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.
Skip Nav Destination
Article navigation
2 October 2000
Report|
October 02 2000
Microtubule and Motor-Dependent Endocytic Vesicle Sorting in Vitro
Eustratios Bananis,
Eustratios Bananis
aMarion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
bDepartment of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Search for other works by this author on:
John W. Murray,
John W. Murray
aMarion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
bDepartment of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Search for other works by this author on:
Richard J. Stockert,
Richard J. Stockert
aMarion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
Search for other works by this author on:
Peter Satir,
Peter Satir
aMarion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
bDepartment of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Search for other works by this author on:
Allan W. Wolkoff
Allan W. Wolkoff
aMarion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
bDepartment of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Search for other works by this author on:
Eustratios Bananis
aMarion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
bDepartment of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
John W. Murray
aMarion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
bDepartment of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Richard J. Stockert
aMarion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
Peter Satir
aMarion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
bDepartment of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Allan W. Wolkoff
aMarion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461
bDepartment of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461
Abbreviations used in this paper: AMP-PNP, 5′-adenylylimido-diphosphate; ASGPR, asialoglycoprotein receptor; ASOR, asialoorosomucoid; MT, microtubule.
Received:
April 24 2000
Revision Requested:
August 24 2000
Accepted:
August 24 2000
Online ISSN: 1540-8140
Print ISSN: 0021-9525
© 2000 The Rockefeller University Press
2000
The Rockefeller University Press
J Cell Biol (2000) 151 (1): 179–186.
Article history
Received:
April 24 2000
Revision Requested:
August 24 2000
Accepted:
August 24 2000
Citation
Eustratios Bananis, John W. Murray, Richard J. Stockert, Peter Satir, Allan W. Wolkoff; Microtubule and Motor-Dependent Endocytic Vesicle Sorting in Vitro. J Cell Biol 2 October 2000; 151 (1): 179–186. doi: https://doi.org/10.1083/jcb.151.1.179
Download citation file:
Sign in
Don't already have an account? Register
Client Account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.
Sign in via your Institution
Sign in via your InstitutionSee also
Email alerts
Advertisement
Advertisement