Molecular determinants of endosome trafficking in hepatocytes
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To understand the molecular determinants of receptor-mediated endocytosis (RME) in hepatocytes, Texas red asialoorosomucoid loaded early endocytic vesicles were prepared from mouse liver. These mouse liver endocytic vesicles moved bidirectionally on microtubules upon ATP addition in vitro, frequently undergoing fission into two daughter vesicles. Studies with specific antibodies revealed that dynein was not associated with these vesicles and that Kif5B and the minus-end kinesin, Kifc1 mediated their plus- and minus-end motility, respectively. Over 90% of vesicles associated with Kifcl also contained Kif5B, and inhibition of Kifc1 with antibody resulted in enhancement of plus-end directed motility. There was reduced vesicle fission when either Kifcl or Kif5B activity was inhibited by antibody, indicating that the opposing forces resulting from activity of both motors are required for fission to occur. Immunoprecipitation of native Kif5B by FLAG antibody following expression of FLAG-Kifc1 in 293T cells indicated that these two motors can interact with each other. Whether they interact directly or through a complex of potential regulatory proteins will need to be clarified in future studies. However, the present studies show that coordinated activity of these kinesins is essential for motility and processing of early endocytic vesicles.;These studies demonstrated that components of RME could vary between species such as rat and mouse. To elucidate mechanistic components of endocytosis in human hepatocytes, six mutants (Trf2-7), defective in RME were isolated from human hepatoma cells (HuH7) by a dual selection protocol. Using time-lapse fluorescence microscopy, endocytosis in living HuH7 cells as well as in these trafficking mutants was followed. The trafficking mutants (Trfl-7) were defective in endosome maturation as fluorescence content of individual vesicles remain unaltered over time whereas in parental HuH7 cells fluorescence content of individual vesicles increases. Identification of proteins that revert these phenotypes to normal should provide insights into novel endocytic regulatory mechanisms.
Source: Dissertation Abstracts International, Volume: 68-08, Section: B, page: 4939.;Advisors: Allan W. Wolkoff.