Abstract. Small GTPases of the rab family are crucial elements of the machinery that controls membrane traffic. In the present study, we examined the distribution and function of rabll. Rabll was shown by confocal immunofluorescence microscopy and EM to colocalize with internalized transferrin in the pericentriolar recycling compartment of CHO and BHK cells. Expression of rabll mutants that are preferentially in the GTP-or GDP-bound state caused opposite effects on the distribution of transferrin-containing elements; rabll-GTP expression caused accumulation of labeled elements in the perinuclear area of the cell, whereas rabll-GDP caused a dispersion of the transferrin labeling. Functional studies showed that the early steps of uptake and recycling for transferrin were not affected by overexpression of rabll proteins. However, recycling from the later recycling endosome was inhibited in cells overexpressing the rabll-GDP mutant. Rab5, which regulates early endocytic trafficking, acted before rabll in the transferrin-recycling pathway as expression of rab5-GTP prevented transport to the rab11-positive recycling endosome. These results suggest a novel role for rabll in controlling traffic through the recycling endosome.

We visualized a fluorescent-protein (FP) fusion to Rab6, a Golgi-associated GTPase, in conjunction with fluorescent secretory pathway markers. FP-Rab6 defined highly dynamic transport carriers (TCs) translocating from the Golgi to the cell periphery. FP-Rab6 TCs specifically accumulated a retrograde cargo, the wild-type Shiga toxin B-fragment (STB), during STB transport from the Golgi to the endoplasmic reticulum (ER). FP-Rab6 TCs associated intimately with the ER, and STB entered the ER via specialized peripheral regions that accumulated FP-Rab6. Microinjection of antibodies that block coatomer protein I (COPI) function inhibited trafficking of a KDEL-receptor FP-fusion, but not FP-Rab6. Additionally, markers of COPI-dependent recycling were excluded from FP-Rab6/STB TCs. Overexpression of Rab6:GDP (T27N mutant) using T7 vaccinia inhibited toxicity of Shiga holotoxin, but did not alter STB transport to the Golgi or Golgi morphology. Taken together, our results indicate Rab6 regulates a novel Golgi to ER transport pathway.

Abstract. MDCKII cells differentiate into a simple columnar epithelium when grown on a permeable support; the monolayer is polarized for transport and secretion. Individual cells within the monolayer continue to divide at a low rate without disturbing the function of the epithelium as a barrier to solutes. This presents an interesting model for the study of mitosis in a differentiated epithelium which we have investigated by confocal immunofluorescence microscopy. We monitored the distribution of microtubules, centrioles, nucleus, tight junctions, and plasma membrane proteins that are specifically targeted to the apical and basolateral domains. The stable interphase microtubule cytoskeleton was rapidly disassembled at prophase onset and reassembled at cytokinesis. As the interphase microtubules disassembled at prophase, the centrioles moved from their interphase position at the apical membrane to the nucleus and acquired the ability to organize microtubule asters. Orientation of the spindle parallel to the plane of the monolayer occurred between late prophase and metaphase and persisted through cytokinesis. The cleavage furrow formed asymmetrically perpendicular to the plane of the monolayer initiating at the basolateral side and proceeding to the apical domain. The interphase microtubule network reformed after the centrioles migrated from the spindle poles to resume their interphase apical position. Tight junctions (ZO-1), which separate the apical from the basolateral domains, remained assembled throughout all phases of mitosis. E-cadherin and a 58-kD antigen maintained their basolateral plasma membrane distributions, and a 114-kD antigen remained polarized to the apical domain. These proteins were useful for monitoring the changes in shape of the mitotic cells relative to neighboring cells, especially during telophase when the cell shape changes dramatically.We discuss the changes in centriole position during the cell cycle, mechanisms of spindle orientation, and how the maintenance of polarized plasma membrane domains through mitosis may facilitate the rapid reformation of the polarized interphase cytoplasm.

. As axons elongate, tubulin, which is synthesized in the cell body, must be transported and assembled into new structures in the axon. The mechanism of transport and the location of assembly are presently unknown . We report here on the use of tubulin tagged with a photoactivatable fluorescent group to investigate these issues . Photoactivatable tubulin, microinjected into frog embryos at the two-cell stage, is incorporated into microtubules in neurons obtained from explants of the neural tube. When activated by light, a fluorescent mark is made on the microtubules in the axon, and transport and turnover can be visualized directly. We find that microtubules are generated in or near the cell body and continually transported distally as a coherent phase of polymer during axon C YTOPLASMIC flux in axons was first demonstrated in the classic experiments of Weiss and Hiscoe (1948) when vesicular elements were found to accumulate at the site of an artificial constriction . Direct visualization of this transport, both in the axon (Burdwood, W 1965. J. Cell Biol. 27:115A ;Matsumoto, 1920;Nakai, 1964) and later in isolated axoplasm , led to a characterization of vesicular movements as occurring on microtubules in both a retrograde and anterograde manner, and later led to the purification of the proteins responsible for generating these movements (for review see Schroer and Sheetz, 1991;Vale, 1987). Although these studies represent important advances in our understanding of axon growth and function, an equally important and unanswered question is to determine how the structural components of the axon, in particular the microtubules, are formed .Previous experiments, focusing on the sites of microtubule assembly in the axon, have generated two diametrically opposed explanations of axonal growth (for review see Bamburg, 1988;Hollenbeck, 1989). In one view, based primarily on metabolic labeling studies, microtubules are thought to be assembled in the cell body and transported down the axon (Hoffman and Lasek, 1975) . In the other view, based on photobleaching experiments (Lim et al ., 1989(Lim et al ., , 1990Okabe and Hirokawa, 1990) and local application of microtubule depolymerizing drugs (Bamburg et al., 1986), tubulin is thought to be transported down the axon as monomer and elongation . This vectorial polymer movement was observed at all levels on the axon, even in the absence of axonal elongation . Measurements of the rate of polymer translocation at various places in the axon suggest that new polymer is formed by intercalary assembly along the axon and assembly at the growth cone in addition to transport of polymer from the cell body. Finally, polymer movement near the growth cone appeared to respond in a characteristic manner to growth cone behavior, while polymer proximally in the axon moved more consistently. These results suggest that microtubule translocation is the principal means of tubulin transport and that translocation plays an important role in generating new axon structure at the growth cone.as...

We have shown that gamma tubulin is localized both in the pericentriolar material and in the core of the mammalian centriole. This result suggests that gamma tubulin has a role in the centriolar duplication process, perhaps as a template for growth of the centriolar microtubules, in addition to its established role in the nucleation of astral microtubules.