Microtubule-Dependent Movement of Late Endocytic Vesicles In Vitro: Requirements for Dynein and Kinesin

• DOI: 10.1091/mbc.e04-04-0278
• Publication Date: 2004-06-08T01:08:40Z
• Authors: Eustratios Bananis; Sangeeta Nath; Kristie Gordon; Peter Satir; Richard J. Stockert; John W. Murray; Allan W. Wolkoff
• Keywords: Kinesin; Dynactin

Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor move undergo fission along microtubules using kinesin I KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare late were devised. addition, flow cytometry was utilized highly purified vesicles, permitting validation microscopy-based experiments as well direct biochemical analysis. These revealed bound moved microtubules, but in contrast did not fission. As compared had reduced association receptor, Rab4, associated dynein, Rab7, dynactin, KIF3A. Dynein KIF3A antibodies inhibited vesicle motility, whereas no effect. Dynamitin prevented microtubules. results indicate acquisition exchange specific motor regulatory proteins characterizes may regulate transition vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis mapping vesicle-associated proteins.