Interaction of the Microtubule Cytoskeleton with Endocytic Vesicles and Cytoplasmic Dynein in Cultured Rat Hepatocytes (*)
- Hitoshi Oda(1),
- Richard J. Stockert(1),
- Christine Collins(3),
- Hali Wang(2),
- Phyllis M. Novikoff(1),
- Peter Satir(2) and
- Allan W. Wolkoff(1)(2)(§)
- From the (1) Marion Bessin Liver Research Center and
- (2)Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461 and the
- (3)Department of Cell, Molecular, and Structural Biology, Northwestern University Medical School, Chicago, Illinois 60611
- § To whom all correspondence should be addressed: Liver Research Ctr., Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2584. Fax: 718-918-0857.
Abstract
In a recent study (Goltz, J. S., Wolkoff, A. W., Novikoff, P. M., Stockert, R. J., and Satir, P.(1992) Proc. Natl. Acad. Sci. U. S. A. 89, 7026-7030), we found that ligand- and receptor-containing endocytic vesicles bind to endogenous microtubules in vitro after 60 min of receptor-mediated endocytosis of asialo-orosomucoid. In the presence of ATP, ligand-containing endocytic vesicles are released from microtubules, while those containing receptor are not. We hypothesized that cytoplasmic dynein may associate with ligand-containing, but not receptor-containing, domains of endocytic vesicles and might be involved in the movement of ligand-containing vesicles along microtubules during sorting of ligand from receptor. Direct evidence in support of this hypothesis has been obtained in the present study. Binding of ligand-containing vesicles to microtubules correlates highly (p < 0.001) with binding of dynein, but not kinesin, under a variety of conditions. Binding of receptor-containing vesicles to microtubules is independent of both cytoplasmic dynein and kinesin binding. Tight association of cytoplasmic dynein with a population of ligand-containing vesicles is seen directly by immunoprecipitation. These results support the view that in receptor-mediated endocytosis, ligand-containing vesicles become bound to microtubules by cytoplasmic dynein. While receptor domains of endosomes remain attached to microtubules in an ATP-independent manner, ligand-containing domains might be moved away toward pericentrosomal lysosomes by this motor molecule.
Footnotes
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↵* This work was supported by Program Project Grant DK-41918 and National Institutes of Health Grants CA-06576 and DK-41296. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- RME
-
receptor-mediated endocytosis
- ASOR
-
asialo-orosomucoid
- MT
-
microtubule
- IC
-
intermediate chain
- HC
-
head chain
- FITC
-
fluorescein isothiocyanate
- AMP-PCP
-
adenosine 5′-(β,
-methylene)triphosphate
- AMP-PNP
-
adenosine −5′(β,
-imino)triphosphate
- PAGE
-
polyacrylamide gel electrophoresis
- PIPES
-
1,4-piperazinediethanesulfonic acid
- TBS
-
Tris-buffered saline
- SFM
-
serum-free medium.
