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Vol. 15, Issue 8, 3688-3697, August 2004
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* Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York 10461;
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461; and
Fluorescence Activated Cell Sorting Facility, Albert Einstein College of Medicine, Bronx, New York 10461
Submitted April 1, 2004;
Revised May 25, 2004;
Accepted May 27, 2004
Monitoring Editor: J. Richard McIntosh
Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.
Abbreviations used: AMP-PNP, 5'-adenylylimido-diphosphate; ASGPR, asialoglycoprotein receptor; ASOR, asialoorosomucoid; HC, heavy chain; IC, intermediate chain; MT, microtubule.
Corresponding author. E-mail address: wolkoff{at}aecom.yu.edu.
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