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(c) Metaphase

Lamin A stain DNA stain

Cells with wild-type human lamin A

▲ EXPERIMENTAL FIGURE 21-17 Transfection experiments demonstrate that phosphorylation of human lamin A is required for lamin depolymerization. Site-directed mutagenesis was used to prepare a mutant human lamin A gene encoding a protein in which alanines replace the serines that normally are phosphorylated in wild-type lamin A (see Figure 21-16b). As a result, the mutant lamin A cannot be phosphorylated. Expression vectors carrying the wild-type or mutant human gene were separately transfected into cultured hamster cells. Because the transfected lamin genes are expressed at much higher levels than the endogenous hamster lamin gene, most of the lamin A produced in transfected cells is human lamin A. Transfected cells at various stages in the cell cycle then were stained with a

Lamin A stain DNA stain

Cells with mutant human lamin A

Lamin A stain DNA stain

Cells with mutant human lamin A

fluorescent-labeled monoclonal antibody specific for human lamin A and with a fluorescent dye that binds to DNA. The bright band of fluorescence around the perimeter of the nucleus in interphase cells stained for human lamin A represents polymerized (unphosphorylated) lamin A (a). In cells expressing the wild-type human lamin A, the diffuse lamin staining throughout the cytoplasm in prophase and metaphase (b and c) and the absence of the bright peripheral band in metaphase (c) indicate depolymerization of lamin A. In contrast, no lamin depolymerization occurred in cells expressing the mutant lamin A. DNA staining showed that the chromosomes were fully condensed by metaphase in cells expressing either wild-type or mutant lamin A. [From R. Heald and F. McKeon, 1990, Cell 61:579.]

In addition, MPF-catalyzed phosphorylation of specific nucleoporins (see Chapter 12) causes nuclear pore complexes to dissociate into subcomplexes during prophase. Similarly, phosphorylation of integral membrane proteins of the inner nuclear membrane is thought to decrease their affinity for chromatin and contribute to disassembly of the nuclear envelope. The weakening of the associations between the inner nuclear membrane and the nuclear lamina and chromatin may allow sheets of inner nuclear membrane to retract into the endoplasmic reticulum, which is continuous with the outer nuclear membrane.

Several lines of evidence indicate that MPF-catalyzed phosphorylation also plays a role in chromosome condensation and formation of the mitotic spindle apparatus. For instance, genetic experiments in the budding yeast cerevisiae identified a family of SMC (structural maintenance of chromosomes) proteins that are required for normal chromosome segregation. These large proteins (»1200 amino acids) contain characteristic ATPase domains at their C-terminus and long regions predicted to participate in coiled-coil structures.

Immunoprecipitation studies with antibodies specific for Xenopus SMC proteins revealed that in cycling egg extracts some SMC proteins are part of a multiprotein complex called condensin, which becomes 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. Other in vitro experiments showed that phosphorylated purified condensin binds to DNA and winds it into supercoils (see Figure 4-7), whereas unphosphorylated condensin does not. These results have lead to the model that individual condensin complexes are activated by phosphorylation catalyzed by MPF or another protein kinase regulated by MPF. Once activated, con-densin complexes 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 micro-tubule dynamics that result in the formation of the mitotic spindle and asters (Chapter 20). In addition, phosphoryla-tion of proteins associated with the endoplasmic reticulum (ER) and Golgi complex, by MPF or other protein kinases activated by MPF-catalyzed phosphorylation, is thought to alter the trafficking of vesicles between the ER and Golgi to favor trafficking in the direction of the ER during prophase. As a result, vesicular traffic from the ER through the Golgi to the cell surface (Chapter 17), seen in interphase cells, does not occur during mitosis.

Unlinking of Sister Chromatids Initiates Anaphase

We saw earlier that in late anaphase, polyubiquitination of mitotic cyclin by the anaphase-promoting complex (APC) leads to the proteasomal destruction of this cyclin (see Figure 21-10). Additional experiments with Xenopus egg extracts provided evidence that degradation of cyclin B, the Xeno-pus mitotic cyclin, and the resulting decrease in MPF activity are required for chromosome decondensation but not for chromosome segregation (Figure 21-18a, b).

^ EXPERIMENTAL FIGURE 21-18 Onset of anaphase depends on polyubiquitination of proteins other than cyclin B in cycling Xenopus egg extracts. The reaction mixtures contained an untreated or RNase-treated Xenopus egg extract and isolated Xenopus sperm nuclei, plus other components indicated below. Chromosomes were visualized with a fluorescent DNA-binding dye. Fluorescent rhodamine-labeled tubulin in the reactions was incorporated into microtubules, permitting observation of the mitotic spindle apparatus. (a, b) After the egg extract was treated with RNase to destroy endogenous mRNAs, an RNase inhibitor was added. Then mRNA encoding either wild-type cyclin B or a mutant nondegradable cyclin B was added. The time at which the condensed chromosomes and assembled spindle apparatus became visible after addition of sperm nuclei is designated 0 minutes. In the presence of wild-type cyclin B (a), condensed chromosomes attached to the spindle microtubules and segregated toward the poles of the spindle. By 40 minutes, the spindle had depolymerized (thus is not visible), and the chromosomes had decondensed (diffuse DNA staining) as cyclin B was degraded.

In the presence of nondegradable cyclin B (b), chromosomes segregated to the spindle poles by 15 minutes, as in (a), but the spindle microtubules did not depolymerize and the chromosomes did not decondense even after 80 minutes. These observations indicate that degradation of cyclin B is not required for chromosome segregation during anaphase, although it is required for depolymerization of spindle microtubules and chromosome decondensation during telophase. (c) Various concentrations of a cyclin B peptide containing the destruction box were added to extracts that had not been treated with RNase; the samples were stained for DNA at 15 or 35 minutes after formation of the spindle apparatus. The two lowest peptide concentrations delayed chromosome segregation, and the higher concentrations completely inhibited chromosome segregation. In this experiment, the added cyclin B peptide is thought to competitively inhibit APC-mediated polyubiquitination of cyclin B as well as another target protein whose degradation is required for chromosome segregation. [From S. L. Holloway et al., 1993, Cell 73:1393; courtesy of A. W. Murray.]

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