Figure 17–38 The initiation of sisterchromatid separation by the APC/C.
The activation of apC/C by Cdc20 leads to the ubiquitylation and destruction of securin, which normally holds separase in an inactive state. The destruction of securin allows separase to cleave scc1, a subunit of the cohesin complex holding the sister chromatids together (see Figure 17–19). The pulling forces of the mitotic spindle then pull the sister chromatids apart. In animal cells, phosphorylation by Cdks also inhibits separase (not shown). Thus, Cdk inactivation in anaphase (resulting from cyclin destruction) also promotes separase activation by allowing its dephosphorylation.
metaphase-to-anaphase transition. When the last sister-chromatid pair is properly bi-oriented, this block is removed, allowing sister-chromatid separation to occur.
The negative checkpoint signal depends on several proteins, including Mad2, which are recruited to unattached kinetochores (Figure 17–39). Detailed structural analyses of Mad2 suggest that the unattached kinetochore acts like an enzyme that catalyzes a change in the conformation of Mad2, so that Mad2, together with other proteins, can bind and inhibit Cdc20–APC/C.
In mammalian somatic cells, the spindle assembly checkpoint determines the normal timing of anaphase. The destruction of securin in these cells begins moments after the last sister-chromatid pair becomes bi-oriented on the spindle, and anaphase begins about 20 minutes later. Experimental inhibition of the checkpoint mechanism causes premature sister-chromatid separation and anaphase. Surprisingly, the normal timing of anaphase does not depend on the spindle assembly checkpoint in some cells, such as yeasts and the cells of early frog and fly embryos. Other mechanisms, as yet unknown, must determine the timing of anaphase in these cells.
The sudden loss of sister-chromatid cohesion at the onset of anaphase leads to sister-chromatid separation, which allows the forces of the mitotic spindle to pull the sisters to opposite poles of the cell—called chromosome segregation. The chromosomes move by two independent and overlapping processes. The first, anaphase A, is the initial poleward movement of the chromosomes, which is accompanied by shortening of the kinetochore microtubules. The second, anaphase B, is the separation of the spindle poles themselves, which begins after the sister chromatids have separated and the daughter chromosomes have moved some distance apart (Figure 17–40).
Chromosome movement in anaphase A depends on a combination of the two major poleward forces described earlier. The first is the force generated by micro-tubule depolymerization at the kinetochore, which results in the loss of tubulin subunits at the plus end as the kinetochore moves toward the pole. The second is provided by microtubule flux, which is the poleward movement of the micro-tubules toward the spindle pole, where minus-end depolymerization occurs. The relative importance of these two forces during anaphase varies in different cell types: in embryonic cells, chromosome movement depends mainly on microtubule flux, for example, whereas movement in yeast and vertebrate somatic cells results primarily from forces generated at the kinetochore.
Spindle-pole separation during anaphase B depends on motor-driven mechanisms similar to those that separate the two centrosomes in early mitosis. Plus-end directed kinesin-5 motor proteins, which cross-link the overlapping plus ends of the interpolar microtubules, push the poles apart. In addition, dynein motors that anchor astral microtubule plus ends to the cell cortex pull the poles apart (see Figure 17–25).