TOGp regulates microtubule assembly and density during mitosis and contributes to chromosome directional instability

Abstract

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.

Specific removal of TACC3/ch-TOG/clathrin at metaphase deregulates kinetochore fiber tension

“…We note that a pool of ch-TOG was retained at the spindle pole after TACC3 KS (supplementary material Fig. S1), which presumably corresponds to a functionally separate ch-TOG population, as previously described Cassimeris et al, 2009;Gergely et al, 2003). Since the discovery of clathrin as a binding partner of TACC3-ch-TOG, it has been debated whether TACC3-ch-TOG can operate independently of this complex Fu et al, 2011;Fu et al, 2010;Hubner et al, 2010;Lin et al, 2010;Royle, 2012).…”

Automatic quantification of microtubule dynamics enables RNAi‐screening of new mitotic spindle regulators

“…Also in vitro studies have suggested that XMAP215 participates in the process of tubulin polymerization by binding to microtubule ends and catalyzing the addition of up to 25 tubulin dimers [Brouhard et al, 2008]. Since depletion of ch-TOG by siRNA in cells was shown to result in disorganized spindle poles and aberrant spindle morphologies [Gergely et al, 2003;Cassimeris and Morabito, 2004;Cassimeris et al, 2009], we can now confirm that such a phenotype is a result of a direct effect on microtubule dynamics. Most ch-TOG-depleted cells were indeed not able to assemble a functional spindle and remained blocked in mitosis (data not shown).…”

Prevention and Correction Mechanisms behind Anaphase Synchrony: Implications for the Genesis of Aneuploidy

“…During mitosis CLASPs are required for the incorporation and translocation/flux of microtubule subunits on kinetochore microtubules as well as to promote microtubule destabilization at kinetochores [Maiato et al, 2005;Maffini et al, 2009]. This is apparently counterintuitive for a family of proteins that promote microtubule stability during interphase [Galjart, 2005] but appears not to be an exception amongst classic microtubule-stabilizing proteins such as ch-TOG/XMAP215, which were also recently shown to induce microtubule destabilization at kinetochores [Cassimeris et al, 2009]. These findings suggest that the normal microtubule stabilizing role of CLASPs (and likely other kinetochore proteins) is being negatively regulated.…”