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    • Albert Einstein College of Medicine: Doctoral Dissertations
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    Vesicular trafficking in hepatocytes: Roles of motor and Rab GTPases in endocytic vesicle processing

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    Date
    2004
    Author
    Bananis, Eustratios
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    Abstract
    Hepatocytes take up the desialylated glycoprotein asialoorosomucoid (ASOR) through receptor-mediated endocytosis via the asialoglycoprotein receptor (ASGPR). ASOR and ASGPR dissociate from each other within early endocytic vesicles that subsequently undergo fission resulting in the segregation of receptor from ligand. Ligands destined for degradation traffic within late endocytic vesicles to lysosomes. Receptor-enriched vesicles are recycled back to the cell surface. The mechanism by which endocytic vesicles undergo fission and sort ligand from receptor is unclear, although previous studies suggested a role for microtubules (MTs) and molecular motors in this process.;To characterize and identify essential components of endocytic processing and to elucidate the roles of motors in ASGPR-mediated endocytosis in hepatocytes, we developed a novel in vitro fluorescent microscopy-based system. Fluorescent endocytic vesicles were prepared from rat livers 5 min (early) and 15 min (late) after portal venous injection of fluorescent ASOR. Vesicles were drawn into a 3 mul motility chamber in which fluorescent MTs had been attached. Motility of vesicles on MTs was recorded in real time after ATP addition. Proteins associated with vesicles were immunolocalized by perfusion of specific antibodies without fixation. In addition, we devised flow-cytometry-based methodology to purify fluorescent ligand-containing early and late vesicles enabling biochemical identification of specific proteins.;These studies revealed that both vesicle populations associate with different cohorts of motor and accessory proteins. Early vesicles containing ASOR and ASGPR moved bi-directionally and underwent fission into two daughter vesicles along microtubules using kinesin I and KIFC2. Following fission, ∼90% of receptor was found in one daughter vesicle, whereas ligand was enriched by ∼300% with respect to receptor in the other daughter vesicle. Conversion of Rab4-GTP, that was bound to these vesicles, to Rab4-GDP, was associated with increased KIFC2-mediated minus-end directed motility and vesicle fission. In contrast to early vesicles, late vesicles had reduced association with ASGPR and Rab4 and did not undergo fission. Bi-directional microtubule-based movement of late vesicles was mediated by dynein and KIF3A through the interaction with the dynactin complex. These studies indicate that during maturation and movement along MTs toward lysosomes, endocytic vesicles acquire and exchange specific motor, regulatory and scaffold proteins.
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    https://yulib002.mc.yu.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:3148808
    https://hdl.handle.net/20.500.12202/736
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    • Albert Einstein College of Medicine: Doctoral Dissertations [1674]

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