CYTOSKELETAL ORGANIZATION IN CLUSTERS OF ISOLATED POLARIZED SKATE HEPATOCYTES - STRUCTURAL AND FUNCTIONAL EVIDENCE FOR MICROTUBULE-DEPENDENT TRANSCYTOSIS

Isolated hepatocytes from the marine vertebrate Raja erinacea (the lit tle skate) retain their structural and functional integrity as cluster s of cells formed around a single tubular bile canaliculus, and theref ore can be used as a model of polarized hepatocytes in situ. In this s tudy we used confocal and conventional epifluorescence microscopy in c onjunction with fluorescent markers and immunocytochemistry to examine the structure and function of the cytoskeleton in these cells. Actin filaments in the hepatocyte clusters were found cortically and also co ncentrated in a pericanalicular array, while microtubules appeared to radiate away from a concentration near the apical membrane of the bili ary pole towards the basolateral sinusoidal surfaces. Treatment of clu sters with the microtubule disrupting agent, nocodazole, resulted in t he microtubules depolymerizing from the basolateral surfaces towards t he apical surface, indicating that the microtubules were oriented with their plus ends at the basolateral surface and their minus ends at th e apical surface. Nocodazole was also found to disrupt the ability of clusters to transcytose a fluorescent bile salt derivative into their canalicular lumens. We detected cytoplasmic dynein in skate hepatocyte homogenates by Western blotting using an anti-dynein intermediate cha in antibody, and immunofluorescent staining of intact hepatocytes reve aled a punctate vesicular pattern. The polarized arrangement of microt ubules, the presence of cytoplasmic dynein, and the inhibition of bile salt secretion by nocodozole are consistent with the microtubule cyto skeleton playing a fundamental role in the mediation of transcytosis, endocytosis, and bile excretory function in these hepatocytes. These p olarized isolated skate hepatocytes represent an excellent experimenta l model for the in vitro study of hepatic transport, and allow for imp ortant comparative studies aimed at elucidating the evolutionarily con served nature of various hepatocyte structures amongst the vertebrates . (C) 1995 Wiley-Liss, Inc.