Vesicular and nonvesicular transport of phosphatidylcholine in polarized HepG2 cells.
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.
Wüstner
D
D
Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie/Biophysik, Invalidenstr. 43, D-10115 Berlin, Germany.
Mukherjee
S
S
Maxfield
F R
FR
Müller
P
P
Herrmann
A
A
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D001647:Bile Acids and Salts / Q000378:metabolism; D001648:Bile Canaliculi / Q000166:cytology / Q000378:metabolism; D001693:Biological Transport, Active; D001896:Boron Compounds / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D003599:Cytoskeleton / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D010713:Phosphatidylcholines / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism; D013997:Time Factors; D014168:Transferrin / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism*; D014407:Tumor Cells, Cultured
C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001647:Bile Acids and Salts; D001896:Boron Compounds; D005452:Fluoresceins; D005456:Fluorescent Dyes; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D014168:Transferrin; C090312:cholylglycylamidofluorescein; C044745:1-palmitoyl-2-(6-(N-(7-nitrobenz)-2-oxa-1,3-diazol-4-yl)aminocaproyl)phosphatidylcholine; C081708:N-(N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-epsilon-aminohexanoyl)sphingosylphosphorylcholine; D009327:4-Chloro-7-nitrobenzofurazan