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Fig. 1 | BMC Biology

Fig. 1

From: Two-step interphase microtubule disassembly aids spindle morphogenesis

Fig. 1

Disassembly of interphase microtubules begins prior to NEP and is accelerated at NEP. a Representative time-lapse confocal images (x-y maximum projection) of a HeLa cell stably expressing H2B-mRFP (to visualise chromosomes) and mEGFP-α-tubulin (to visualise microtubules and NEP) and transiently overexpressing Rap1* (to keep cell flat in mitosis). Boxed areas show regions zoomed in b and c. b Higher magnification (sum projection of mEGFP-α-tubulin sections around the centrosome, pseudo-color, spectra look-up table (LUT)) of boxed region 2 indicated in a showing changes of mEGFP-α-tubulin levels at the centrosome relative to NEP. Insets indicate regions used for quantifications: green (centrosomal microtubules), red (nuclear tubulin). c Higher magnification (maximum projection of mEGFP-α-tubulin basal sections, inverted greyscale) of region 1 in a showing that non-centrosomal microtubule disassembly is triggered before NEP and accelerates during loss of the nuclear-cytoplasmic compartment boundary. Boxed area indicates region used for quantifications. d Changes in median centrosomal and non-centrosomal microtubule intensity relative to NEP for H2B-mRFP mEGFP-α-tubulin HeLa cell transiently overexpressing Rap1* (shown in ac, left) and for five equivalent cells from two independent experiments (right). Median intensity of mEGFP-α-tubulin signal was calculated within a 15 × 15 pixel circle around the centrosome (green line, as indicated in b), a 10 × 10 pixel box within the nucleus (red, as indicated in b) and a 30 × 30 pixel box at the cell periphery (blue, as indicated in c). Time point 0 represents NEP. Graph shows mean and standard deviation (SD). e Changes in non-centrosomal microtubule levels relative to NEP. Measurements show median of mEGFP-α-tubulin signal in a 30 × 30 pixel box at two locations at the periphery of a cell as shown in Additional file 1A at –30, –20, –2 and 4, 6 min relative to NEP in H2B-mRFP mEGFP-α-tubulin HeLa stable cell line transiently overexpressing Rap1* (13 cells (including 5 cells from d), four independent experiments). Repeated measures analysis of variance (ANOVA), Tukey's multiple comparisons test with a single pooled variance, ****P < 0.0001. Scale bars represent 10 μm

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