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, 182 (4), 623-9

Microtubules Do Not Promote Mitotic Slippage When the Spindle Assembly Checkpoint Cannot Be Satisfied

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Microtubules Do Not Promote Mitotic Slippage When the Spindle Assembly Checkpoint Cannot Be Satisfied

Daniela A Brito et al. J Cell Biol.

Abstract

When the spindle assembly checkpoint (SAC) cannot be satisfied, cells exit mitosis via mitotic slippage. In microtubule (MT) poisons, slippage requires cyclin B proteolysis, and it appears to be accelerated in drug concentrations that allow some MT assembly. To determine if MTs accelerate slippage, we followed mitosis in human RPE-1 cells exposed to various spindle poisons. At 37 degrees C, the duration of mitosis in nocodazole, colcemid, or vinblastine concentrations that inhibit MT assembly varied from 20 to 30 h, revealing that different MT poisons differentially depress the cyclin B destruction rate during slippage. The duration of mitosis in Eg5 inhibitors, which induce monopolar spindles without disrupting MT dynamics, was the same as in cells lacking MTs. Thus, in the presence of numerous unattached kinetochores, MTs do not accelerate slippage. Finally, compared with cells lacking MTs, exit from mitosis is accelerated over a range of spindle poison concentrations that allow MT assembly because the SAC becomes satisfied on abnormal spindles and not because slippage is accelerated.

Figures

Figure 1.
Figure 1.
In the absence of spindle MTs, RPE-1 cells spend 20–30 h in mitosis. Images from video records of cells entering and exiting mitosis in 3.2 μM nocodazole (A), 250 nM colcemid (B), 25 nM vinblastine (C), or 1 μM vinblastine (D). In each sequence, the second frame defines the start of mitosis, the third the end, and the last depicts the chromosome (DNA) and MT pattern in a mitotic cell from a similarly treated culture fixed and stained for the immunofluorescence localization of MTs. Time from addition of the drug to the medium is in minutes. Arrows note the cell that is followed in subsequent frames. Bars: (IMF images) 5 μm; (phase images) 20 μm.
Figure 2.
Figure 2.
When RPE-1 cells cannot satisfy the SAC, the formation of spindle MTs does not accelerate mitotic slippage. (A and B) Selected images from video recordings of cells entering and exiting mitosis in the presence of 500 nM taxol (A), which stabilizes spindle MTs, or 2.5 μM of the Eg5 inhibitor dimethylenastron (B). In each sequence the second frame defines the start of mitosis, the third the end, and the last depicts the chromosome (DNA) and MT pattern in a mitotic cell from a similarly treated culture fixed and stained for the immunofluorescence localization of MTs. Time from addition of drug to the medium is in minutes. See text for details. Arrows note the cell that is followed in subsequent frames. Bars: (IMF images) 5 μm; (phase images) 20 μm.
Figure 3.
Figure 3.
In low concentrations of MT poisons or stabilizers, RPE-1 cells form aberrant spindles that segregate chromosomes into two or more daughter cells. Each row depicts a cell entering (second frame) and exiting (third frame) mitosis in either 50 nM nocodazole (A), 5 nM vinblastine (B), or 5 nM taxol (C). The split-screen fluorescence images at the end of each row depict the distribution of chromosomes (DNA) and MTs in cells fixed during spindle assembly (fifth frame) and telophase/cytokinesis (sixth). Time from addition of drug to the medium is in minutes. Arrows note the cell of interest. Bars: (IMF images) 5 μm; (phase images) 20 μm.
Figure 4.
Figure 4.
Exit from mitosis in the presence of low concentrations of spindle MT poisons occurs from SAC satisfaction and not slippage. (A and B) Selected phase-contrast (top) and fluorescence (bottom) frames from video recordings of cyclin B/GFP-expressing RPE-1 cells as they enter and exit mitosis after no treatment (A) or in the presence of 50 nM nocodazole (B). Numbers define the time in minutes from NEB. (C) GFP intensity versus time plots reveal that the fluorescence intensity of control cells (e.g., no drug = cell shown in A) begins to rapidly decay at metaphase (18 min) after the SAC is satisfied and, once initiated, the chromatids disjoin ∼5 min later (23 min; arrow in C). A similar behavior is seen in cells treated with 50 nM nocodazole, except that the rapid decay of fluorescence intensity is delayed for a variable period during the prolonged mitosis (cell in B = cell 3). The cyclin B/GFP intensity in cells that enter and exit mitosis in 3.2 μM nocodazole exhibits a slow steady decline, with a slope reflecting the duration of mitosis, until the cell exits mitosis. Bars, 10 μm.

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