Cytoskeletal organization in clusters of isolated polarized skate hepatocytes: Structural and functional evidence for microtubule‐dependent transcytosis

Abstract

Isolated hepatocytes from the marine vertebrate Raja erinacea (the little skate) retain their structural and functional integrity as clusters of cells formed around a single tubular bile canaliculus, and therefore can be used as a model of polarized hepatocytes in situ. In this study we used confocal and conventional epifluorescence microscopy in conjunction 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 concentrated in a pericanalicular array, while microtubules appeared to radiate away from a concentration near the apical membrane of the biliary pole towards the basolateral sinusoidal surfaces. Treatment of clusters with the microtubule disrupting agent, nocodazole, resulted in the microtubules depolymerizing from the basolateral surfaces towards the apical surface, indicating that the microtubules were oriented with their plus ends at the basolateral surface and their minus ends at the 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 chain antibody, and immunofluorescent staining of intact hepatocytes revealed a punctate vesicular pattern. The polarized arrangement of microtubules, the presence of cytoplasmic dynein, and the inhibition of bile salt secretion by nocodozole are consistent with the microtubule cytoskeleton playing a fundamental role in the mediation of transcytosis, endocytosis, and bile excretory function in these hepatocytes. These polarized isolated skate hepatocytes represent an excellent experimental model for the in vitro study of hepatic transport, and allow for important comparative studies aimed at elucidating the evolutionarily conserved nature of various hepatocyte structures amongst the vertebrates.

How to induce non-polarized cells of hepatic origin to express typical hepatocyte polarity: generation of new highly polarized cell models with developed and functional bile canaliculi

“…Thus, Can 10 and Can 9 are the first models with functional highly developed BC reminiscent of those found in vivo. Developed functional BC are not often observed in hepatocyte primary cultures, except for hepatocytes from the marine vertebrate Raja erinacea (Henson et al 1995). However, in this last model, as in hepatocyte couplets, BC are preserved but never newly formed.…”

David S. Miller: Scientist, Mentor, Friend—a tribute and thank you

“…With Jim Boyer, Yale Medical School, New Haven, CT, David also studied isolated hepatocytes from the marine vertebrate Raja erinacea (little skate; [52]). Using confocal fluorescence microscopy, David and colleagues elucidated the cellular organization of actin filaments and observed that nocodazole disrupted transcytosis of a fluorescent bile salt derivative into canalicular lumens.…”

Cell and molecular biology of the spiny dogfish Squalus acanthias and little skate Leucoraja erinacea: insights from in vitro cultured cells

“…Leucoraja erinacea has been used for a number of years in studies of liver function (Barnes et al , 2005). The cells are interesting because, among other phenomena, they do not synthesize albumin, they produce bile alcohols instead of bile acids and they are a useful system for comparative studies on evolutionarily conservation of vertebrate hepatocyte structures (Henson et al , 1995). In addition to studies with perfused liver and molecular studies of genes cloned from the liver cells, a relatively simple culture system has been devised to allow studies of isolated L. erinacea hepatocytes (Smith et al , 1987).…”

“…The cultures also have been used to characterize chloride and manganese uptake and release (Davis‐Amaral et al , 1996, 1997; Madejczyk et al , 2009). Ultrastructural and functional studies in vitro have shown that the microtubule cytoskeleton is involved in an essential way in transcytosis, endocytosis and bile excretory function (Henson et al , 1995; Miller et al , 1996).…”