Excessive expression of the plant kinesin TBK5 converts cortical and perinuclear microtubules into a radial array emanating from a single focus.
TBK5 is a plant-specific kinesin constantly expressed in tobacco BY-2 cells. An analysis of the distribution of green fluorescent protein-tagged TBK5 (GFP-TBK5) transiently expressed in BY-2 protoplasts revealed that TBK5 could associate with microtubules in vivo. GFP-TBK5 often assembled to form a single particle when accumulated in cells. The particle was located in close proximity to the nucleus, and its formation was accompanied by the development of a radial array of microtubules emanating from it and the loss of cortical microtubules. Microtubule depolymerization by treatment with propyzamide inhibited particle formation and stimulated the formation of dispersed aggregates of GFP-TBK5. Through expression of different TBK5 mutants as GFP fusions, the motor domain, two separated coiled-coil domains and the C-terminal domain of TBK5 were identified as the domains playing essential roles in particle formation. Mutants with putatively non-motile motor domains or lacking the C-terminal domain were localized to cortical and perinuclear microtubules, whereas those lacking either of the coiled-coil domains were preferentially distributed around the nucleus and along perinuclar microtubules. Further, the deletion of one of the coiled-coil domains or the C-terminal domain was sufficient to inhibit the propyzamide-induced formation of dispersed aggregates, whereas the mutation in the motor domain was not. These results led us to propose a model in which the particle is formed through the microtubule-based movement of GFP-TBK5 toward the nucleus and subsequent microtubule-independent aggregation based on coiled-coil interactions. The dramatic microtubule rearrangement would be explained if GFP-TBK5 relocated and gathered newly formed microtubules and/or microtubule-nucleating units.
Goto
Yuhei
Y
Department of Biological Science, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043 Japan.
Asada
Tetsuhiro
T
D001549:Benzamides / Q000494:pharmacology; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003572:Cytochalasins / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D018506:Gene Expression Regulation, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D011523:Protoplasts / Q000166:cytology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D001549:Benzamides; D003572:Cytochalasins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C001059:pronamide; C007215:cytochalasin E; D016547:Kinesins