Mutants of the Drosophila ncd microtubule motor protein cause centrosomal and spindle pole defects in mitosis.
Nonclaret disjunctional (ncd) is a kinesin-related microtubule motor protein required for meiotic and early mitotic chromosome distribution in Drosophila. ncd translocates on microtubules with the opposite polarity to kinesin, toward microtubule minus ends, and is associated with spindles in chromosome/spindle preparations. Here we report a new mutant of ncd caused by partial deletion of the predicted coiled-coil central stalk. The mutant protein exhibits a velocity of translocation and ability to generate torque in motility assays comparable to near full-length ncd, but only partially rescues a null mutant for chromosome mis-segregation. Antibody staining experiments show that the partial loss-of-function and null mutants cause centrosomal and spindle pole defects, including centrosome splitting and loss of centrosomes from spindle poles, and localize ncd to centrosomes as well as spindles of wild-type embryos. Association of ncd with spindles and centrosomes is microtubule- and cell cycle-dependent: inhibition of microtubule assembly with colchicine abolishes ncd staining and centrosomal staining is observed in prometaphase, metaphase and anaphase, but diminishes in late anaphase/telophase. The cell cycle dependence of centrosomal staining and the defects of mutants provide clear evidence for activity of the ncd motor protein near or at the spindle poles in mitosis. The ncd motor may interact with centrosomal microtubules and spindle fibers to attach centrosomes to spindle poles, and mediate poleward translocation (flux) of kinetochore fibers, a process that may underlie poleward movement of chromosomes in mitosis. Together with previous work, our findings indicate that ncd is important in maintaining spindle poles in mitosis as well as in meiosis.
Endow
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SA
Department of Microbiology, Duke University Medical Center, Durham, North Carolina 27710.
Chandra
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Komma
D J
DJ
Yamamoto
A H
AH
Salmon
E D
ED
D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D002869:Chromosome Aberrations / Q000235:genetics; D025063:Chromosome Disorders; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000166:cytology / Q000235:genetics*; D005260:Female; D016547:Kinesins; D008297:Male; D008868:Microtubule Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008938:Mitosis; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009630:Nondisjunction, Genetic; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D008941:Spindle Apparatus / Q000737:chemistry* / Q000648:ultrastructure
D029721:Drosophila Proteins; D008868:Microtubule Proteins; D011993:Recombinant Fusion Proteins; C066696:ncd protein, Drosophila; D016547:Kinesins