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Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000.

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Molecular Cell Biology. 4th edition.

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Section 13.4Molecular Mechanisms for Regulating Mitotic Events

So far, we have seen that a regulated increase in MPF activity induces entry into mitosis. Presumably, the entry into mitosis is a consequence of the phosphorylation of specific proteins by the protein kinase activity of MPF. For the most part, however, the proteins phosphorylated by MPF in vivo have not been identified. The active phosphorylated forms of these proteins are thought to mediate the many remarkable events of mitosis including chromosome condensation, formation of the mitotic spindle, and breakdown of the nuclear envelope (see Figure 19-34).

As demonstrated in the studies with Xenopus egg cycling extracts described earlier (see Figure 13-7), a decrease in mitotic cyclins and the associated inactivation of MPF coincides with the later stages of mitosis (late anaphase and telophase). Just before this, in early anaphase, sister chromatids separate and move to opposite spindle poles. During telophase, microtubule dynamics return to interphase conditions, the chromosomes decondense, the nuclear envelope reforms, the endoplasmic reticulum and Golgi complex are remodeled, and cytokinesis occurs. Some of these processes are triggered by dephosphorylation; others, by protein degradation.

In this section, we discuss the molecular mechanisms and specific proteins associated with some of the events that characterize early and late mitosis.

Phosphorylation of Nuclear Lamins by MPF Leads to Nuclear-Envelope Breakdown

The nuclear envelope is a double-membrane extension of the rough endoplasmic reticulum containing many nuclear pore complexes (see Figure 5-42). The lipid bilayer of the inner nuclear membrane is supported by the nuclear lamina, a meshwork of lamin filaments located adjacent to the inside face of the nuclear envelope (Figure 13-15a). The three nuclear lamins (A, B, and C) present in vertebrate cells belong to the class of cytoskeletal proteins, the intermediate filaments, that are critical in supporting cellular membranes (Chapter 19). Lamins A and C, which are encoded by the same transcription unit and produced by alternative splicing of a single pre-mRNA, are identical except for a 133-residue region at the C-terminus of lamin A, which is absent in lamin C. Lamin B, encoded by a different transcription unit, is post-transcriptionally modified by the addition of a hydrophobic isoprenyl group near its carboxyl-terminus. This fatty acid is incorporated into the inner leaflet of the lipid bilayer that forms the inner nuclear membrane, thereby anchoring the nuclear lamina to the membrane. All three nuclear lamins form dimers containing a rodlike α-helical coiled-coil central section and globular head and tail domains; polymerization of these dimers through head-to-head and tail-to-tail associations generates the intermediate filaments that compose the nuclear lamina (see Figure 19-51).

Figure 13-15. The nuclear lamina and its depolymerization.

Figure 13-15

The nuclear lamina and its depolymerization. (a) Electron micrograph of the nuclear lamina. A nuclear membrane from a hand-dissected Xenopus oocyte was fixed to an electron microscope grid and then extracted with a nonionic detergent to remove the lipid (more...)

Early in mitosis, MPF phosphorylates specific serine residues in all three nuclear lamins, causing depolymerization of the lamin intermediate filaments (Figure 13-15b). The phosphorylated lamin A and C dimers are released into solution, whereas the phosphorylated lamin B dimers remain associated with the nuclear membrane via their isoprenyl anchor. Depolymerization of the nuclear lamins leads to disintegration of the nuclear lamina meshwork and contributes to the breakdown of the nuclear envelope into small vesicles. The experiment summarized in Figure 13-16 shows that the breakdown of the nuclear envelope, which normally occurs early in mitosis, depends on phosphorylation of lamin A.

Figure 13-16. Experimental demonstration that phosphorylation of human nuclear lamin A is required for lamin depolymerization, which contributes to nuclear-envelope breakdown during mitosis.

Figure 13-16

Experimental demonstration that phosphorylation of human nuclear lamin A is required for lamin depolymerization, which contributes to nuclear-envelope breakdown during mitosis. Site-directed mutagenesis was used to prepare a mutant human lamin A gene (more...)

Other Early Mitotic Events May Be Controlled Directly or Indirectly by MPF

The demonstration that nuclear-envelope breakdown depends on phosphorylation of nuclear lamins suggests that MPF-catalyzed phosphorylation of other proteins may play a role in other early mitotic events, such as chromosome condensation. For instance, genetic experiments in the budding yeast S. cerevisiae identified a family of SMC (structural maintenance of chromosomes) proteins that are required for normal chromosome segregation. Biochemical studies of the homologous Xenopus proteins showed that these large proteins (≈1200 amino acids) contain long regions predicted to participate in coiled-coil structures (see Figure 3-9) and characteristic ATPase domains at their C-terminus. Homologs of yeast SMC proteins were cloned from a Xenopus cDNA library, and antibodies were raised against the encoded proteins. Immunoprecipitation studies with these antibodies revealed that in a Xenopus egg extract the SMC proteins are part of a protein complex, called condensin, that includes three additional proteins, which become phosphorylated as cells enter mitosis. When the anti-SMC antibodies were used to deplete condensin from an egg extract, the extract lost its ability to condense added sperm chromatin.

In experiments with purified condensin and DNA, phosphorylated condensin binds to DNA and winds it into supercoils in a reaction requiring the hydrolysis of ATP. These results have lead to the model that individual condensin complexes, activated by MPF or another protein kinase regulated by MPF, bind to DNA at intervals along the chromosome scaffold. Self-association of the bound complexes via their coiled-coil domains and supercoiling of the DNA segments between them is proposed to cause chromosome condensation.

Phosphorylation of microtubule-associated proteins by MPF probably is required for the dramatic changes in microtubule dynamics that result in the formation of the mitotic spindle and asters (Section 19.5). In addition, all vesicular traffic in the cell ceases during mitosis, and the endoplasmic reticulum and Golgi complex break down into small vesicles as the nuclear membrane does. Phosphorylation of proteins associated with these membranous organelles, by MPF or other protein kinases activated by MPF-catalyzed phosphorylation, likely is responsible for these mitotic events as well.

APC-Dependent Unlinking of Sister Chromatids Initiates Anaphase

We saw earlier that in the late anaphase and telophase stages of mitosis, APC-mediated polyubiquitination of cyclin B targets it for destruction (see Figure 13-9). Additional experiments with RNase-treated Xenopus egg extracts provided evidence that polyubiquitination and subsequent degradation of noncyclin proteins also is required to initiate anaphase. In these studies, the mitotic spindle, which is formed from tubulin-containing microtubules, was visualized by including fluorescent-labeled tubulin in the reaction mixtures. When RNase-treated egg extracts and sperm chromatin were incubated in the presence of mRNA encoding wild-type cyclin B, the mitotic spindle apparatus and condensed sperm chromosomes aligned between the spindle poles were visible, similar to their appearance during metaphase in intact cells. After 15 minutes of incubation, the chromosomes were seen to move toward the spindle poles, just as they do during anaphase in intact cells. Cyclin B degradation and the resulting precipitous decrease in MPF activity began after this point, and over the next half hour the spindle depolymerized and the chromosomes decondensed (Figure 13-17a).

Figure 13-17. Experimental evidence that onset of anaphase depends on polyubiquitination of proteins other than cyclin B.

Figure 13-17

Experimental evidence that onset of anaphase depends on polyubiquitination of proteins other than cyclin B. The reaction mixtures contained an untreated or RNase-treated Xenopus egg extract and isolated Xenopus sperm nuclei, plus other components indicated (more...)

When mRNA encoding a nondegradable cyclin B was substituted for wild-type mRNA, MPF activity remained high as in the experiments described earlier (see Figure 13-7d). As before, chromosome decondensation did not occur, but chromosome segregation was observed to occur normally, indicating that chromosome segregation during anaphase does not require MPF inactivation (Figure 13-17b).

Researchers then prepared a peptide corresponding to residues 13 – 110 of cyclin B, which contains the destruction-box sequence and the site of polyubiquitination. When this peptide was added to a reaction mixture containing untreated egg extract and sperm chromatin, movement of chromosomes toward the spindle poles was greatly delayed at peptide concentrations of 20 – 40 μg/ml and blocked altogether at higher concentrations (Figure 13-17c). 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 proteasomes.

As discussed in Chapter 19, each sister chromatid of a metaphase chromosome is attached to microtubules via its kinetochore, a complex of proteins assembled at the centromere (see Figure 19-39); the opposite ends of these kinetochore microtubules associate with one of the spindle poles. At metaphase, the spindle is in a state of tension with forces pulling the two kinetochores towards the opposite spindle poles balanced by forces pushing the spindle poles apart. Sister chromatids do not separate because they are held together at their centromeres and multiple positions along the chromosome arms by recently discovered multiprotein complexes called cohesins. Among the proteins composing the cohesin complex are members of the SMC protein family discussed earlier. Thus SMC proteins function in multiprotein complexes with other proteins in both chromosome condensation and the cohesion of sister chromatids. The function of the cohesin complex was demonstrated in experiments in which antibodies specific for the cohesin SMC proteins were used to deplete the cohesin complex from Xenopus egg extracts. The depleted extract was able to replicate the DNA in added sperm chromatin, but the resulting sister chromatids were defective in their association with each other.

Cohesin function is regulated by the anaphase inhibitor, a protein that is a target of APC-directed polyubiquitination. In yeast, anaphase inhibitor functions together with another protein to stimulate the proper association of cohesin with daughter chromosomes. As cells enter anaphase, anaphase inhibitor is polyubiquitinated by the APC and degraded by proteasomes (see Figure 13-2). As a consequence, cohesin function is inactivated, allowing the poleward force exerted on kinetochores to move sister chromatids toward opposite spindle poles (Figure 13-18).

Figure 13-18. Model for induction of anaphase by regulation of cohesin complexes.

Figure 13-18

Model for induction of anaphase by regulation of cohesin complexes. Arrows indicate direction of the forces acting on the kinetochores. Cohesin complexes are shown connecting centromeres, but they also occur along the arms of sister chromatids. Cohesin (more...)

After APC is activated, it initially acts on certain target proteins such as the anaphase inhibitor, but it does not act on cyclin B until late in anaphase (Figure 13-19). This is necessary in order to maintain MPF activity until late anaphase, keeping chromosomes in their condensed state until they have segregated to opposite spindle poles. Recent genetic studies in budding yeast indicate that this stage-dependent targeting of APC activity is due to the regulated activity of two APC-associated proteins.

Figure 13-19. Control of entry into anaphase and exit from mitosis by the anaphase-promoting complex (APC), which directs the degradation of at least two classes of proteins.

Figure 13-19

Control of entry into anaphase and exit from mitosis by the anaphase-promoting complex (APC), which directs the degradation of at least two classes of proteins. Inactive APC (light orange) is activated (dark purple) directly or indirectly by MPF (Cdc2 – cyclin (more...)

Phosphatase Activity Is Required for Reassembly of the Nuclear Envelope and Cytokinesis

Earlier we saw that phosphorylation of nuclear lamins results in their depolymerization, leading to breakdown of the nuclear envelope, a crucial event of early mitosis. Removal of these phosphates coincides with lamin repolymerization and re-formation of the nuclear lamina associated with the daughter-cell nuclei during telophase. Studies with Xenopus egg extracts and analyses of various organisms with temperature-sensitive mutations in protein phosphatases indicate that specific protein phosphatases indeed are required for reassembly of the nuclear lamina and the nuclear envelope. When MPF is inactivated by the degradation of cyclin B late in anaphase, the action of these phosphatases, which remove the lamin regulatory phosphates, is unopposed; consequently, the lamins are rapidly dephosphorylated.

Figure 13-20 schematically depicts reassembly of the nuclear envelope, which occurs late in mitosis. Vesicles derived from the breakdown of the nuclear envelope during prophase associate with the surface of the decondensing chromosomes during telophase. These vesicles fuse to form continuous double membranes around each chromosome. Nuclear pore complexes, which disassemble into subpore complexes during prophase, reassemble into the nuclear membrane around each chromosome, forming individual mininuclei called karyomeres. Subsequent fusion of the karyomeres associated with each spindle pole generates the two daughter-cell nuclei, each one containing a full set of chromosomes. 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 nuclear-envelope vesicles throughout mitosis via the isoprenyl anchors covalently linked to the C-terminal region of lamin B.

Figure 13-20. Assembly of the nuclear envelope during telophase.

Figure 13-20

Assembly of the nuclear envelope during telophase. Nuclear envelope vesicles, generated by the breakdown of the envelope during prophase, associate with decondensing chromosomes and then fuse. Subpore complexes reassemble into nuclear pores, forming individual (more...)

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 diameter (see Figure 18-37). As MPF activity rises early in mitosis, it phosphorylates the myosin light chain, thereby inhibiting its ability to associate with actin filaments (Figure 13-21). Inactivation of MPF toward the end of anaphase, due to the degradation of cyclin B, permits the unopposed action of protein phosphatases to dephosphorylate myosin light chain. 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 before the completion of anaphase when mitotic cyclins are degraded and MPF activity falls.

Figure 13-21. Regulation of myosin light chain by MPF.

Figure 13-21

Regulation of myosin light chain by MPF. Phosphorylation of inhibitory sites on the myosin light chain by MPF early in mitosis prevents active myosin heavy chains from interacting with and sliding along actin filaments, a process required for cytokinesis. (more...)


  •  Although most substrates of MPF remain to be identified, nuclear lamins, subunits of condensin, and myosin light chain are three identified substrates.
  • MPF-catalyzed phosphorylation of specific lamin serines early in mitosis causes depolymerization of lamin filaments, leading to breakdown of the nuclear envelope (see Figure 13-15). In addition, phosphorylation of condensin complexes by MPF or a kinase regulated by MPF is thought to promote chromosome condensation.
  •  When MPF activity falls in late anaphase and telophase, protein phosphatases remove the regulatory phosphates from lamins A, B, and C, permitting reassembly of the nuclear lamina in the two daughter cell nuclei.
  • MPF-catalyzed phosphorylation of the myosin light chain prevents cytokinesis. Since MPF activity does not fall until the completion of anaphase, cytokinesis is delayed until sister chromatids have been segregated to opposite poles of the spindle apparatus.
  •  The APC-directed degradation of the anaphase inhibitor causes inactivation of the cohesin complexes that connect sister chromatids. This unlinking of sister chromatids heralds the onset of anaphase and allows sister chromatids to move apart (see Figure 13-19). Later, the same APC targets cyclin B for destruction, causing the decrease in MPF activity that marks the onset of telophase.
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By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2000, W. H. Freeman and Company.
Bookshelf ID: NBK21704


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