Figure 3: Effects of gatastatin on interphase MT network, interphase MTs dynamics, mitotic entry and mitotic spindle formation.
(a) The effect of gatastatin on the interphase MT network. HeLa cells were treated with 1% DMSO, 30 μM AG1 or 100 μM gatastatin for 2.5 h. The MT network was observed using a LSM 700 laser-scanning confocal microscope. Scale bar, 10 μm. (b) The effects of AG1 and gatastatin on MT dynamics. HeLa cells expressing EB3-EGFP were treated with DMSO, 3 μM AG1 or 30 μM gatastatin for 15 min. EB3 tracks are displayed in rainbow colours as maximum intensity projection of all time points from a 60-s sequence of imaging. Scale bar, 10 μm. MT parameters are summarized in Table 3. (c,d) γ-Tubulin function is not required for the cell cycle progression from S phase to mitotic entry. (c) Scheme of the experiment in d. After HeLa cells were arrested at S phase with 2 mM thymidine for 15 h, thymidine was removed and the timings of nuclear envelope breakdown and chromosome condensation in the presence or absence of 30 μM gatastatin were observed by time-lase microscopy. (d) The time required for mitotic entry was calculated. Error bars represent s.d. Two-tailed, unpaired Student’s t-test was used to obtain P value. (e) The effect of gatastatin on the mitotic spindle structure of cells from the experimental set-up of c. Green and orange represent α-tubulin and DNA, respectively. Scale bar, 5 μm. (f) Spindles in e were analysed from 39 (control) or 47 (gatastatin) cells. (g) Gatastatin reduces spindle length. The distance between two pericentrin signals was measured as the length of the mitotic spindle in the presence or absence of 30 μM gatastatin (spindle length=
). The length of 20 spindles per experiment was analysed. Three independent experiments were performed and one representative experiment is shown. Error bars represent s.d. Two-tailed, unpaired Student’s t-test was used to obtain P value. (h) BubR1 localized on misaligned chromosome around poles in 30 μM gatastatin-treated cells. Green, red and blue represent BubR1, α-tubulin and DNA, respectively. Scale bar, 5 μm.
