To determine if ubiquitin-dependent degradation of another protein is required for chromosome segregation, researchers prepared a peptide containing the cyclin destruction-box sequence and the site of polyubiquitination. When this peptide was added to a reaction mixture containing untreated egg extract and sperm nuclei, decondensation of the chromosomes and, more interestingly, movement of chromosomes toward the spindle poles were greatly delayed at peptide concentrations of 20-40 ^g/ml and blocked altogether at higher concentrations (Figure 21-18c). The added excess destruction-box peptide is thought to act as a substrate for the APC-directed polyubiquitination system, competing with the normal endogenous target proteins and thereby delaying or preventing their degradation by protea-somes. Competition for cyclin B accounts for the observed inhibition of chromosome decondensation. The observation that chromosome segregation also was inhibited in this experiment but not in the experiment with mutant nondegrad-able cyclin B (see Figure 21-18b) indicated that segregation depends on proteasomal degradation of a different target protein.

As mentioned earlier, each sister chromatid of a metaphase chromosome is attached to microtubules via its kinetochore, a complex of proteins assembled at the centromere. The opposite ends of these kinetochore micro-tubules associate with one of the spindle poles (see Figure 20-31). At metaphase, the spindle is in a state of tension with forces pulling the two kinetochores toward the opposite spindle poles balanced by forces pushing the spindle poles apart. Sister chromatids do not separate because they are

▲ FIGURE 21-19 Model for control of entry into anaphase by APC-regulated degradation of the cohesin link between sister chromatids. (Left) The multiprotein cohesin complex contains SMC1 and SMC3 (purple), dimeric proteins that bind DNA of each sister chromatid through globular domains at one end. Scc1 (orange) and two other cohesin subunits (not shown) bind to the SMC proteins associated with each chromatid, thus cross-linking the chromatids. (Center) Once all chromosome held together at their centromeres and multiple positions along the chromosome arms by multiprotein complexes called cohesins. Among the proteins composing the cohesin complexes are members of the SMC protein family discussed in the previous section. When Xenopus egg extracts were depleted of cohesin by treatment with antibodies specific for the cohesin SMC proteins, the depleted extracts were able to replicate the DNA in added sperm nuclei, but the resulting sister chromatids did not associate properly with each other. These findings demonstrate that cohesin is necessary for cross-linking sister chromatids.

Recent genetic studies in the budding yeast ,S. cerevisiae have led to the model depicted in Figure 21-19 for how the APC regulates sister chromatid separation to initiate anaphase. Cohesin SMC proteins bind to each sister chro-matid; other subunits of cohesin, including Scc1, then link the SMC proteins, firmly associating the two chromatids. The cross-linking activity of cohesin depends on securin, which is found in all eukaryotes. Prior to anaphase, securin binds to and inhibits separase, a ubiquitous protease related to the caspase proteases that regulate programmed cell death (Chapter 22). Once all chromosome kinetochores have attached to spindle microtubules, the APC is directed by a specificity factor called Cdc20 to polyubiquitinate securin, leading to the onset of anaphase. (This specificity factor is distinct from Cdh1, which directs the APC to polyubiquiti-nate B-type cyclins.) Polyubiquitinated securin is rapidly degraded by proteasomes, thereby releasing separase. Free from its inhibitor, separase cleaves Scc1, breaking the protein cross-link between sister chromatids. Once this link is bro-

kinetochores have bound to spindle microtubules, the APC specificity factor Cdc20 targets APC to polyubiquitinate securin which is then degraded by the proteasome (not shown). (Right) The released separase then cleaves Scc1, severing the cross-link between sister chromatids. See text for discussion. [Adapted from F Uhlmann, 2001, Cum Opin. Cell Biol. 13:754; and A. Tomans, Nature Milestones, full/milestone23.html.]

ken, the poleward force exerted on kinetochores can move sister chromatids toward the opposite spindle poles.

Because Cdc20—the specificity factor that directs APC to securin—is activated before Cdhl—the specificity factor that directs APC to mitotic cyclins—MPF activity does not decrease until after the chromosomes have segregated (see Figure 21-2, steps 8 and 9). As a result of this temporal order in the activation of the two APC specificity factors (Cdc20 and Cdhl), the chromosomes remain in the condensed state and reassembly of the nuclear envelope does not occur until chromosomes are moved to the proper position. We consider how the timing of Cdhl activation is regulated in a later section.

Reassembly of the Nuclear Envelope and Cytokinesis Depend on Unopposed Constitutive Phosphatase Activity

Earlier we discussed how MPF-mediated phosphorylation of nuclear lamins, nucleoporins, and proteins in the inner nuclear membrane contributes to the dissociation of nuclear pore complexes and retraction of the nuclear membrane into the reticular ER. Once MPF is inactivated in late anaphase by the degradation of mitotic cyclins, the unopposed action of phosphatases reverses the action of MPF. The dephosphor-ylated inner nuclear membrane proteins are thought to bind to chromatin once again. As a result, multiple projections of regions of the ER membrane containing these proteins are thought to associate with the surface of the decondensing chromosomes and then fuse with each other to form a continuous double membrane around each chromosome (Figure 21-20). Dephosphorylation of nuclear pore subcomplexes is thought to allow them to reassemble nuclear pore complexes traversing the inner and outer membranes soon after fusion of the ER projections (see Figure 12-20).

The fusion of ER projections depicted in Figure 2l-20 occurs by a mechanism similar to that described for the fusion of vesicles and target membranes in the secretory pathway (see Figure 17-11). Proteins with activities similar to those of NSF and a-SNAP in the secretory pathway have been shown to function in the fusion of experimentally produced nuclear envelope vesicles in vitro and are thought to mediate the fusion of ER projections around chromosomes during telophase in the intact cell. These same proteins also function in the fusion events that reassemble the Golgi apparatus. The membrane-associated SNARE proteins that direct the fusion of nuclear envelope extensions from the ER have not been identified, but syntaxin 5 has been shown to function as both the V-SNARE and T-SNARE during reassembly of the Golgi. During fusion of nuclear envelope vesicles in vitro, the same Ran GTPase that functions in transport through nuclear pore complexes (Chapter 12) functions similarly to Rab GTPases in vesicle fusion in the secretory pathway. Because the Ran-specific guanosine nucleotide-exchange factor (Ran-GEF) is associated with chromatin, a high local concentration of Ran • GTP is produced around the chromosomes, directing membrane fusions at the chromosome surface.

The reassembly of nuclear envelopes containing nuclear pore complexes around each chromosome forms individual mininuclei called karyomeres (see Figure 21-20). Subsequent fusion of the karyomeres associated with each spindle pole generates the two daughter-cell nuclei, each containing a full set of chromosomes. Dephosphorylated lamins A and C appear to be imported through the reassembled nuclear pore complexes during this period and reassemble into a new nuclear lamina. Reassembly of the nuclear lamina in the daughter nuclei probably is initiated on lamin B molecules, which remain associated with the ER membrane via their isoprenyl anchors throughout mitosis and become localized to the inner membrane of the reassembled nuclear envelopes of karyomeres.

During cytokinesis, the final step in cell division, the actin and myosin filaments composing the contractile ring slide past each other to form a cleavage furrow of steadily decreasing

Membrane fusion, NPC assembly

▲ FIGURE 21-20 Model for reassembly of the nuclear envelope during telophase. Extensions of the endoplasmic reticulum (ER) associate with each decondensing chromosome and then fuse with each other, forming a double membrane around the chromosome. Nuclear pore subcomplexes reassemble into nuclear pores, forming individual mininuclei called karyomeres. The enclosed chromosome further decondenses, and subsequent fusion of the nuclear envelopes of all the karyomeres at each spindle pole forms a single nucleus containing a full set of chromosomes. Reassembly of the nuclear lamina is not shown. [Adapted from B. Burke and J. Ellenberg, 2002, Nature Rev. Mol. Cell Biol. 3:487]

diameter (see Figure 19-20). As MPF activity rises early in mitosis, it phosphorylates the regulatory myosin light chain, thereby inhibiting the ability of myosin to associate with actin filaments. The inactivation of MPF at the end of anaphase permits protein phosphatases to dephosphorylate myosin light chain (see Figure 20-42). As a result, the contractile machinery is activated, the cleavage furrow can form, and cytokinesis proceeds. This regulatory mechanism assures that cytokinesis does not occur until the daughter chromosomes have segregated sufficiently toward the opposite poles to assure that each daughter cell receives the proper number of chromosomes.

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