TOGp regulates microtubule assembly and density during mitosis and contributes to chromosome directional instability.
TOGp, a member of the XMAP215 MAP family, is required for bipolar mitotic spindle assembly. To understand how TOGp contributes to spindle assembly, we examined microtubule dynamics after depleting TOGp by siRNA. Fluorescence recovery after photobleaching of GFP-tubulin demonstrated that spindle microtubule turnover is slowed two-fold in the absence of TOGp. Consistent with photobleaching results, microtubule regrowth after washout of the microtubule depolymerizing drug nocodazole was slower at the centrosomes and in the vicinity of mitotic chromatin in cells depleted of TOGp. The slower microtubule turnover is likely due to either nucleation or the transitions of dynamic instability because TOGp depletion did not effect the rate of plus end growth, measured by tracking EB1-GFP at microtubule ends. In contrast, microtubule regrowth after nocodazole washout was unaffected by prior depletion of TACC3, a centrosomal protein that interacts with TOGp. Kinetochore fibers in both untreated and TOGp-depleted cells were stable to incubation at 4 degrees C or lysis in buffer containing calcium indicating that stable kinetochore-microtubule attachments are formed in the absence of TOGp. Depletion of TOGp, but not TACC3, reduced kinetochore oscillations during prometaphase/metaphase. Defects in oscillations are not due simply to multipolarity or loss of centrosome focus in the TOGp-depleted cells, since kinetochore oscillations appear normal in cells treated with the proteosome inhibitor MG132, which also results in multipolar spindles and centrosome fragmentation. We hypothesize that TOGp is required for chromosome motility as a downstream consequence of reduced microtubule dynamics and/or density. Cell Motil. Cytoskeleton 2009. (c) 2009 Wiley-Liss, Inc.
Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania, USA. lc07@lehigh.edu
Lynne
L
Cassimeris
Bret
B
Becker
Bruce
B
Carney
D043171:Chromosomal Instability / Q000235:genetics / Q000502:physiology*; D036681:Fluorescence Recovery After Photobleaching; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis / Q000235:genetics / Q000502:physiology*; D034741:RNA, Small Interfering; D014404:Tubulin / Q000235:genetics / Q000378:metabolism
C454180:CKAP5 protein, human; D008869:Microtubule-Associated Proteins; D034741:RNA, Small Interfering; D014404:Tubulin; D049452:Green Fluorescent Proteins