Bile canaliculus formation in cultured HEPG2 cells.
BACKGROUND
The plasma membrane of hepatocytes can be divided in sinusoidal, lateral and apical membrane, each with functionally and structurally distinct features. The apical domain consists of the bile canalicular structures. The morphogenesis and the polarization of hepatocytes is still poorly known.


METHODS
We used HepG2 cells, a hepatoma cell line to study the formation of the bile canaliculi in the apical part of the cells. The cells were synchronized by using nocodazole. The formation of the bile canaliculi was monitored by using immunofluorescence microscopy, confocal laser scanning microscopy, and immunoelectron microscopy. Antibodies to alpha-fodrin and villin were used. Actin was visualized with rhodamine phalloidin.


RESULTS
Confocal laser scanning microscopy showed accumulations of actin, villin and fodrin at the cell membranes 8 to 12 hours after the release of the nocodazole block. These sites probably represent areas destined to develop into bile canaliculi. Later, immature bile canaliculi were discerned that were located asymmetrically between adjacent cells. Transmission electron microscopy of serial sections showed that they were always connected with the surface of the cell. Mature bile canaliculi appeared between adjacent cells 48 hours after the release of the nocodazole block. They were round, vesicle-like structures lined with microvilli and sealed by tight junctions and desmosomes. They were usually seen between two juxtaposed cells, and often several cells contributed to their formation. Typically, mature bile canaliculi were delineated by a subplasmalemmal filamentous meshwork of fodrin and actin, resembling a terminal web of enterocytes. Actin and villin were also found in microvillar cores.


CONCLUSIONS
The results show that (i) bile canaliculi are formed de novo between two or more juxtaposed cells; (ii) canalicular-formation is accompanied by a distinct accumulation of the membrane skeletal and microvillar proteins fodrin, actin and villin at the apical surfaces of the cells, suggesting that they play an important role in bile canaliculus morphogenesis, and that (iii) apical membrane differentiation in the cells contributing to the formation of a single canaliculus is an asymmetric process.
Sormunen
R
R
Biocenter, University of Oulu, Finland.
Eskelinen
S
S
Lehto
V P
VP
D001648:Bile Canaliculi / Q000737:chemistry / Q000254:growth & development* / Q000648:ultrastructure; D006528:Carcinoma, Hepatocellular / Q000737:chemistry / Q000648:ultrastructure*; D006801:Humans; D008113:Liver Neoplasms / Q000737:chemistry / Q000648:ultrastructure*; D008840:Microfilament Proteins / Q000032:analysis*; D008854:Microscopy, Electron; D016253:Microscopy, Immunoelectron; D014407:Tumor Cells, Cultured
D008840:Microfilament Proteins