Microtubule motor Ncd induces sliding of microtubules in vivo.
The mitotic spindle is a microtubule (MT)-based molecular machine that serves for equal segregation of chromosomes during cell division. The formation of the mitotic spindle requires the activity of MT motors, including members of the kinesin-14 family. Although evidence suggests that kinesins-14 act by driving the sliding of MT bundles in different areas of the spindle, such sliding activity had never been demonstrated directly. To test the hypothesis that kinesins-14 can induce MT sliding in living cells, we developed an in vivo assay, which involves overexpression of the kinesin-14 family member Drosophila Ncd in interphase mammalian fibroblasts. We found that green fluorescent protein (GFP)-Ncd colocalized with cytoplasmic MTs, whose distribution was determined by microinjection of Cy3 tubulin into GFP-transfected cells. Ncd overexpression resulted in the formation of MT bundles that exhibited dynamic "looping" behavior never observed in control cells. Photobleaching studies and fluorescence speckle microscopy analysis demonstrated that neighboring MTs in bundles could slide against each other with velocities of 0.1 microm/s, corresponding to the velocities of movement of the recombinant Ncd in in vitro motility assays. Our data, for the first time, demonstrate generation of sliding forces between adjacent MTs by Ncd, and they confirm the proposed roles of kinesins-14 in the mitotic spindle morphogenesis.
Oladipo
Abiola
A
Department of Cell Biology and Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06032-1507, USA.
Cowan
Ann
A
Rodionov
Vladimir
V
D000818:Animals; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D016547:Kinesins / Q000378:metabolism*; D007700:Kinetics; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D020409:Molecular Motor Proteins / Q000378:metabolism*; D017239:Paclitaxel / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D029721:Drosophila Proteins; D020409:Molecular Motor Proteins; D011993:Recombinant Fusion Proteins; C066696:ncd protein, Drosophila; D049452:Green Fluorescent Proteins; D016547:Kinesins; D017239:Paclitaxel