L Distribution and Size of HRP-labeled Endocytic Organelles in MDCK Cells after Treatment with Various Acidification and Alkalinization Media

The interaction between late endocytic structures and microtubules in polarized cells was studied using a procedure previously shown to cause microtubule-dependent redistribution of lysosomes in fibroblasts and macrophages (Heuser, J. 1989. J. Cell Biol. 108:855-864). In cultured rat hippocampal neurons, low cytoplasmic pH caused cation-independent mannose-6-phosphate receptor-enriched structures to move out of the cell body and into the processes. In filter grown MDCK cells lowering the cytosolic pH to ,06.5 caused late endosomes to move to the base of the cell and this process was shown to be microtubule dependent. Alkalinization caused a shift in distribution towards the apical pole of the cell. The results are consistent with low pH causing the redistribution of late endosomes towards the plus ends of the microtubules. In MDCK cells the microtubules orientated vertically in the cell may play a role in this process. The shape changes that accompanied the redistribution of the late endosomes in MDCK cells were examined by electron microscopy. On low pH treatment fragmentation of the late endosomes was observed whereas after microtubule depolymerization individual late endosomal structures appeared to fuse together. The late endosomes of the MDCK cell appear to be highly pleomorphic and dependent on microtubules for their form and distribution in the cell. I N fibroblasts, microtubules radiate out from the centrally located microtubule organizing center towards the periphery of the cell. The minus ends of the microtubules are associated with the centrioles while the plus (fastgrowing) ends are peripherally located. This organization is important for maintaining the intracellular organization of the membrane-bound compartments of the exocytic and endocytic pathways and for directed membrane traffic between these compartments (for review see Kelly, 1990). On the biosynthetic pathway, the endoplasmic reticulum is extended out along microtubules to the cell periphery (Terasaki et al., 1986) and the involvement of a mechanochemical ATPase, such as kinesin, which drives movement towards the plus ends of the microtubules has been suggested (reviewed by Terasaki, 1990). The Golgi apparatus is maintained in a central position apparently by interaction with minus-end directed motors (Ho et al., 1989). The interactions of both these compartments with microtubules are highly dynamic as shown by observations of living cells (Lee and Chen, 1988; Cooper et al., 1990). On the endocytic pathway, vesicles containing internalized markers move from the cell periphery to the perinuclear region of the cell in a microtubule-dependent fashion (Herman and Albertini, 1984; De Brabander et al., 1988). Possible candidates for these K. Prydz's present address is Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, 0310 Oslo 3, Norway. vesicles are large spherical structures, termed endosome carrier vesicles, in which internalized markers accumulate after microtubule depolymerization (Gruenberg et al., 1989). Lysosomes also interact with microtubules and so are maintained in a perinuclear position in the cell (Matteoni and Kreis, 1987). In macrophages, the lysosomes are aligned on microtubules to form long tubules (Swanson et al., 1987; Knapp and Swanson, 1990) and experimentally induced cytosolic acidification causes them to move out along microtubules to the cell periphery (Heuser, 1989). Polarized ceils show specialized microtubule organization. For example, in neuronal cells, which have been extensively studied as a model system for microtubule-dependent movement of organelles (Adams, 1982; Allen et al., 1982; Vale et al., 1985), the microtubules are organized into bundles of uniform polarity (Heidemann et al., 1981). These microtubules mediate the vesicular transport of newly synthesized material from the cell body to the synapses at the tips of the axons whereas material destined for degradation is routed in the opposite direction (reviewed by Grafstein and Forman, 1980). In polarized epithelial cells the microtubule network shows a different organization. The microtubules do not radiate out from a single nucleating center but the majority run vertically through the cell (Gorbsky and Borisy, 1985; AchIer et al., 1989; Tucker et al., 1986; Bacallao et al., 1989). The plus ends of the microtubules are predominantly basal while the minus ends are close to the apical surface (Mogen© The Rockefeller University Press, 0021-9525/91/O4/261/14 $2.00 The Journal of Cell Biology, Volume 113, Number 2, April 1991 261-274 261 on July 9, 2017 jcb.rress.org D ow nladed fom sen et al., 1989; Bacallao et al., 1989). In addition, a dense cap of microtubules of mixed polarity lies underneath the apical surface and a sparse network lies at the base of the MDCK cell (Bacallao et al., 1989). The bulk of these microtubules are not organized by the centrioles, which lie directly under the apical surface (Buendia et al., 1990). Microtubules seem to play a role in the maintenance of the polarized phenotype by directing exocytic and endocytic traffic. For example, delivery of exocytic vesicles to the apical surface of epithelial cells appears to be mediated by microtubules (Rindler et al., 1987; Achier et al., 1989; Eilers et al., 1989) and microtubules are involved in endocytic transport from the two sets of early endosomes in the MDCK cell to the late endosomes (Bomsel et al., 1990). The positioning of endocytic organelles in differentiating epithelial cells also appears to involve microtubules (reviewed by Fleming and Johnson, 1988). The role of the vertically orientated microtubules in these processes is at present unclear. In the present study we examined the interaction of endocytic organelles with microtubules in these two types of polarized cells using cultured rat hippocampal neurons and MDCK epithelial cells. In neurons, low cytoplasmic pH caused a dramatic redistribution of cation-independent mannose-6-phosphate receptor (C1-MPR)~-positive late endocytic structures that moved out of the cell body and into the processes. We studied this in more detail in MDCK cells. Late endosomes, and to a partial extent, lysosomes moved to the base of the cells. The Golgi apparatus and early endosomes showed no apparent change in distribution. The late endosomes, but not the lysosomes, also underwent fragmentation at low pH. Movement was shown to be microtubule dependent and towards the plus ends of the microtubules in MDCK cells. On alkalinization the distribution of late endosomes was shifted towards the apical pole of the cell. On microtubule depolymerization, individual late endosomal structures appeared to fuse with each other. These results suggest that under normal conditions the distribution of late endosomes in both these polarized ceils is maintained through interactions with microtubules and that they can move retrogradely (apically) or anterogradely (basally) upon manipulating the cytosolic pH. Materials and Methods