13:  13.1 Overview of the Cell Cycle and Its Control

The Cell Cycle Is an Ordered Series of Events Leading to Replication of Cells

As illustrated in Figure 13-1, the cell cycle is divided into four major phases. In cycling (replicating) somatic cells, chromosomes are replicated during the S (synthesis) phase. After progressing through the G₂ phase, cells begin the complicated process of mitosis, also called the M phase, which is divided into several stages (see Figure 19-34). Chromosomes condense during the prophase period of mitosis, by tightly folding loops of the 30-nm chromatin fiber attached to the chromosome scaffold (see Figure 9-35). Sister chromatids, produced by DNA replication during the S phase, remain attached at the centromere and multiple points along their length and become aligned in the center of the cell during metaphase. During the anaphase portion of mitosis, sister chromatids separate and move to opposite poles of the mitotic apparatus, or spindle (see Figure 19-36), segregating one of the two sister chromatids to each daughter cell.

In most cells from higher eukaryotes, the nuclear envelope breaks down into multiple small vesicles early in mitosis and re-forms around the segregated chromosomes as they decondense during telophase, the last mitotic stage. The physical division of the cytoplasm, called cytokinesis, then yields two daughter cells. The Golgi complex and endoplasmic reticulum also vesiculate during mitosis and re-form in the two daughter cells after cell division. In yeasts and other fungi, the nuclear envelope does not break down. In these organisms, the mitotic spindle forms within the nuclear envelope, which then pinches off, forming two nuclei at the time of cytokinesis. Following mitosis, cycling cells enter the G₁ phase, the period before DNA synthesis is reinitiated in the S phase.

In vertebrates and diploid yeasts, cells in G₁ have a diploid number of chromosomes (2n), one inherited from each parent. In haploid yeasts, cells in G₁ have one of each chromosome (1n). Rapidly replicating human cells progress through the full cell cycle in about 24 hours: mitosis takes ≈30 minutes; G₁, 9 hours; the S phase, 10 hours; and G₂, 4.5 hours. In contrast, the full cycle takes only ≈90 minutes in rapidly growing yeast cells.

Postmitotic cells in multicellular organisms can “exit” the cell cycle and remain for days, weeks, or in some cases (e.g., nerve cells and cells of the eye lens) even the lifetime of the organism without proliferating further. Most postmitotic cells in vertebrates exit the cell cycle in G₁, entering a phase called G₀ (see Figure 13-1). G₀ cells returning to the cell cycle enter into the S phase; this reentry is regulated, thereby providing control of cell proliferation.

Regulated Protein Phosphorylation and Degradation Control Passage through the Cell Cycle

As mentioned in the chapter introduction, the complex macromolecular events of the eukaryotic cell cycle are regulated by a small number of heterodimeric protein kinases. The concentrations of the regulatory subunits of these kinases, called cyclins, increase and decrease in phase with the cell cycle. Their catalytic subunits are called cyclin-dependent kinases (Cdks) because they have no kinase activity unless they are associated with a cyclin. Each Cdk catalytic subunit can associate with different cyclins, and the associated cyclin determines which proteins are phosphorylated by the Cdk-cyclin complex.

Figure 13-2 outlines the role of the three classes of cyclin-Cdk complexes that control passage through the cell cycle: the G₁, S-phase, and mitotic Cdk complexes. When cells are stimulated to replicate, G₁ Cdk complexes are expressed first. These prepare the cell for the S phase by activating transcription factors that cause expression of enzymes required for DNA synthesis and the genes encoding S-phase Cdk complexes. The activity of S-phase Cdk complexes is initially held in check by a specific inhibitor. Then, in late G₁, G₁ Cdk complexes induce the degradation of the S-phase inhibitor, releasing the activity of the S-phase Cdk complexes, which stimulate entry into the S phase.

Once activated by degradation of the S-phase inhibitor, the S-phase Cdk complexes phosphorylate regulatory sites in the proteins that form DNA pre-replication complexes, which are assembled on replication origins during G₁. Phosphorylation of these proteins by S-phase Cdk complexes not only activates initiation of DNA replication but also prevents re-assembly of new pre-replication complexes. Because of this inhibition, each chromosome is replicated just once during passage through the cell cycle, ensuring that the proper chromosome number is maintained in the daughter cells.

Mitotic Cdk complexes are synthesized during the S phase and G₂, but their activities are held in check until DNA synthesis is completed. Once activated, mitotic Cdk complexes induce chromosome condensation, breakdown of the nuclear envelope, assembly of the mitotic spindle apparatus, and alignment of condensed chromosomes at the metaphase plate (see Figure 19-34). After the proper association of all chromosomes with spindle microtubules has occurred, the mitotic Cdk complexes activate the anaphase-promoting complex (APC). This multiprotein complex directs the ubiquitin-mediated proteolysis of anaphase inhibitors, leading to inactivation of the protein complexes that connect sister chromatids at metaphase. Degradation of these inhibitors thus permits the onset of anaphase, during which sister chromatids segregate to opposite spindle poles. Later in anaphase, the APC also directs proteolytic degradation of the mitotic cyclins. The resulting decrease in mitotic Cdk activity permits the now separated chromosomes to decondense, the nuclear envelope to re-form around daughter-cell nuclei during telophase, and the cytoplasm to divide at cytokinesis, yielding the two daughter cells.

During early G₁ of the next cell cycle, phosphatases dephosphorylate the proteins that form pre-replication complexes. As a result, these complexes can assemble at replication origins in preparation for the next S phase. Phosphorylation of APC by G₁ Cdk complexes in late G₁ inactivates it, allowing the subsequent accumulation of mitotic cyclins during the S phase and G₂ of the ensuing cycle.

Passage through three critical cell-cycle transitions, G₁ → S phase, metaphase → anaphase, and anaphase → telophase and cytokinesis, is irreversible because these transitions are triggered by the regulated degradation of proteins, an irreversible process. As a consequence, cells are forced to traverse the cell cycle in one direction only.

In higher organisms, control of the cell cycle is achieved primarily by regulating the synthesis and activity of G₁ Cdk complexes. Extracellular growth factors, called mitogens, induce the synthesis of G₁ Cdk complexes. The activity of these and other Cdk complexes is regulated by phosphorylation at specific inhibitory and activating sites in the catalytic subunit. Once mitogens have acted for a sufficient period, the cell cycle continues through mitosis even when they are removed. The point in late G₁ where passage through the cell cycle becomes independent of mitogens is called the restriction point (see Figure 13-2).

Diverse Experimental Systems Have Been Used to Identify and Isolate Cell-Cycle Control Proteins

The first evidence that diffusible factors regulate the cell cycle came from cell-fusion experiments with cultured mammalian cells. When interphase cells in the G₁, S, or G₂ stage of the cell cycle were fused to cells in mitosis, their nuclear envelopes vesiculated and their chromosomes condensed (Figure 13-3). This finding indicates that some diffusible component or components in the cytoplasm of the mitotic cells forced interphase nuclei to undergo many of the processes associated with early mitosis. We now know that these factors are the mitotic Cdk complexes. Similarly, when cells in G₁ were fused to cells in the S phase and the fused cells exposed to radiolabeled thymidine, the label was incorporated into the DNA of the G₁ nucleus, indicating that DNA synthesis began in the G₁ nucleus shortly after fusion. However, when cells in G₂ were fused to S-phase cells, no incorporation of labeled thymidine occurred in the G₂ nuclei. Thus diffusible factors in an S-phase cell can enter the nucleus of a G₁ cell and stimulate DNA synthesis, but these factors cannot induce DNA synthesis in a G₂ nucleus. We now know that these factors are S-phase Cdk complexes, which can activate the pre-replication complexes assembled on DNA replication origins in early G₁ nuclei. Although these cell-fusion experiments demonstrated that diffusible factors control entry into the S and M phases of the cell cycle, genetic and biochemical experiments were needed to identify these factors.

The budding yeast Saccharomyces cerevisiae and the distantly related fission yeast Schizosaccharomyces pombe have been especially useful for isolation of mutants that are blocked at specific steps in the cell cycle or that exhibit altered regulation of the cycle. In both of these yeasts, temperature-sensitive mutants with defects in specific proteins required to progress through the cell cycle are readily recognized microscopically and therefore easily isolated. S. cerevisiae daughter cells form from a growing bud, whose size relative to the parental cell increases during the cell cycle. Mutant S. cerevisiae cells with a cell-cycle defect are easily identified because at the nonpermissive temperature they are arrested in the budding process (see Figure 8-9). Such cells are called cdc (cell-division cycle) mutants. S. pombe cells, in contrast, increase in length and then divide in the middle to form daughter cells. In this yeast, cdc mutants grow without dividing and form enormously elongated cells at the nonpermissive temperature. Other S. pombe mutants, called wee (from the Scottish word for small), divide before the parental cell has grown to the normal length, forming cells that are shorter than normal.

Temperature-sensitive mutations that block progression through the cell cycle at the nonpermissive temperature obviously prevent colony formation from a single haploid yeast cell. The wild-type alleles of recessive temperature-sensitive mutant alleles can be isolated readily by transforming haploid mutant cells with a plasmid library prepared from wild-type cells and then plating the transformed cells at the nonpermissive temperature (Figure 13-4). Complementation of the recessive mutation by the wild-type allele on one of the plasmids in the library allows a transformed mutant cell to grow into a colony; the plasmid bearing the wild-type allele can then be recovered from those cells. Because many of the proteins that regulate the cell cycle are highly conserved, human cDNAs cloned into yeast expression vectors often can complement yeast cell-cycle mutants, leading to the rapid isolation of human genes encoding cell-cycle control proteins.

Biochemical studies require the preparation of cell extracts from many cells. For biochemical studies of the cell cycle, the eggs and early embryos of amphibians and marine invertebrates are particularly suitable. In these organisms, multiple synchronous cell cycles follow fertilization of a large egg. By isolating large numbers of eggs from females and fertilizing them simultaneously by addition of sperm (or treating them in ways that mimic fertilization), researchers can obtain extracts for analysis of proteins and enzymatic activities that occur at specific points in the cell cycle.

In the following sections we describe critical experiments that led to the current model of eukaryotic cell-cycle regulation summarized in Figure 13-2 and present further details of the various regulatory events. As we will see, results obtained with different experimental systems and approaches have provided insights about each of the key transition points in the cell cycle.

SUMMARY

• The eukaryotic cell cycle is divided into four phases: M (mitosis), G₁ (the period between mitosis and the initiation of nuclear DNA replication), S (the period of nuclear DNA replication), and G₂ (the period between the completion of nuclear DNA replication and mitosis) (see Figure 13-1).

• Cdk complexes, composed of a regulatory cyclin subunit and a catalytic cyclin-dependent kinase subunit, regulate progress of a cell through the cell cycle (see Figure 13-2). Large protein complexes also mark specific inhibitors of cell-cycle events for proteolytic degradation by proteasomes.

• Diffusible factors in mitotic cells, now known to be mitotic Cdk complexes, cause chromosome condensation and vesiculation of the nuclear envelope in G₁ and G₂ cells when they are fused to mitotic cells. Similarly, S-phase Cdk complexes stimulate DNA replication in the nuclei of G₁ cells when they are fused to S-phase cells.

• Amphibian and invertebrate eggs and early embryos from synchronously fertilized eggs provide sources of extracts for biochemical studies of cell-cycle events.

• The isolation of yeast cell-division cycle (cdc) mutants led to the identification of genes that regulate the cell cycle (see Figure 13-4).