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Vol. 11, Issue 2, 419-433, February 2000
Marion Bessin Liver Research Center, Albert Einstein College of
Medicine, Bronx, New York 10461
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.
Online version of this article contains video
material for Figures 6-8 and 11. Online version available at
www.molbiolcell.org.
*
Corresponding author. E-mail address:
wolkoff{at}aecom.yu.edu.
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