Reconstitution of ATP-dependent Movement of Endocytic Vesicles Along Microtubules In Vitro: An Oscillatory Bidirectional Process

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Abstract
We have previously used the asialoglycoprotein receptor system to elucidate the pathway of hepatocytic processing of ligands such as asialoorosomucoid (ASOR). These studies suggested that endocytic vesicles bind to and travel along microtubules under the control of molecular motors such as cytoplasmic dynein. We now report reconstitution of this process in vitro with the use of a microscope assay to observe the interaction of early endocytic vesicles containing fluorescent ASOR with fluorescent microtubules. We find that ASOR-containing endosomes bind to microtubules and translocate along them in the presence of ATP. This represents the first time that mammalian endosomes containing a well-characterized ligand have been directly observed to translocate on microtubules in vitro. The endosome movement does not require cytosol or exogenous motor protein, is oscillatory, and is directed toward the plus and minus ends at equal frequencies. We also observe endosomes being stretched in opposite directions along microtubules, suggesting that microtubules could provide a mechanical basis for endocytic sorting events. The movement of endosomes in vitro is consistent with the hypothesis that microtubules actively participate in the sorting and distribution of endocytic contents.