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Published online 2 October 2000. doi:10.1083/jcb.151.1.179
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© The Rockefeller University Press, 0021-9525/2000//179 $5.00
The Journal of Cell Biology, Volume 151, Number 1, , 2000 179-186

Report

Microtubule and Motor-Dependent Endocytic Vesicle Sorting in Vitro



Eustratios Bananisa,b, John W. Murraya,b, Richard J. Stockerta, Peter Satira,b, and Allan W. Wolkoffa,b

a Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
b Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Marion Bessin Liver Research Center, 611 Ullmann Bldg., Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461.(718) 430-8975(718) 430-2584

Endocytic vesicles undergo fission to sort ligand from receptor. Using quantitative immunofluorescence and video imaging, we provide the first in vitro reconstitution of receptor–ligand sorting in early endocytic vesicles derived from rat liver. We show that to undergo fission, presegregation vesicles must bind to microtubules (MTs) and move upon addition of ATP. Over 13% of motile vesicles elongate and are capable of fission. After fission, one vesicle continues to move, whereas the other remains stationary, resulting in their separation. On average, almost 90% receptor is found in one daughter vesicle, whereas ligand is enriched by ~300% with respect to receptor in the other daughter vesicle. Although studies performed on polarity marked MTs showed approximately equal plus and minus end–directed motility, immunofluorescence microscopy revealed that kinesins, but not dynein, were associated with these vesicles. Motility and fission were prevented by addition of 1 mM 5'-adenylylimido-diphosphate (AMP-PNP, an inhibitor of kinesins) or incubation with kinesin antibodies, but were unaffected by addition of 5 µM vanadate (a dynein inhibitor) or dynein antibodies. These studies indicate an essential role of kinesin-based MT motility in endocytic vesicle sorting, providing a system in which factors required for endocytic vesicle processing can be identified and characterized.

Key Words: endocytosis • microtubules • kinesin • dynein • motility

© 2000 The Rockefeller University Press

Abbreviations used in this paper: AMP-PNP, 5'-adenylylimido-diphosphate; ASGPR, asialoglycoprotein receptor; ASOR, asialoorosomucoid; MT, microtubule.


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