Fig. 2

Removal of Ensconsin/MAP7 by Cdk1/cyclin B activation triggers non-centrosomal microtubule depolymerisation before NEP. a Representative time-lapse confocal images (x-y maximum projection) of HeLa cells during mitotic entry stably expressing H2B-mRFP and mEGFP-α-tubulin and transiently overexpressing Rap1* treated with control small interfering RNA (siRNA) (upper panel) or Cep192 siRNA (lower panel). b Changes in centrosomal and non-centrosomal microtubule levels relative to NEP for control siRNA (upper panel, five cells) and for Cep192 siRNA (lower panel, five cells) (see Methods for measurement details). Two independent experiments. Graphs show mean and SD. c Changes in non-centrosomal microtubule levels relative to NEP for cells in b. Median of α-tubulin-GFP signal was measured as described in Fig. 1e. d Schematic representation of Ensconsin/MAP7 structure. e Representative confocal images (x-y maximum projection) of fixed HeLa cells stained to show that Ensconsin/MAP7 is removed from microtubules in prophase. Ensconsin/MAP7 in red, α-tubulin in green and 4’,6-diamidino-2-phenylindole (DAPI) in blue (11 prophase, > 30 interphase cells, two independent experiments). f Representative confocal images of fixed HeLa cells overexpressing Rap1* stained to show that Ensconsin/MAP7 relocalises to the microtubules upon Cdk1 inhibition with RO-3306. Ensconsin/MAP7 in red, α-tubulin in green and DAPI in blue (9 dimethyl sulfoxide (DMSO) and 10 RO-3306 cells, two independent experiments). g Representative confocal images of fixed HeLa cells overexpressing Rap1* stained to show that overexpressed Wt-EMTB-mCherry (9 cells) and a non-phosphorylatable mutant (A-EMTB-mCherry, 14 cells) remain bound to microtubules in interphase, whereas the phospho-mimetic (E-EMTB-mCherry, 10 cells) remains largely cytoplasmic. α-Tubulin in green, mCherry in red (one experiment). h Representative time-lapse confocal images (x-y maximum projection) of flat (Rap1*) HeLa cells stably expressing GFP-α-tubulin and Wt-EMTBmCherry (upper panel, 18 cells) or its non-phosphorylatable mutant (A-EMTB-mCherry, lower panel, 15 cells) to show that the A-EMTB-mCherry stays associated with microtubules during prophase, leading to delay in microtubule disassembly at mitotic entry. White arrows indicate microtubule clumps. Black arrows indicate interphase microtubules just before or after NEP (five independent experiments). Boxed areas show regions zoomed in inverted greyscale. Scale bars represent 10 μm