Figure 3.
Figure 3. Outer KT relaxation upon double ablation and 9 µM NOC treatment in PtK1 and fission yeast cells. (A) Maximum intensity projections of a merotelic KT in PtK1 cell expressing Hec1-GFP, shown in green, and injected with α-tubulin–X-rhodamine, shown in magenta. Time 0 indicates the frame before ablation. The white lightning signs indicate ablation sites. Images were acquired with a time resolution Δt = 2.2 s. Close-up views show lagging KTs (2.5×). (B) Time projection of the area containing the merotelic KT from the cell in A. The boxed region indicates the time interval shown in A. (D) Maximum intensity projections of a merotelic KT in fission yeast cell expressed Ndc80-GFP, shown in green, and α-tubulin–mCherry, shown in magenta. The white lightning signs indicate ablation sites. The time 0 is the frame before ablation. The spindle breakage occurred immediately after ablation. Images were acquired with a time resolution Δt = 3 s. Close-up views show lagging KTs (1.65×). (E) Time projection of an area containing the merotelic KT from the cell in D. The boxed region indicates the time interval shown in D. (C and F) Normalized KT length as a function of time, for the inner (magenta) and outer (green) KT in PtK1 (C) and S. pombe cells (F) after single ablation. Normalized outer KT length as a function of time after double ablation is shown in light blue. Data for each KT were normalized setting the value before ablation to 1 and the minimum value reached by the individual KT to 0, binned, and averaged. Error bars represent standard deviation, n = 25/18 for Hec1/Ndc80 single ablation, n = 15/24 for CenpA/Cnp1 single ablation, and n = 9/10 for Hec1/Ndc80 double ablation. All the data were fitted with a single exponential equation, L(t) = A × exp(−t × ln(2)/t1/2). Half-lives are shown in the insets. (G) Maximum intensity projections of a merotelic KT in PtK1 cell expressing Hec1-GFP, shown in green, and injected with α-tubulin–X-rhodamine, shown in magenta. Time 0 indicates the frame before addition of 9 µM NOC. Images were acquired with a time resolution Δt = 20s. Close-up views show lagging KTs (1.5×). (H) Time projection of an area containing the merotelic KT from the cell in panel G. The boxed region indicates the time interval shown in G. (I) Normalized KT length (outer KT) as a function of time. PtK1 cells were treated with 9 µM NOC. Comparison between ablation experiments and 9 µM NOC treatment for the first 200 s is shown in I (inset). The length change was initially slow (I, inset), most likely because of the slow kinetics of NOC-dependent MT depolymerization as compared with a sudden MT severing.

Outer KT relaxation upon double ablation and 9 µM NOC treatment in PtK1 and fission yeast cells. (A) Maximum intensity projections of a merotelic KT in PtK1 cell expressing Hec1-GFP, shown in green, and injected with α-tubulin–X-rhodamine, shown in magenta. Time 0 indicates the frame before ablation. The white lightning signs indicate ablation sites. Images were acquired with a time resolution Δt = 2.2 s. Close-up views show lagging KTs (2.5×). (B) Time projection of the area containing the merotelic KT from the cell in A. The boxed region indicates the time interval shown in A. (D) Maximum intensity projections of a merotelic KT in fission yeast cell expressed Ndc80-GFP, shown in green, and α-tubulin–mCherry, shown in magenta. The white lightning signs indicate ablation sites. The time 0 is the frame before ablation. The spindle breakage occurred immediately after ablation. Images were acquired with a time resolution Δt = 3 s. Close-up views show lagging KTs (1.65×). (E) Time projection of an area containing the merotelic KT from the cell in D. The boxed region indicates the time interval shown in D. (C and F) Normalized KT length as a function of time, for the inner (magenta) and outer (green) KT in PtK1 (C) and S. pombe cells (F) after single ablation. Normalized outer KT length as a function of time after double ablation is shown in light blue. Data for each KT were normalized setting the value before ablation to 1 and the minimum value reached by the individual KT to 0, binned, and averaged. Error bars represent standard deviation, n = 25/18 for Hec1/Ndc80 single ablation, n = 15/24 for CenpA/Cnp1 single ablation, and n = 9/10 for Hec1/Ndc80 double ablation. All the data were fitted with a single exponential equation, L(t) = A × exp(−t × ln(2)/t1/2). Half-lives are shown in the insets. (G) Maximum intensity projections of a merotelic KT in PtK1 cell expressing Hec1-GFP, shown in green, and injected with α-tubulin–X-rhodamine, shown in magenta. Time 0 indicates the frame before addition of 9 µM NOC. Images were acquired with a time resolution Δt = 20s. Close-up views show lagging KTs (1.5×). (H) Time projection of an area containing the merotelic KT from the cell in panel G. The boxed region indicates the time interval shown in G. (I) Normalized KT length (outer KT) as a function of time. PtK1 cells were treated with 9 µM NOC. Comparison between ablation experiments and 9 µM NOC treatment for the first 200 s is shown in I (inset). The length change was initially slow (I, inset), most likely because of the slow kinetics of NOC-dependent MT depolymerization as compared with a sudden MT severing.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal