Reciprocal localization of Nod and kinesin fusion proteins indicates microtubule polarity in the Drosophila oocyte, epithelium, neuron and muscle.
Polarization of the microtubule cytoskeleton is an early event in establishment of anterior-posterior polarity for the Drosophila oocyte. During stages 8-9 of oogenesis, when oskar mRNA is transported to the posterior pole of the oocyte, a fusion protein consisting of the plus-end-directed microtubule motor kinesin and beta-galactosidase (Kin:beta gal) similarly localizes to the posterior pole, thereby suggesting that plus ends of microtubules are pointed to the posterior. In this paper, we have substituted the motor domain of Kin:beta gal with the putative motor domain (head) from the kinesin-related protein Nod. In cells with defined microtubule polarity, the Nod:beta gal fusion protein is an in vivo minus-end reporter for microtubules. Nod:beta gal localizes to apical cytoplasm in epithelial cells and to the poles of mitotic spindles in dividing cells. In stage 8-10 oocytes, the Nod fusion localizes to the anterior margin, thus supporting the hypothesis that minus ends of microtubules at these stages are primarily at the anterior margin of the oocyte. The fusion protein also suggests a polarity to the microtubule cytoskeleton of dendrites and muscle fibers, as it accumulates at the ends of dendrites in the embryonic PNS and is excluded from terminal cytoplasm in embryonic muscle. Finally, the reciprocal in vivo localization of Nod:beta gal and Kin:beta gal suggests that the head of Nod may be a minus-end-directed motor.
Clark
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Department of Biochemistry and HHMI, University of California at San Francisco, 94143, USA.
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D000818:Animals; D030801:Animals, Genetically Modified; D003433:Crosses, Genetic; D017931:DNA Primers; D003712:Dendrites / Q000502:physiology*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster; D004625:Embryo, Nonmammalian / Q000502:physiology; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology; D005260:Female; D016547:Kinesins / Q000096:biosynthesis*; D008297:Male; D008868:Microtubule Proteins / Q000096:biosynthesis*; D008870:Microtubules / Q000502:physiology* / Q000648:ultrastructure; D009132:Muscles / Q000166:cytology / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000502:physiology*; D009865:Oocytes / Q000166:cytology / Q000502:physiology*; D009866:Oogenesis; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D011995:Recombination, Genetic; D008941:Spindle Apparatus; D001616:beta-Galactosidase / Q000096:biosynthesis
D017931:DNA Primers; D029721:Drosophila Proteins; D008868:Microtubule Proteins; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase; C080298:Nod protein, Drosophila; D016547:Kinesins