Abstract
We have investigated the transport and canalicular enrichment of fluorescent phosphatidylcholine (PC) in HepG2 cells using the fluorescent analogs of PC C6-NBD-PC and beta-BODIPY-PC. Fluorescent PC was efficiently transported to the biliary canaliculus (BC) and became enriched on the lumenal side of the canalicular membrane as shown for C6-NBD-PC. Some fluorescent PC was transported in vesicles to a subapical compartment (SAC) or apical recycling compartment (ARC) in polarized HepG2 cells as shown by colocalization with fluorescent sphingomyelin (C6-NBD-SM) and fluorescent transferrin, respectively. Extensive trafficking of vesicles containing fluorescent PC between the basolateral domain, the SAC/ARC and the BC as well as endocytosis of PC analogs from the canalicular membrane were found. Evidence for nonvesicular transport included enrichment of the PC-analog beta-BODIPY-PC in the BC (t1/2 = 3.54 min) prior to its accumulation in the SAC/ARC (t1/2 = 18.5 min) at 37 degrees C. Transport of fluorescent PC to the canalicular membrane also continued after disruption of the actin or microtubule cytoskeleton and at 2 degrees C. These results indicate that: (i) a nonvesicular transport pathway significantly contributes to the canalicular enrichment of PC in hepatocytic cells, and (ii) vesicular transport of fluorescent PC occurs from both membrane domains via the SAC/ARC.
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