13:  13.2 Biochemical Studies with Oocytes, Eggs, and Early Embryos

MPF Promotes Maturation of Oocytes and Mitosis in Somatic Cells

The process of oocyte maturation, from G₂-arrested oocyte to the egg arrested in metaphase of meiosis II, can be studied in vitro by surgically removing G₂-arrested oocytes from the ovary of an adult female frog and treating them with progesterone (Figure 13-5a). When cytoplasm from eggs arrested in metaphase of meiosis II is microinjected into G₂-arrested oocytes, the oocytes mature into eggs in the absence of progesterone (Figure 13-5b). This system not only led to the initial identification of a factor in egg cytoplasm that stimulates maturation of oocytes in vitro in the absence of progesterone but also provided an assay for this factor, called maturation-promoting factor (MPF). As we will see shortly, MPF turned out to be the key factor that regulates the initiation of mitosis in all eukaryotic cells.

Using the microinjection system to assay MPF activity at different times during oocyte maturation in vitro, researchers found that untreated G₂-arrested oocytes have low levels of MPF activity; treatment with progesterone induces MPF activity as the cells enter meiosis I (Figure 13-6). As the cells enter the interphase between meiosis I and II, MPF activity falls; it then rises as the cells enter meiosis II and are arrested. Following fertilization, MPF activity falls again until the zygote (fertilized egg) enters the first mitosis of embryonic development. All the cells in early frog embryos undergo 12 synchronous cycles of mitosis. Throughout these cycles 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 cytosolic 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 broke down into small vesicles and their chromosomes condensed. Thus MPF is the diffusible factor, first revealed in cell-fusion experiments (see Figure 13-3), 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 assay for MPF is cumbersome, several years passed before MPF was purified by column chromatography and the MPF proteins were characterized. MPF is in fact one of the heterodimeric complexes composed of a cyclin and cyclin-dependent protein kinase (Cdk) now known to regulate the cell cycle (see Figure 13-2). Each MPF subunit 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 Cdk catalytic 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 (see Figure 13-6). Biochemical studies with sea urchin eggs and embryos led to identification of the cyclin component of MPF. As in early frog embryos, the initial cell cycles in the early sea urchin embryo occur synchronously, with all the embryonic cells entering mitosis simultaneously. In these studies, synchronously fertilized sea urchin eggs were incubated with a radiolabeled amino acid and samples were removed every 10 minutes. Protein was isolated from each sample and analyzed by gel electrophoresis followed by autoradiography. The amount of radiolabel in the vast majority of proteins increased steadily through several cell cycles. However, one protein peaked in intensity early in mitosis, fell abruptly during anaphase, and then slowly accumulated during the following interphase to peak early in the next mitosis. Careful analysis showed that this protein, named cyclin B, is synthesized continuously during the embryonic cell cycles and is abruptly destroyed at the onset of anaphase.

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. Western blotting of MPF purified from Xenopus eggs (see Figure 3-44), using antibody prepared against the protein encoded by cyclin B cDNA, showed that one subunit of MPF is indeed cyclin B. The other subunit is the catalytic Cdk subunit, first identified in genetic experiments with yeasts discussed later.

Cyclin B Levels and MPF Activity Change Together in Cycling Xenopus Egg Extracts

Some unusual aspects of the rapid cell cycles in early animal embryos provided a way to study the role of mitotic cyclin in controlling MPF activity. Of particular importance, in the 12 rapid, synchronous cell cycles that occur following fertilization of Xenopus eggs, the G₁ and G₂ periods are minimized, and the cell cycle consists of alternating M and S phases. Once mitosis is complete, the early embryonic cells proceed immediately into the S phase, and once DNA replication is complete, the cells progress almost immediately into the next mitosis.

Remarkably, the oscillation in MPF activity that occurs as early frog embryos enter and exit mitosis (see Figure 13-6) is observed even when the nucleus is removed from a fertilized egg. This finding shows that a cell-cycle clock operates in the cytoplasm of early frog embryos completely independently of nuclear events. This phenomenon occurs only in synchronously dividing cells of early animal embryos. No transcription occurs during these rapid cell cycles, indicating that all the cellular components required for progress through the truncated cell cycles 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 animal embryos, all the mRNAs necessary for the early cell divisions are present in the unfertilized egg. Extracts prepared from unfertilized frog 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 lipids required in formation of the nuclear envelope. These egg extracts also synthesize proteins encoded by mRNAs in the extract, including cyclin B.

When chromatin prepared from interphase frog sperm is added to a Xenopus egg extract, a nuclear envelope develops around the chromatin, forming a haploid nucleus. Following formation of a nuclear envelope, the sperm DNA replicates one time. Following DNA replication, the sperm chromosomes condense and the nuclear envelope breaks down into vesicles, just as it does in intact cells entering mitosis. About 10 minutes after the nuclear envelope breaks down, all the cyclin B in the extract suddenly is degraded, as it is in intact cells during 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.

Using this experimental system, researchers found that MPF activity, assayed by its ability to phosphorylate histone H1, rises and falls in synchrony with the concentration of cyclin B (Figure 13-7a). The early events of mitosis — chromosome condensation and nuclear envelope breakdown — 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 breakdown.

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 chromatin was added to the RNase-treated extracts, nuclear envelopes assembled around the sperm chromatin and the resulting 1n nuclei replicated their DNA, but the increase in MPF activity and the early mitotic events (chromosome condensation and nuclear envelope breakdown), which the untreated extract supports, did not occur (Figure 13-7b). Addition of cyclin B mRNA, produced in vitro from cloned cyclin B cDNA, to the RNase-treated egg extract and sperm chromatin restored the parallel oscillations in MPF activity and cyclin B level and the characteristic early and late mitotic events as observed with the untreated egg extract (Figure 13-7c). Since cyclin B is the only protein synthesized under these conditions, these results demonstrate that it is the crucial protein whose synthesis is required to regulate MPF activity and the cycles of chromosome condensation and nuclear envelope breakdown mediated by cycling egg extracts.

In these experiments, chromosome decondensation and nuclear envelope formation (late mitotic events) coincided with decreases in MPF activity and the cyclin B level. As mentioned earlier and described in detail below, mitotic cyclins can be polyubiquitinated and subsequently degraded. To determine whether degradation of cyclin B is required for exit from mitosis, researchers added a mutant mRNA encoding a nondegradable cyclin B to a mixture of RNase-treated Xenopus egg extract and sperm chromatin. As shown in Figure 13-7d, MPF activity increased in parallel with the level of the mutant cyclin B, triggering condensation of the sperm chromatin and nuclear envelope breakdown (early mitotic events). However, the mutant cyclin B synthesized in this reaction never was degraded as in the reaction with wild-type cyclin B mRNA (see Figure 13-7c). As a consequence, MPF activity continued to increase 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 depends on degradation of cyclin B.

Ubiquitin-Mediated Degradation of Mitotic Cyclins Promotes Exit from Mitosis

Animal cells actually contain three cyclins that can function like cyclin B to stimulate Xenopus oocyte maturation: cyclin A (which was the first cyclin shown to have this function) and two closely related cyclin Bs. Sequencing of cDNAs encoding several mitotic cyclins from various eukaryotes has shown that all the encoded proteins contain a homologous sequence near the N-terminus called the destruction box (Figure 13-8a). In intact cells, cyclin degradation begins shortly after the onset of anaphase (late anaphase), the period of mitosis when sister chromatids are separated and pulled toward opposite spindle poles.

Biochemical studies with Xenopus egg extracts showed that after their synthesis, wild-type mitotic cyclins are modified by addition of ubiquitin, a highly conserved, 76-residue protein. As discussed in Chapter 3, covalent attachment of chains of ubiquitin, a process called polyubiquitination, marks proteins for rapid degradation in eukaryotic cells by proteasomes, multiprotein cylindrical structures containing numerous proteases (see Figure 3-18).

Addition of ubiquitin to a mitotic cyclin or other target protein requires three types of enzymes (Figure 13-8b). Ubiquitin is first activated at its carboxyl-terminus by formation of a thioester bond with the cystine residue of ubiquitinactivating enzyme, E1. Ubiquitin is subsequently transferred from E1 to the cystine 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 multiple ubiquitins will be covalently linked via a lysine residue, marking the substrate protein for rapid degradation by a proteasome. E3 proteins are frequently complex, multisubunit proteins; for instance, the E3 for cyclin B purified from Xenopus eggs contains at least eight different subunits. This E3 that targets mitotic cyclins for polyubiquitination is the anaphase-promoting complex (APC) mentioned earlier (see Figure 13-2). The APC targets E2-ubiquitin complexes to the destruction box in mitotic cyclins, and then stimulates transfer of the ubiquitin to a lysine residue on the C-terminal side of the destruction box. Further cycles of ubiquitination result in chains of polyubiquitin, which are recognized by proteasomes (see Figure 13-8b). Mutant cyclins that lack a destruction box have been constructed using recombinant DNA techniques; because they lack a destruction box, these mutant proteins are not rapidly degraded.

Regulation of APC Activity Controls Degradation of Cyclin B

The degradation of cyclin B in late anaphase is regulated by controlling APC activity. The APC that is isolated from Xenopus eggs arrested in metaphase has low activity for stimulating polyubiquitination of cyclin B. In contrast, APC isolated from eggs stimulated to complete mitosis has high ubiquitination-stimulating activity. Several of the subunits in APC with high activity are phosphorylated; removal of these phosphates with a protein phosphatase decreases APC activity. These findings led to the model for regulating APC activity depicted in Figure 13-9.

When MPF activity reaches its peak at metaphase, it phosphorylates and thereby activates APC. Polyubiquitination of cyclin B then occurs, leading to the degradation of cyclin B. Since cyclin B is an essential subunit of MPF, its degradation causes inactivation of MPF activity. APC is deactivated late in G₁, permitting a rise in the cyclin B level and the concomitant increase in MPF activity needed to enter another mitotic cycle. Since cyclin B is synthesized continuously during the cell cycle, this mechanism accounts for the rise in the cyclin B levels following mitosis (during interphase) and the sudden fall in cyclin B levels late in mitosis.

SUMMARY

• MPF is a heterodimer composed of a mitotic cyclin and a cyclin-dependent protein kinase (Cdk). The protein kinase activity of MPF stimulates the onset of mitosis by phosphorylating multiple specific protein substrates, most of which remain to be identified.

• In the synchronously dividing cells of early Xenopus embryos, the concentration of mitotic cyclins (e.g., cyclin B) and MPF activity increase as cells enter mitosis and then fall precipitously during late anaphase (see Figure 13-7).

• Proteolysis of mitotic cyclins, which leads to a decrease in MPF activity, is required for the completion of mitosis.

• Mitotic cyclins contain a nine-residue sequence, the destruction box, that is recognized by ubiquitinating enzymes. The multisubunit anaphase-promoting complex (APC) directs specific ubiquitin-conjugating enzymes to polyubiquitinate mitotic cyclins, marking the proteins for rapid degradation by proteasomes.

• The concentration of mitotic cyclins, which are synthesized continuously in early Xenopus embryos, is regulated by controlling APC activity. APC activity rises in response to elevated MPF activity, possibly due to direct phosphorylation of APC subunits by MPF. Activated APC then promotes the ubiquitin-dependent degradation of mitotic cyclins in late anaphase (see Figure 13-9). Deactivation of APC in late G₁ permits accumulation of mitotic cyclins.

• The cyclical increases and decreases in MPF activity, resulting in entry into and exit from mitosis in early Xenopus embryos, depends on cyclical decreases and increases in the rate of mitotic cyclin degradation.