M Experimental Figure 217

activity in Xenopus oocytes, eggs, and early embryos peaks as cells enter meiosis and mitosis. Diagrams of the cell structures corresponding to each stage are shown in Figure 21-5. MPF activity was determined by the microinjection assay shown in Figure 21-6 and quantitated by making dilutions of cell extracts. See text for discussion. [See J. Gerhart et al., 1984, J. Cell Biol. 98:1247; adapted from A. Murray and M. W. Kirschner, 1989, Nature 339:275.]

Second embryonic mitosis

fertilization, MPF activity falls again until the zygote (fertilized egg) enters the first mitosis of embryonic development. Throughout the following 11 synchronous cycles of mitosis in the early embryo, MPF activity is low in the interphase periods between mitoses and then rises as the cells enter mitosis.

Although initially discovered in frogs, MPF activity has been found in mitotic cells from all species assayed. For example, cultured mammalian cells can be arrested in mitosis by treatment with compounds (e.g., colchicine) that inhibit assembly of microtubules. When cytoplasm from such mitotically arrested mammalian cells was injected into G2-arrested Xenopus oocytes, the oocytes matured into eggs; that is, the mammalian somatic mitotic cells contained a cy-tosolic factor that exhibited frog MPF activity. This finding suggested that MPF controls the entry of mammalian somatic cells into mitosis as well as the entry of frog oocytes into meiosis. When cytoplasm from mitotically arrested mammalian somatic cells was injected into interphase cells, the interphase cells entered mitosis; that is, their nuclear membranes disassembled and their chromosomes condensed. Thus MPF is the diffusible factor, first revealed in cell-fusion experiments (see Figure 21-3), that promotes entry of cells into mitosis. Conveniently, the acronym MPF also can stand for mitosis-promoting factor, a name that denotes the more general activity of this factor.

Because the oocyte injection assay initially used to measure MPF activity is cumbersome, several years passed before MPF was purified by column chromatography and characterized. MPF is in fact one of the heterodimeric complexes composed of a cyclin and CDK now known to regulate the cell cycle. Each MPF subunit initially was recognized through different experimental approaches. First we discuss how the regulatory cyclin subunit was identified and then describe how yeast genetic experiments led to discovery of the catalytic CDK subunit.

Mitotic Cyclin Was First Identified in Early Sea Urchin Embryos

Experiments with inhibitors showed that new protein synthesis is required for the increase in MPF during the mitotic phase of each cell cycle in early frog embryos. As in early

▲ EXPERIMENTAL FIGURE 21-8 Experimental detection of cyclical synthesis and destruction of mitotic cyclin in sea urchin embryos. A suspension of sea urchin eggs was synchronously fertilized by the addition of sea urchin sperm, and 35S-methionine was added. At 10-minute intervals beginning 16 minutes after fertilization, samples were taken for protein analysis on an SDS polyacrylamide gel and for detection of cell cleavage by microscopy. (a) Autoradiograms of the SDS gels at each sampling time. Most proteins, such as X and Y, continuously increased in intensity. In contrast, cyclin suddenly decreased in intensity at 76 minutes after fertilization and then began increasing again at 86 minutes. The cyclin band peaked again at 106 min and decreased again at 126 min. (b) Plot of the intensity of the cyclin band (red line) and the fraction of cells that had undergone cleavage during the previous 10-minute interval (cyan line). Note that the amount of cyclin fell precipitously just before cell cleavage. [From T Evans et al., 1983, Cell 33:389; courtesy of R. Timothy Hunt, Imperial Cancer Research Fund.]

frog embryos, the initial cell cycles in the early sea urchin embryo occur synchronously, with all the embryonic cells entering mitosis simultaneously. Biochemical studies with sea urchin eggs and embryos led to identification of the cyclin component of MPF. These studies with synchronously fertilized sea urchin eggs revealed that the concentration of one protein peaked early in mitosis, fell abruptly just before cell cleavage, and then accumulated during the following interphase to peak early in the next mitosis and fall abruptly just before the second cleavage (Figure 21-8). Careful analysis showed that this protein, named cyclin B, is synthesized continuously during the embryonic cell cycles but is abruptly destroyed following anaphase. Since its concentration peaks in mitosis, cyclin B functions as a mitotic cyclin.

In subsequent experiments, a cDNA clone encoding sea urchin cyclin B was used as a probe to isolate a homologous cyclin B cDNA from Xenopus laevis. Pure Xenopus cyclin B, obtained by expression of the cDNA in E. coli, was used to produce antibody specific for cyclin B. Using this antibody, a polypeptide was detected in Western blots that co-purified with MPF activity from Xenopus eggs, demonstrating that one subunit of MPF is indeed cyclin B.

Cyclin B Levels and Kinase Activity of Mitosis-Promoting Factor (MPF) Change Together in Cycling Xenopus Egg Extracts

Some unusual aspects of the synchronous cell cycles in early Xenopus embryos provided a way to study the role of mitotic cyclin in controlling MPF activity. First, following fertilization of Xenopus eggs, the G1 and G2 periods are minimized during the initial 12 synchronous cell cycles. That is, once mitosis is complete, the early embryonic cells proceed immediately into the next S phase, and once DNA replication is complete, the cells progress almost immediately into the next mitosis. Second, the oscillation in MPF activity that occurs as early frog embryos enter and exit mitosis is observed in the cytoplasm of a fertilized frog egg even when the nucleus is removed and no transcription can occur. This finding indicates that all the cellular components required for progress through the truncated cell cycles in early Xenopus embryos are stored in the unfertilized egg. In somatic cells generated later in development and in yeasts considered in later sections, specific mRNAs must be produced at particular points in the cell cycle for progress through the cycle to proceed. But in early Xenopus embryos, all the mRNAs necessary for the early cell divisions are present in the unfertilized egg.

Extracts prepared from unfertilized Xenopus eggs thus contain all the materials required for multiple cell cycles, including the enzymes and precursors needed for DNA replication, the histones and other chromatin proteins involved in assembling the replicated DNA into chromosomes, and the proteins and phospholipids required in formation of the nuclear envelope. These egg extracts also synthesize proteins encoded by mRNAs in the extract, including cyclin B.

When nuclei prepared from Xenopus sperm are added to such a Xenopus egg extract, the highly condensed sperm chromatin decondenses, and the sperm DNA replicates one time. The replicated sperm chromosomes then condense and the nuclear envelope disassembles, just as it does in intact cells entering mitosis. About 10 minutes after the nuclear envelope disassembles, all the cyclin B in the extract suddenly is degraded, as it is in intact cells following anaphase. Following cyclin B degradation, the sperm chromosomes decondense and a nuclear envelope re-forms around them, as in an intact cell at the end of mitosis. After about 20 minutes, the cycle begins again. DNA within the nuclei formed after the first mitotic period (now 2n) replicates, forming 4n nuclei. Cyclin B, synthesized from the cyclin B mRNA present in the extract, accumulates. As cyclin B approaches peak levels, the chromosomes condense once again, the nuclear envelopes break down, and about 10 minutes later cyclin B is once again suddenly destroyed. These remarkable Xenopus egg extracts can mediate several of these cycles, which mimic the rapid synchronous cycles of an early frog embryo.

Studies with this egg extract experimental system were aided by development of a new assay for MPF activity. Using MPF purified with the help of the oocyte injection assay (see Figure 21-6), researchers had found that MPF phosphory-lates histone H1. This H1 kinase activity provided a much simpler and more easily quantitated assay for MPF activity than the oocyte injection assay. Armed with a convenient assay, researchers tracked the MPF activity and concentration of cyclin B in cycling Xenopus egg extracts. These studies showed that MPF activity rises and falls in synchrony with the concentration of cyclin B (Figure 21-9a). The early events of mitosis—chromosome condensation and nuclear envelope disassembly—occurred when MPF activity reached its highest levels in parallel with the rise in cyclin B concentration. Addition of cycloheximide, an inhibitor of protein synthesis, prevented cyclin B synthesis and also prevented the rise in MPF activity, chromosome condensation, and nuclear envelope disassembly.

To test the functions of cyclin B in these cell-cycle events, all mRNAs in the egg extract were degraded by digestion with a low concentration of RNase, which then was inactivated by addition of a specific inhibitor. This treatment destroys mRNAs without affecting the tRNAs and rRNAs required for protein synthesis, since their degradation requires much higher concentrations of RNase. When sperm nuclei were added to the RNase-treated extracts, the 1 n nuclei replicated their DNA, but the increase in MPF activity and the early mitotic events (chromosome condensation and nuclear envelope disassembly), which the untreated extract supports, did not occur (Figure 21-9b). Addition of cyclin B mRNA, produced in vitro from cloned cyclin B cDNA, to the RNase-treated egg extract and sperm nuclei restored the parallel oscillations in the cyclin B concentration and MPF activity and the characteristic early and late mitotic events as observed with the untreated egg extract (Figure 21-9c). Since cyclin B is the only protein synthesized under these conditions, these results demonstrate that it is the

(a) Untreated extract

Mitotic events

= MPF activity = Cyclin B concentration

(a) Untreated extract

Mitotic events

= MPF activity = Cyclin B concentration

Time

(b) RNase-treated extract

Addition of sperm nuclei

Time

Time

(c) RNase-treated extract + wild-type cyclin B mRNA Mitotic events

Time

(d) RNase-treated extract + nondegradable cyclin B mRNA

Addition of sperm nuclei

Addition of sperm nuclei

Mitotic arrest -

Time

▲ EXPERIMENTAL FIGURE 21-9 Cycling of MPF activity and mitotic events in Xenopus egg extracts depend on synthesis and degradation of cyclin B. In all cases, MPF activity and cyclin B concentration were determined at various times after addition of sperm nuclei to a Xenopus egg extract treated as indicated in each panel. Microscopic observations determined the occurrence of early mitotic events (blue shading), including chromosome condensation and nuclear envelope disassembly, and of late events (orange shading), including chromosome decondensation and nuclear envelope reassembly. See text for discussion. [See A. W. Murray et al., 1989, Nature 339:275; adapted from A. Murray and T Hunt, 1993, The Cell Cycle: An Introduction, W. H. Freeman and Company.]

crucial protein whose synthesis is required to regulate MPF activity and the cycles of chromosome condensation and nuclear envelope breakdown mediated by cycling Xenopus egg extracts.

In these experiments, chromosome decondensation and nuclear envelope assembly (late mitotic events) coincided with decreases in the cyclin B level and MPF activity. To determine whether degradation of cyclin B is required for exit from mitosis, researchers added a mutant mRNA encoding a non-degradable cyclin B to a mixture of RNase-treated Xenopus egg extract and sperm nuclei. As shown in Figure 21-9d, MPF activity increased in parallel with the level of the mutant cy-clin B, triggering condensation of the sperm chromatin and nuclear envelope disassembly (early mitotic events). However, the mutant cyclin B produced in this reaction never was degraded. As a consequence, MPF activity remained elevated, and the late mitotic events of chromosome decondensation and nuclear envelope formation were both blocked. This experiment demonstrates that the fall in MPF activity and exit from mitosis depend on degradation of cyclin B.

The results of the two experiments with RNase-treated extracts show that entry into mitosis requires the accumula-

tion of cyclin B, the Xenopus mitotic cyclin, to high levels, and that exit from mitosis requires the degradation of this mitotic cyclin. Since MPF kinase activity varied in parallel with the concentration of the mitotic cyclin, the results implied that high MPF kinase activity results in entry into mitosis and that a fall in MPF kinase activity is required to exit mitosis.

Anaphase-Promoting Complex (APC) Controls Degradation of Mitotic Cyclins and Exit from Mitosis

Further studies revealed that vertebrate cells contain three proteins that can function like cyclin B to stimulate Xeno-pus oocyte maturation: two closely related cyclin Bs and cy-clin A. Collectively called B-type cyclins, these proteins exhibit regions of high sequence homology. (B-type cyclins are distinguished from G1 cyclins described in Section 21.5.) In intact cells, degradation of all the B-type cyclins begins after the onset of anaphase, the period of mitosis when sister chromatids are separated and pulled toward opposite spindle poles.

M FIGURE 21-10 Regulation of mitotic cyclin levels in cycling Xenopus early embryonic cells. In late anaphase, the anaphase-promoting complex (APC) polyubiquitinates mitotic cyclins. As the cyclins are degraded by proteasomes, MPF kinase activity declines precipitously, triggering the onset of telophase. APC activity is directed toward mitotic cyclins by a specificity factor, called Cdh1, that is phosphorylated and thereby inactivated by G-i cyclin-CDK complexes. A specific phosphatase called Cdc14 removes the regulatory phosphate from the specificity factor late in anaphase. Once the specificity factor is inhibited in G-^ the concentration of mitotic cyclin increases, eventually reaching a high enough level to stimulate entry into the subsequent mitosis.

Metaphase

High mitotic cyclin High MPF activity

Metaphase

High mitotic cyclin High MPF activity

Low mitotic cyclin Low MPF activity

Telophase

Low mitotic cyclin Low MPF activity

Telophase

Biochemical studies with Xenopus egg extracts showed that at the time of their degradation, wild-type B-type cyclins are modified by covalent addition of multiple ubiquitin molecules. This process of polyubiquitination marks proteins for rapid degradation in eukaryotic cells by proteasomes, multiprotein cylindrical structures containing numerous proteases. Addition of a ubiquitin chain to a B-type cyclin or other target protein requires three types of enzymes (see Figure 3-13). Ubiquitin is first activated at its carboxyl-terminus by formation of a thioester bond with a cysteine residue of ubiquitin-activating enzyme, E1. Ubiquitin is subsequently transferred from E1 to a cysteine of one of a class of related enzymes called ubiquitin-conjugating enzymes, E2. The specific E2 determines, along with a third protein, ubiquitin ligase (E3), the substrate protein to which a ubiquitin chain will be covalently linked. Many ubiquitin ligases are multisubunit proteins.

Sequencing of cDNAs encoding several B-type cyclins from various eukaryotes showed that all contain a homologous nine-residue sequence near the N-terminus called the destruction box. Deletion of this destruction box, as in the mutant mRNA used in the experiment depicted in Figure 21-8, prevents the polyubiquitination of B-type cyclins and thus makes them nondegradable. The ubiquitin ligase that recognizes the mitotic cyclin destruction box is a multisubunit protein called the anaphase-promoting complex (APC), which we introduced earlier in the chapter (see Figure 21-2, steps 8 and 9).

Figure 21-10 depicts the current model that best explains the changes in mitotic cyclin levels seen in cycling Xenopus early embryonic cells. Cyclin B, the primary mi totic cyclin in multicellular animals (metazoans), is synthesized throughout the cell cycle from a stable mRNA. The observed fall in its concentration in late anaphase results from its APC-stimulated degradation at this point in the cell cycle. As we discuss in Section 21.5, genetic studies with yeast led to identification of an APC specificity factor, called Cdh1, that binds to APC and directs it to polyu-biquitinate mitotic cyclins. This specificity factor is active only in late anaphase when the segregating chromosomes have moved far enough apart in the dividing cell to assure that both daughter cells will contain one complete set of chromosomes. Phosphorylation of Cdh1 by other cyclin-CDK complexes during G1 inhibits its association with the APC and thus degradation of mitotic cyclin (see Figure 21-2, step 2). This inhibition permits the gradual rise in mitotic cyclin levels observed throughout interphase of the next cell cycle.

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