J Cell Biol. the AZD8835 coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways. INTRODUCTION Rab proteins constitute a family of small, monomeric GTPases that are essential components of virtually all membrane trafficking pathways (Nuoffer and Balch, 1994 ). The complexity of vesicular transport in eukaryotic cells is emphasized by the fact that more than 40 members of the Rab family have been identified to date in mammals, and many of these have been implicated in the regulation of specific stages of either exocytic or endocytic transport. A surprising number of Rabs have been associated with compartments of the endocytic pathway, suggesting a higher degree of complexity than had originally been appreciated. Accumulating evidence indicates that early endosomes can be functionally subdivided into two distinct subcompartments. Sorting endosomes, which are located in the peripheral cytoplasm, are the site at which fluid-phase cargo and ligands dissociated from their receptors are segregated for transport to lysosomes. Sorting endosomes are characterized by the presence of Rab5, which is required for the import of endocytic material from the cell surface (Bucci (1996) . Similarly, for production of Rab25T26N, the two complementary oligonucleotide method was used to alter the rabbit Rab25 sequence in pCB7. For prokaryotic expression of Rab25, the rabbit Rab25 sequence was recloned into pGEX-2T to construct a glutathione-supernatant, dialyzed against reaction buffer) (Zahraoui (1994) . RESULTS Localization of Endogenous Rab11a in MDCK Cells Previous investigations in nonpolarized cells indicated that Rab11a was concentrated on a population of endosomes in close proximity to the centrosome (Ullrich (1994) for apical recycling endosomes in MDCK cells expressing the pIgR receptor and support the hypothesis that Rab11a is associated with the apical recycling system in MDCK cells. Open in a separate window Figure 3 Effects of microtubule integrity on Rab11a distribution. (A) MDCK cells cultured on permeable supports were treated with 33 Mouse monoclonal to KLHL11 M nocodazole for 2 h and then fixed in 4% paraformaldehyde. Cells were dual-stained with antibodies against Rab11a and ZO-1 with detection by secondary antibodies conjugated with Cy5 and Cy2, respectively. The top two panels show confocal projections of 40 XCY optical sections AZD8835 (0.3 m each) from the apical regions of the cells. Arrowheads at the right mark the region used to construct the XCZ projections shown in the bottom two panels. Punctate Rab11a immunoreactivity is dispersed throughout the cell with some concentration at the lateral margins. (B) Polarized MDCK cells cultured on permeable supports were AZD8835 treated with 5 M taxol for 4 h at 37C. Cells were dual-stained with antibodies against Rab11a and ZO-1. Top two panels show maximum intensity projection reconstructions of 40 confocal XCY optical sections (0.3 m each). Arrowheads at left indicate the region used to construct XCZ projections shown in bottom panels. Taxol caused the redistribution of Rab11a immunoreactivity to the region of the tight junctions (arrows) as well as a diffuse subapical staining pattern. Bar, 4 m. In light of the prominent effect of nocodazole on Rab11a distribution, we also.