Quantification of polarized trafficking of transferrin and comparison with bulk membrane transport in hepatic cells.
Transport of the recycling marker transferrin was analysed in polarized hepatic HepG2 cells using quantitative fluorescence microscopy and mathematical modelling. A detailed map and kinetic model for transport of transferrin in hepatic cells was developed. Fluorescent transferrin was found to be transported sequentially through basolateral SE (sorting endosomes) to a SAC/ARC (subapical compartment/apical recycling compartment). DiI (di-indocarbocyanine) lipid probes of different acyl chain length (DiIC12 and DiIC16) co-localized with transferrin in basolateral SE and in the SAC/ARC. By kinetic comparison of hepatic transport of transferrin and labelled HDL (high-density lipoprotein), it is shown that transport of transferrin from SE to the SAC/ARC follows a default pathway together with HDL. Kinetic modelling of fluorescence data provides an identical half-time for SE-to-SAC/ARC transport of transferrin and fluorescent HDL (t(1/2)=4.2 min). Fluorescent transferrin was found to recycle with a half-time of t(1/2)=12.9 min from the SAC/ARC to the basolateral cell surface of HepG2 cells. In contrast with HDL, targeting of labelled transferrin from the SAC/ARC to the apical biliary canaliculus was negligible. The results indicate that transport from basolateral hepatic SE to the SAC/ARC represents a bulk flow process and that polarized sorting occurs mainly at the level of the SAC/ARC.
Wüstner
Daniel
D
Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark. wuestner@bmb.sdu.dk
D001692:Biological Transport; D002232:Carbocyanines / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology; D011992:Endosomes / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D006834:Hydrazines / Q000378:metabolism; D007700:Kinetics; D008075:Lipoproteins, HDL / Q000378:metabolism; D008113:Liver Neoplasms; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D014168:Transferrin / Q000378:metabolism*
C417561:Alexa 488 hydrazide; D002232:Carbocyanines; D005456:Fluorescent Dyes; D006834:Hydrazines; D008075:Lipoproteins, HDL; D014168:Transferrin