We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

We have previously shown that aPKC interacts with cell polarity proteins PAR-3 and PAR-6 and plays an indispensable role in cell polarization in the C. elegans one-cell embryo as well as in mammalian epithelial cells. Here, to clarify the molecular basis underlying this aPKC function in mammalian epithelial cells, we analyzed the localization of aPKC and PAR-3 during the cell repolarization process accompanied by wound healing of MTD1-A epithelial cells. Immunofluorescence analysis revealed that PAR-3 and aPKCλ translocate to cell-cell contact regions later than the formation of the primordial spot-like adherens junctions (AJs) containing E-cadherin and ZO-1. Comparison with three tight junction (TJ) membrane proteins, JAM,occludin and claudin-1, further indicates that aPKCλ is one of the last TJ components to be recruited. Consistently, the expression of a dominant-negative mutant of aPKCλ (aPKCλkn) in wound healing cells does not inhibit the formation of the spot-like AJs; rather, it blocks their development into belt-like AJs. These persistent spot-like AJs in aPKCλ-expressing cells contain all TJ membrane proteins and PAR-3,indicating that aPKC kinase activity is not required for their translocation to these premature junctional complexes but is indispensable for their further differentiation into belt-like AJs and TJs. Cortical bundle formation is also blocked at the intermediate step where fine actin bundles emanating from premature cortical bundles link the persistent spot-like AJs at apical tips of columnar cells. These results suggest that aPKC contributes to the establishment of epithelial cell polarity by promoting the transition of fibroblastic junctional structures into epithelia-specific asymmetric ones.

It is well established that intraepithelial T lymphocytes (IELs) are derived from conventional single-positive (SP) thymocytes, as well as unconventional double-negative (DN) thymocytes and CD103+CD8αβ recent thymic emigrants (RTEs). We show that IELs can be divided into two groups according to their dependency on sphingosine 1-phosphate (S1P) for trafficking into the intestines. CD4 or CD8αβ naive lymphocytes originating from SP thymocytes express high levels of type 1 S1P receptor (S1P1), and their preferential migration into the large intestine is regulated by S1P. In contrast, RTEs migrate exclusively into the small intestine, whereas DN thymic IEL precursors expressing either TCRαβ or TCRγδ migrate into both the small and large intestines. S1P does not play a role in the migration pathways of these unconventional thymic IEL precursors. Thus, down-regulation of S1P1 expression or disruption of the S1P gradient halted conventional CD4 or CD8αβ IEL trafficking into the intestines, but did not affect the trafficking of unconventional thymic IEL precursors. These data are the first to demonstrate that a lipid-mediated system discriminates IELs originating from conventional and unconventional thymic precursors.