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• W2160376121 abstract "The transport of vesicular organelles along microtubules has been well documented in a variety of systems, but the molecular mechanisms underlying this process are not well understood. We have developed a method for preparing extracts from Dictyostelium discoideum which supports high levels of bidirectional, microtubule-based vesicle transport in vitro. This organelle transport assay was also adapted to observe specifically the motility of vesicles in the endocytic pathway. Vesicle transport can be reconstituted by recombining a high-speed supernatant with KI-washed organelles, which do not move in the absence of supernatant. Furthermore, a microtubule affinity-purified motor fraction supports robust bidirectional movement of the salt-washed organelles. The plus and minus end-directed transport activities can be separated by exploiting differences in their affinities for microtubules in the presence of 0.3 M KCl. We also used our assay to examine organelle transport in a strain of Dictyostelium overexpressing a 380-kDa C-terminal fragment of the cytoplasmic dynein heavy chain, which displays an altered microtubule pattern (380-kDa cells; [Koonce and Samso, Mol. Biol. Cell 7:935–948, 1996]). We have found that the frequency and velocity of minus end-directed membrane organelle movements were significantly reduced in 380-kDa cells relative to wild-type cells, while the frequency and velocity of plus end-directed movements were equivalent in the two cell types. The 380-kDa C-terminal fragment cosedimented with membrane organelles, although its affinity was significantly lower than that of native dynein. An impaired membrane-microtubule interaction may be responsible for the altered microtubule patterns in the 380-kDa cells. Cell Motil. Cytoskeleton 40:304–314, 1998. © 1998 Wiley-Liss, Inc." @default.
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• W2160376121 date "1998-01-01" @default.
• W2160376121 modified "2023-10-15" @default.
• W2160376121 title "In vitro microtubule-based organelle transport in wild-typeDictyostelium and cells overexpressing a truncated dynein heavy chain" @default.
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• W2160376121 doi "https://doi.org/10.1002/(sici)1097-0169(1998)40:3<304::aid-cm8>3.0.co;2-c" @default.
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