Figure 3.
Figure 3. Spindle microtubule stability is enhanced by depletion of GTSE1. (A, top) Select frames from live-cell imaging of tubulin photoactivation in metaphase U2OS cells stably expressing PAGFP-tubulin after transfection with either control or GTSE1 siRNA. Only cells entering mitosis during imaging were photoactivated to ensure tubulin turnover was not affected by prolonged mitotic arrest. Time is in s. (Bottom) Fluorescence dissipation after photoactivation. The filled and unfilled circles represent the mean values recorded at each time point after photoactivation. The bars represent SEM. Control n = 22 cells; GTSE1 n = 18 cells from four independent experiments. Lines indicate fitted curves (control R2 = 0.998; GTSE1 R2 = 0.995). F = A1 × exp(−k1 × t) + A2 × exp(−k2 × t) where A1 and A2 represent the percent total fluorescence, and k1 and k2 represent the respective decay rate constants. (B, left) Immunofluorescence of α-tubulin levels in methanol-fixed HeLa cells transfected with either control or GTSE1 siRNA. Images represent maximum-intensity projections. α-Tubulin levels are scaled equivalently. (Right) Inner spindle microtubule (MT) fluorescence intensity quantification of α-tubulin after control or GTSE1 siRNA transfection. The fluorescence intensity from a region encompassing only inner spindle microtubules was analyzed (the dashed circle in the top left image marks region of interest). GTSE1 depletion enhanced the levels of α-tubulin found in the inner spindle when compared with control cells. SD is plotted for the mean from each condition. n = 24 cells from two independent experiments for each condition. **, P < 0.01. (C) Immunofluorescence of GTSE1 in HeLa cells that were untreated or cold treated to examine localization to the more stable microtubule population, or transfected with Hec1 siRNA to examine localization in cells unable to make kinetochore–microtubule interactions. GTSE1 labeling of spindle microtubules was largely unaffected by cold treatment, but it failed to accumulate on microtubules after depletion of Hec1. Images represent maximum-intensity projections. Bars, 10 µm.

Spindle microtubule stability is enhanced by depletion of GTSE1. (A, top) Select frames from live-cell imaging of tubulin photoactivation in metaphase U2OS cells stably expressing PAGFP-tubulin after transfection with either control or GTSE1 siRNA. Only cells entering mitosis during imaging were photoactivated to ensure tubulin turnover was not affected by prolonged mitotic arrest. Time is in s. (Bottom) Fluorescence dissipation after photoactivation. The filled and unfilled circles represent the mean values recorded at each time point after photoactivation. The bars represent SEM. Control n = 22 cells; GTSE1 n = 18 cells from four independent experiments. Lines indicate fitted curves (control R2 = 0.998; GTSE1 R2 = 0.995). F = A1 × exp(−k1 × t) + A2 × exp(−k2 × t) where A1 and A2 represent the percent total fluorescence, and k1 and k2 represent the respective decay rate constants. (B, left) Immunofluorescence of α-tubulin levels in methanol-fixed HeLa cells transfected with either control or GTSE1 siRNA. Images represent maximum-intensity projections. α-Tubulin levels are scaled equivalently. (Right) Inner spindle microtubule (MT) fluorescence intensity quantification of α-tubulin after control or GTSE1 siRNA transfection. The fluorescence intensity from a region encompassing only inner spindle microtubules was analyzed (the dashed circle in the top left image marks region of interest). GTSE1 depletion enhanced the levels of α-tubulin found in the inner spindle when compared with control cells. SD is plotted for the mean from each condition. n = 24 cells from two independent experiments for each condition. **, P < 0.01. (C) Immunofluorescence of GTSE1 in HeLa cells that were untreated or cold treated to examine localization to the more stable microtubule population, or transfected with Hec1 siRNA to examine localization in cells unable to make kinetochore–microtubule interactions. GTSE1 labeling of spindle microtubules was largely unaffected by cold treatment, but it failed to accumulate on microtubules after depletion of Hec1. Images represent maximum-intensity projections. Bars, 10 µm.

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