Identification and immunolocalization of cytoplasmic dynein in Dictyostelium.
A high molecular weight microtubule binding protein has been isolated from homogenates of Dictyostelium. Because of its sedimentation velocity (20s), ATP-sensitive binding to microtubules, UV-vanadate-ATP mediated fragmentation, prominent CTPase activity, and its ability to produce limited microtubule movement in vitro, we consider this protein to be a form of cytoplasmic dynein. A polyclonal antibody monospecific to this protein was produced, and dynein's intracellular distribution in ameboid cells was examined by immunofluorescence. The antibody labels a punctate cytoplasmic pattern, localizes to a spherical region adjacent to the nucleus, and also appears to label the nuclei. The punctate staining pattern is consistent with cytoplasmic dynein's proposed function in organelle transport. The spherical juxtanuclear object stained is coincident with this cell's microtubule organizing center, an obvious termination point for minus-end directed microtubule motors. By immunofluorescence, there does not appear to be a substantial amount of dynein in the intranuclear mitotic spindles of Dictyostelium. These data provide evidence for localization of cytoplasmic dynein in cells, and suggest that Dictyostelium will be a useful system in which to study the molecular biology of microtubule-associated motor enzymes.
Koonce
M P
MP
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder 80309.
McIntosh
J R
JR
D000251:Adenosine Triphosphatases / Q000032:analysis*; D003593:Cytoplasm / Q000032:analysis; D004023:Dictyostelium / Q000032:analysis* / Q000648:ultrastructure; D004398:Dyneins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D008870:Microtubules / Q000378:metabolism; D008970:Molecular Weight; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011755:Pyrophosphatases; D008941:Spindle Apparatus / Q000032:analysis
D010744:Phosphoric Monoester Hydrolases; D000251:Adenosine Triphosphatases; C019950:CTPase; D011755:Pyrophosphatases; D004398:Dyneins