Vesicular and nonvesicular transport of phosphatidylcholine in polarized HepG2 cells

We have investigated the transport and canalicular enrichment of fluorescen t phosphatidylcholine (PC) in HepG2 cells using the fluorescent analogs of PC C6-NBD-PC and beta -BODIPY-PC. Fluorescent PC was efficiently transporte d to the biliary canaliculus (BC) and became enriched on the lumenal side o f the canalicular membrane as shown for C6-NBD-PC. Some fluorescent PC was transported in vesicles to a subapical compartment (SAC) or apical recyclin g compartment (ARC) in polarized HepG2 cells as shown by colocalization wit h fluorescent sphingomyelin (C6-NBD-SM) and fluorescent transferrin, respec tively. Extensive trafficking of vesicles containing fluorescent PC between the basolateral domain, the SAC/ARC and the BC as well as endocytosis of P C analogs from the canalicular membrane were found. Evidence for nonvesicul ar transport included enrichment of the PC-analog beta -BODIPY-PC in the BC (t(1/2)=3.54 min) prior to its accumulation in the SAC/ARC (t(1/2)=18.5 mi n) at 37 degreesC. Transport of fluorescent PC to the canalicular membrane also continued after disruption of the actin or microtubule cytoskeleton an d at 2 degreesC. These results indicate that: (i) a nonvesicular transport pathway significantly contributes to the canalicular enrichment of PC in he patocytic cells, and (ii) vesicular transport of fluorescent PC occurs from both membrane domains via the SAC/ARC.