cells, but distinct from splenic [Gamma][Delta] T cells that develop and expand in Syk-deficient mice. The characteristic associations of certain T-cell receptor V[Gamma]/V[Delta] gene rearrangements with specific epithelia are also disrupted by Syk deficiency.

Gap junctions are made up of connexin proteins, which comprise a multigene family in mammals. Targeted mutagenesis of connexin43 (Cx43), one of the most prevalent connexin proteins, showed that its absence was compatible with survival of mouse embryos to term, even though mutant cell lines showed reduced dye coupling in vitro. However, mutant embryos died at birth, as a result of a failure in pulmonary gas exchange caused by a swelling and blockage of the right ventricular outflow tract from the heart. This finding suggests that Cx43 plays an essential role in heart development but that there is functional compensation among connexins in other parts of the developing fetus., The connexins are a family of at least 12 proteins, each the product of a distinct gene, that make up the intercellular membrane channels of gap junctions [1]. Gap junction [...]

An asymmetric synthesis of anti-aldol segments via a nonaldol route is described, where the strategy has involved a highly diastereoselective synthesis of functionalized tetrahydrofuran derivatives from optically active 4-phenylbutyrolactone. The utility of the method is shown by preparing statine derivatives and pancreatic lipase inhibitor, (-)-tetrahydrolipstatin.

We have used cell lines deficient in p120 Ras GTPase activating protein (Ras-GAP) to investigate the roles of Ras-GAP and the associated p190 Rho-GAP (p190) in cell polarity and cell migration. Cell wounding assays showed that Ras-GAP--deficient cells were incapable of establishing complete cell polarity and migration into the wound. Stimulation of mutant cells with growth factor rescued defects in cell spreading, Golgi apparatus fragmentation, and polarized vesicular transport and partially rescued migration in a Ras-dependent manner. However, for directional movement, the turnover of stress fibers and focal adhesions to produce an elongate morphology was dependent on the constitutive association between Ras-GAP and p190, independent of Ras regulation. Disruption of the phosphotyrosine-mediated Ras-GAP/p190 complex by microinjecting synthetic peptides derived from p190 sequences in wild-type cells caused a suppression of actin filament reorientation and migration. From these observations we suggest that although Ras-GAP is not directly required for motility per se, it is important for cell polarization by regulating actin stress fiber and focal adhesion reorientation when complexed with 190. This observation suggests a specific function for Ras-GAP separate from Ras regulation in cell motility. Key words: cytoskeleton * cell polarity * migration * actin * p190 Rho-GAP

A study was undertaken to investigate the biological activity of the mammalian Grb2, a subset of the SH2 domain-containing proteins of protein-tyrosine kinases, to relate it to the genetic analysis of an SH2/SH3 adaptor. The mammalian Grb2 was believed to bind phosphoproteins, docking proteins and cytoplasmic tyrosine kinases. Results revealed that Grb2 manifested multiple activities in normal and oncogenic signaling pathways. It was suggested that the Grb2 signaling pathway regulated cell differentiation in the early embryonic development.