Figure 17–36 How opposing forces may drive chromosomes to the metaphase plate. (a) Evidence for a polar ejection force that pushes chromosomes away from the spindle poles toward the spindle equator. In this experiment, a laser beam severs a prometaphase chromosome that is attached to a single pole by a kinetochore microtubule. The part of the severed chromosome without a kinetochore is pushed rapidly away from the pole, whereas the part with the kinetochore moves toward the pole, reflecting a decreased repulsion. (B) a model of how two opposing forces may cooperate to move chromosomes to the metaphase plate. plus-end-directed motor proteins (kinesin-4 and kinesin-10) on the chromosome arms are thought to interact with microtubules to generate the polar ejection force, which pushes chromosomes toward the spindle equator (see Figure 17–25). poleward forces generated by depolymerization at the kinetochore, together with microtubule flux, are thought to pull chromosomes toward the pole.
Figure 17–37 Sister-chromatid separation at anaphase. In the transition from metaphase (a) to anaphase (B), sister chromatids suddenly and synchronously separate and move toward opposite poles of the mitotic spindle—as shown in these light micrographs of Haemanthus (lily) endosperm cells that were stained with gold-labeled antibodies against tubulin. (Courtesy of andrew Bajer.) binds to and inhibits the activity of a protease called separase. The destruction of securin at the end of metaphase releases separase, which is then free to cleave one of the subunits of cohesin. The cohesins fall away, and the sister chromatids separate (Figure 17–38).
In addition to securin, the APC/C also targets the Sand M-cyclins for destruction, leading to the loss of most Cdk activity in anaphase. Cdk inactivation allows phosphatases to dephosphorylate the many Cdk target substrates in the cell, as required for the completion of mitosis and cytokinesis.
If the APC/C triggers anaphase, what activates the APC/C? The answer is only partly known. As mentioned earlier, APC/C activation requires binding to the protein Cdc20 (see Figure 17–15A). At least two processes regulate Cdc20 and its association with the APC/C. First, Cdc20 synthesis increases as the cell approaches mitosis, owing to an increase in the transcription of its gene. Second, phosphorylation of the APC/C helps Cdc20 bind to the APC/C, thereby helping to create an active complex. Among the kinases that phosphorylate and thus activate the APC/C is M-Cdk. Thus, M-Cdk not only triggers the early mitotic events leading up to metaphase, but it also sets the stage for progression into anaphase. The ability of M-Cdk to promote Cdc20–APC/C activity creates a negative feedback loop: M-Cdk sets in motion a regulatory process that leads to cyclin destruction and thus its own inactivation.
Unattached Chromosomes Block sister-Chromatid separation: The spindle assembly Checkpoint
Drugs that destabilize microtubules, such as colchicine or vinblastine (discussed in Chapter 16), arrest cells in mitosis for hours or even days. This observation led to the identification of a spindle assembly checkpoint mechanism that is activated by the drug treatment and blocks progression through the metaphase-toanaphase transition. The checkpoint mechanism ensures that cells do not enter anaphase until all chromosomes are correctly bi-oriented on the mitotic spindle.
The spindle assembly checkpoint depends on a sensor mechanism that monitors the strength of microtubule attachment at the kinetochore, possibly by sensing tension as described earlier (see Figure 17–34). Any kinetochore that is not properly attached to the spindle sends out a diffusible negative signal that blocks Cdc20–APC/C activation throughout the cell and thus blocks the