In vitro microtubule-based organelle transport in wild-type Dictyostelium and cells overexpressing a truncated dynein heavy chain.
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.
Pollock
N
N
Department of Pharmacology, University of California San Francisco, 94143, USA.
Koonce
M P
MP
de Hostos
E L
EL
Vale
R D
RD
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D004398:Dyneins / Q000096:biosynthesis*; D008081:Liposomes / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D009154:Mutation; D015388:Organelles / Q000378:metabolism*
D008081:Liposomes; D004398:Dyneins