Figure 4. Kinetochores with persistent... | Rockefeller University Press

Figure 4.

$Figure 4. Kinetochores with persistent Plk1 activity accumulate microtubule attachment errors and fail to silence the spindle checkpoint. (A) Cells expressing either Hec1 or Hec1-Plk1T210D were selected at metaphase and followed for 60 min to determine the time of anaphase onset (n > 20). The Mps1 inhibitor reversine was added, as indicated, at t = 0 to override the spindle checkpoint. (B and C) Cells expressing Hec1 or Hec1-Plk1T210D were briefly permeabilized and treated with calcium to remove nonkinetochore microtubules, fixed, and stained for microtubules. Images (B) are maximal intensity projections of confocal z series. Insets show sister kinetochore pairs in optical sections. Images are scaled differently in the insets to show merotelic attachments (arrows) more clearly. The number of kinetochores with microtubules attached from both directions (merotelic errors) was determined (n = 20 cells; C). (D–F) For cells treated with reversine at metaphase, as in A, the fraction of cells with lagging kinetochores in anaphase (D) and the number of laggers per cell (E) were determined (n > 20). Images (F) show a representative cell expressing Hec1-Plk1T210D with lagging kinetochores in anaphase. Insets show lagging kinetochores at higher magnification. Note that Hec1-Plk1T210D localizes to kinetochores and spindle poles and to the anaphase spindle midzone. DIC, differential interference contrast. (B and F) Bars, 5 µm.$

Kinetochores with persistent Plk1 activity accumulate microtubule attachment errors and fail to silence the spindle checkpoint. (A) Cells expressing either Hec1 or Hec1-Plk1^T210D were selected at metaphase and followed for 60 min to determine the time of anaphase onset (n > 20). The Mps1 inhibitor reversine was added, as indicated, at t = 0 to override the spindle checkpoint. (B and C) Cells expressing Hec1 or Hec1-Plk1^T210D were briefly permeabilized and treated with calcium to remove nonkinetochore microtubules, fixed, and stained for microtubules. Images (B) are maximal intensity projections of confocal z series. Insets show sister kinetochore pairs in optical sections. Images are scaled differently in the insets to show merotelic attachments (arrows) more clearly. The number of kinetochores with microtubules attached from both directions (merotelic errors) was determined (n = 20 cells; C). (D–F) For cells treated with reversine at metaphase, as in A, the fraction of cells with lagging kinetochores in anaphase (D) and the number of laggers per cell (E) were determined (n > 20). Images (F) show a representative cell expressing Hec1-Plk1^T210D with lagging kinetochores in anaphase. Insets show lagging kinetochores at higher magnification. Note that Hec1-Plk1^T210D localizes to kinetochores and spindle poles and to the anaphase spindle midzone. DIC, differential interference contrast. (B and F) Bars, 5 µm.