Figure 6.
Figure 6. Hook2 regulate MT nucleation at the centrosome. (A) Representative images of metaphase HeLa cells transfected with indicated siRNA zoomed to visualize astral MTs. Bars, 2 µm. (B) Quantification of the intensity of astral MTs in A (n = 3; 40 metaphase cells/experiment). (C) Representative image of MT regrowth upon cold depolymerization in mitotic HeLa cells transfected with indicated siRNA. Bar, 5 µm. (D) Quantification of MT length as shown in C (n = 3; 30 cells/experiment). (E–J) Maximum intensity projections of z-stacks of live-cell time-lapse imaging of early prophase HeLa cells stably expressing EB1-GFP and treated with indicated siRNA and transfected with respective siRNA-resistant Hook2 (WT/mutants) constructs as indicated. Images were acquired every 5 s for a total duration of 5 m. Bars, image, 10 µm; zoomed insets, 2 µm. (K) Quantification of the rate of MT nucleation from centrosomes as determined from the time-lapse videos shown in E through J from 40 cells (two centrosomes/cell). (L) Representative maximum intensity projection of airy-scan images of metaphase-arrested HeLa cells treated with indicated siRNA. MTs were stained with α-tubulin, KTs were stained with CREST and Hec1 antibodies, and chromatin was visualized with DAPI. Bars, upper images, 5 µm; lower insets, 2 µm. (M) Quantification of inter-KT distance from 10 metaphase cells (30 KT pairs/cell, representative image shown in L). (N) Representative images of metaphase-arrested HeLa cells treated with indicated siRNA. KTs were stained with Hec1 antibody, and silencing of the mitotic checkpoint was confirmed by the Mad1 antibody. Prometaphase-arrested (100 µM nocodazole) control siRNA-treated cells were taken as a positive control in the experiment. (O) Quantification of silencing of mitotic checkpoint upon Hook2 siRNA from cells depicted in N from 20 cells (10–15 KTs/cell). (P) Quantification of Hec1 levels at the KTs in HeLa cells from 10 metaphase cells (30 KTs/cell, representative image shown in L). Data represent mean ± SD (ns, not significant; *, P < 0.1; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test).

Hook2 regulate MT nucleation at the centrosome. (A) Representative images of metaphase HeLa cells transfected with indicated siRNA zoomed to visualize astral MTs. Bars, 2 µm. (B) Quantification of the intensity of astral MTs in A (n = 3; 40 metaphase cells/experiment). (C) Representative image of MT regrowth upon cold depolymerization in mitotic HeLa cells transfected with indicated siRNA. Bar, 5 µm. (D) Quantification of MT length as shown in C (n = 3; 30 cells/experiment). (E–J) Maximum intensity projections of z-stacks of live-cell time-lapse imaging of early prophase HeLa cells stably expressing EB1-GFP and treated with indicated siRNA and transfected with respective siRNA-resistant Hook2 (WT/mutants) constructs as indicated. Images were acquired every 5 s for a total duration of 5 m. Bars, image, 10 µm; zoomed insets, 2 µm. (K) Quantification of the rate of MT nucleation from centrosomes as determined from the time-lapse videos shown in E through J from 40 cells (two centrosomes/cell). (L) Representative maximum intensity projection of airy-scan images of metaphase-arrested HeLa cells treated with indicated siRNA. MTs were stained with α-tubulin, KTs were stained with CREST and Hec1 antibodies, and chromatin was visualized with DAPI. Bars, upper images, 5 µm; lower insets, 2 µm. (M) Quantification of inter-KT distance from 10 metaphase cells (30 KT pairs/cell, representative image shown in L). (N) Representative images of metaphase-arrested HeLa cells treated with indicated siRNA. KTs were stained with Hec1 antibody, and silencing of the mitotic checkpoint was confirmed by the Mad1 antibody. Prometaphase-arrested (100 µM nocodazole) control siRNA-treated cells were taken as a positive control in the experiment. (O) Quantification of silencing of mitotic checkpoint upon Hook2 siRNA from cells depicted in N from 20 cells (10–15 KTs/cell). (P) Quantification of Hec1 levels at the KTs in HeLa cells from 10 metaphase cells (30 KTs/cell, representative image shown in L). Data represent mean ± SD (ns, not significant; *, P < 0.1; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Student’s t test).

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