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. Author manuscript; available in PMC: 2017 Oct 24.
Published in final edited form as: Nat Cell Biol. 2017 Apr 24;19(5):480–492. doi: 10.1038/ncb3511

Figure 8. Dissociation of katanin from ASPM impairs spindle orientation and poleward flux.

Figure 8

(a) Western blotting with the indicated antibodies of extracts of wild type (WT) and p80Y571A HeLa and U2OS cells. Katanin protein levels in WT and p80Y571A mutant cell lines are similar.

(b-d) Immunofluorescence staining and quantification of p80 and ASPM intensities at spindle poles in WT and p80Y571A HeLa cells. For p80 intensity, n=204 spindle poles, WT; n=220, p80Y571A; for ASPM intensity, n = 244, WT; n=240, p80Y571A. Scale bars, 5 μm.

(e,f) Orthogonal view (x-z) of metaphase HeLa cells stained for CEP135 and DAPI in WT, p80Y571A HeLa cells (e) and quantification of spindle angles (f; n=40 cells, WT and p80Y571A). Scale bar, 5 μm.

(g) Immunostaining for α-tubulin, CEP135 and DNA (DAPI) in WT and p80Y571A HeLa cells. Maximum intensity projections of Z series with ~40 stacks and 200 nm step were shown here. Scale bar, 5 μm.

(h) Quantification of astral microtubule intensity shown in g. n=39 (WT) and 41 (p80Y571A) cells. Intensity was normalized to the average value in the WT cells.

(i) Live cell images of spindle flux in WT and p80Y571A U2OS cells stably expressing PA-GFP-α-tubulin. Time displayed as min:sec. Plots of the distance between photoactivated GFP stripe and the spindle pole over time are shown on the right. The flux speed was derived from the plot slopes. Scale bar, 5 μm.

(j) Flux speed in WT and p80Y571A U2OS cells. n=24 (WT) and 28 (p80Y571A) cells.

(k) Model for regulation of microtubule minus-end dynamics at spindle poles by the ASPM/katanin complex. (1) Katanin recruited to microtubule lattice by ASPM can oligomerize through the p60 ATPase domain and sever microtubules. (2) Minus-end binding of the ASPM/katanin complex promotes its accumulation near the pole. Through its end-binding activity, katanin enhances blocking of microtubule minus-end growth by ASPM, which likely primes the minus-ends for depolymerization by depolymerases.

Data represent mean ± SD. ***, P<0.001, Mann-Whitney U test. Unprocessed original scans of blots are shown in Supplementary Figure 8. Source data for panels d, f, h and j can be found in Supplementary Table 5.