Actin Filaments and Microtubules are Involved in Different Membrane Traffic Pathways That Transport Sphingolipids to the Apical Surface of Polarized HepG2 Cells

In polarized HepG2 hepatoma cells, sphingolipids are transported to the apical, bile canalicular membrane by two different transport routes, as revealed with fluorescently tagged sphingolipid analogs. One route involves direct, transcytosis-independent transport of Golgi-derived glucosylceramide and sphingomyelin, whereas the other involves basolateral to apical transcytosis of both sphingolipids. We show that these distinct routes display a different sensitivity toward nocodazole and cytochalasin D, implying a specific transport dependence on either microtubules or actin filaments, respectively. Thus, nocodazole strongly inhibited the direct route, whereas sphingolipid transport by transcytosis was hardly affected. Moreover, nocodazole blocked “hyperpolarization,” i.e., the enlargement of the apical membrane surface, which is induced by treating cells with dibutyryl-cAMP. By contrast, the transcytotic route but not the direct route was inhibited by cytochalasin D. The actin-dependent step during transcytotic lipid transport probably occurs at an early endocytic event at the basolateral plasma membrane, because total lipid uptake and fluid phase endocytosis of horseradish peroxidase from this membrane were inhibited by cytochalasin D as well. In summary, the results show that the two sphingolipid transport pathways to the apical membrane must have a different requirement for cytoskeletal elements

Actin Dependence of Polarized Receptor Recycling in Madin-Darby Canine Kidney Cell Endosomes

Mammalian epithelial cell plasma membrane domains are separated by junctional complexes supported by actin. The extent to which actin acts elsewhere to maintain cell polarity remains poorly understood. Using latrunculin B (Lat B) to depolymerize actin filaments, several basolateral plasma membrane proteins were found to lose their polarized distribution. This loss of polarity did not reflect lateral diffusion through junctional complexes because a low-density lipoprotein receptor mutant lacking a functional endocytosis signal remained basolateral after Lat B treatment. Furthermore, Lat B treatment did not facilitate membrane diffusion across the tight junction as observed with ethylenediaminetetraacetic acid or dimethyl sulfoxide treatment. Detailed analysis of transferrin recycling confirmed Lat B depolarized recycling of transferrin from endosomes to the basolateral surface. Kinetic analysis suggested sorting was compromised at both basolateral early endosomes and perinuclear recycling endosomes. Despite loss of function, these two endosome populations remained distinct from each other and from early endosomes labeled by apically internalized ligand. Furthermore, apical and basolateral early endosomes were functionally distinct populations that directed traffic to a single common recycling endosomal compartment even after Lat B treatment. Thus, filamentous actin may help to guide receptor traffic from endosomes to the basolateral plasma membrane

Myosin Motors and Not Actin Comets Are Mediators of the Actin-based Golgi-to-Endoplasmic Reticulum Protein Transport

We have previously reported that actin filaments are involved in protein transport from the Golgi complex to the endoplasmic reticulum. Herein, we examined whether myosin motors or actin comets mediate this transport. To address this issue we have used, on one hand, a combination of specific inhibitors such as 2,3-butanedione monoxime (BDM) and 1-[5-isoquinoline sulfonyl]-2-methyl piperazine (ML7), which inhibit myosin and the phosphorylation of myosin II by the myosin light chain kinase, respectively; and a mutant of the nonmuscle myosin II regulatory light chain, which cannot be phosphorylated (MRLC2(AA)). On the other hand, actin comet tails were induced by the overexpression of phosphatidylinositol phosphate 5-kinase. Cells treated with BDM/ML7 or those that express the MRLC2(AA) mutant revealed a significant reduction in the brefeldin A (BFA)-induced fusion of Golgi enzymes with the endoplasmic reticulum (ER). This delay was not caused by an alteration in the formation of the BFA-induced tubules from the Golgi complex. In addition, the Shiga toxin fragment B transport from the Golgi complex to the ER was also altered. This impairment in the retrograde protein transport was not due to depletion of intracellular calcium stores or to the activation of Rho kinase. Neither the reassembly of the Golgi complex after BFA removal nor VSV-G transport from ER to the Golgi was altered in cells treated with BDM/ML7 or expressing MRLC2(AA). Finally, transport carriers containing Shiga toxin did not move into the cytosol at the tips of comet tails of polymerizing actin. Collectively, the results indicate that 1) myosin motors move to transport carriers from the Golgi complex to the ER along actin filaments; 2) nonmuscle myosin II mediates in this process; and 3) actin comets are not involved in retrograde transport