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T-cell polarization is required for cell migration and cell-cell interactions, 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 CD44(lo/-)CD25(+) DN3 cells, evidenced by the accumulation of early CD44(int)CD25(+) DN3 cells. As a consequence, a limited cellular expansion and a reduced frequency of intracellular T-cell receptor β-positive DN3 cells are observed among Scribble-deficient differentiating T cells. Moreover, whereas purified Scribble-depleted DN2 and DN3 cells do not exhibit compromised spontaneous motility, T-cell clustering and prolonged homotypic interactions among such cells are reduced. This deficiency correlates with a lack of polarization of the integrin LFA-1 during T-cell migration or on the initiation of T-cell-T-cell interactions. Scribble is therefore a critical contributor to the clustering of immature T cells, an event shown here to be necessary for efficient developmental progression.

Using functional and proteomic screens of proteins that regulate the Cdc42 GTPase, we have identified a network of protein interactions that center around the Cdc42 RhoGAP Rich1 and organize apical polarity in MDCK epithelial cells. Rich1 binds the scaffolding protein angiomotin (Amot) and is thereby targeted to a protein complex at tight junctions (TJs) containing the PDZ-domain proteins Pals1, Patj, and Par-3. Regulation of Cdc42 by Rich1 is necessary for maintenance of TJs, and Rich1 is therefore an important mediator of this polarity complex. Furthermore, the coiled-coil domain of Amot, with which it binds Rich1, is necessary for localization to apical membranes and is required for Amot to relocalize Pals1 and Par-3 to internal puncta. We propose that Rich1 and Amot maintain TJ integrity by the coordinate regulation of Cdc42 and by linking specific components of the TJ to intracellular protein trafficking.

The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions.