Live-cell analysis of mitotic spindle formation in taxol-treated cells.
Taxol functions to suppress the dynamic behavior of individual microtubules, and induces multipolar mitotic spindles. However, little is known about the mechanisms by which taxol disrupts normal bipolar spindle assembly in vivo. Using live imaging of GFP-alpha tubulin expressing cells, we examined spindle assembly after taxol treatment. We find that as taxol-treated cells enter mitosis, there is a dramatic re-distribution of the microtubule network from the centrosomes to the cell cortex. As they align there, the cortical microtubules recruit NuMA to their embedded ends, followed by the kinesin motor HSET. These cortical microtubules then bud off to form cytasters, which fuse into multipolar spindles. Cytoplasmic dynein and dynactin do not re-localize to cortical microtubules, and disruption of dynein/dynactin interactions by over-expression of p50 "dynamitin" does not prevent cytaster formation. Taxol added well before spindle poles begin to form induces multipolarity, but taxol added after nascent spindle poles are visible-but before NEB is complete-results in bipolar spindles. Our results suggest that taxol prevents rapid transport of key components, such as NuMA, to the nascent spindle poles. The net result is loss of mitotic spindle pole cohesion, microtubule re-distribution, and cytaster formation.
Hornick
Jessica E
JE
Department of Biological Sciences and Notre Dame Integrated Imaging Facility, University of Notre Dame, Notre Dame, Indiana 46556, USA. Hinchcliffe.3@nd.edu
Bader
Jason R
JR
Tribble
Emily K
EK
Trimble
Kayleigh
K
Breunig
J Scott
JS
Halpin
Elizabeth S
ES
Vaughan
Kevin T
KT
Hinchcliffe
Edward H
EH
D000818:Animals; D002460:Cell Line; D018385:Centrosome / Q000187:drug effects / Q000378:metabolism; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D017239:Paclitaxel / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000187:drug effects / Q000378:metabolism*; D014162:Transfection; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D050257:Tubulin Modulators / Q000494:pharmacology
D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D050257:Tubulin Modulators; D049452:Green Fluorescent Proteins; D004398:Dyneins; D017239:Paclitaxel