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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.

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.

T-cell polarization is required for cell migration and cell-cell inter- actions, cellular behaviors crucial for lymphocyte differentiation. Despite expression of the epithelial polarity network in T cells, neither its contribution to thymocyte polarity nor its requirement during development is known. We report here that depletion of the polarity protein Scribble in hematopoietic progenitor cells results in inefficient T-cell development characterized by a partial developmental block during the early double-negative (DN) stage of differentiation. Scribble-depleted hematopoietic progenitor cells exhibit a delayed transition into late CD44lo/-CD25+ DN3 cells, evidenced by the accumulation of early CD44intCD25+ DN3 cells. As a consequence, a limited cellular expansion and a reduced frequency of intracellular T-cell receptor β-positive DN3 cells are ob- served among Scribble-deficient differentiating T cells. Moreover, whereas purified Scribble-depleted DN2 and DN3 cells do not ex- hibit compromised spontaneous motility, 1-cell clustering and pro- longed homotypic interactions among such cells are reduced. This deficiency correlates with a lack of polarization of the integrin LFA-1 during 1-cell migration or on the initiation of T-cell-T-cell interactions. Scribble is therefore a critical contributor to the clus- tering of immature T cells, an event shown here to be necessary for efficient developmental progression. [ABSTRACT FROM AUTHOR]