A Long Twentieth Century of the Cell Cycle and Beyond

Only those exacting editors at Cell could seriously ask you to review the past century of cell cycle research and to predict the course of research for the next century, and to do it all in 10 pages! It is unrealistic to try to be comprehensive in such a review, and so I will focus on what I consider to be the important principles underlying the cell cycle, with less emphasis on detailed descriptions of molecular mechanisms which would degenerate into lists of genes and proteins. Referencing will be minimal, and will be restricted to reviews, a few key primary publications, and to books written in English for summaries of the earlier literature. Let us begin at the end of the century by summarizing what is now known about the cell cycle. We know that the cell cycle is the universal process by which cells reproduce, and that it underlies the growth and development of all living organisms. The most important events of the cell cycle are those concerned with the copying and partitioning of the hereditary material, that is replicating the chromosomal DNA during S phase and separating the replicated chromosomes during mitosis. Controls operate that regulate onset of these events and compensate for errors in their execution. The molecular basis of these controls is highly conserved from simple unicellular eukaryotes such as yeast to complex metazoans such as ourselves. The precision with which cell cycle events are executed ensures the survival of living organisms, while loss of this precision increases genomic instability, an important factor in the formation of cancer. The mitotic cell cycle is modified to a meiotic cycle during gamete formation, leading to a reduction in chromosome number that is essential for sexual reproduction, and to an increase in genetic variation that is a driving force for evolution. Thus, the cell cycle plays a central role in the operation and development of all life, and in ensuring the continuity of life across generations. These discoveries were made over a period that extends into the previous century, and so the scope of this review will be similarly extended, hence “A Long Twentieth Century of the Cell Cycle.” Study of the cell cycle began with the discovery of cell division. The concept of a cell was well established by the mid-nineteenth century, but understanding of how cells were reproduced remained confused, partly because Schleiden and Schwann, the major proponents of the cell theory, thought that cells arose from within preexisting cells by a process somewhat similar to precipitation or crystallization (43Schwann T Microscopical researches into the accordance in the structure and growth of animals and plants. Syndenham Society, London1857Google Scholar). Greater clarity came with Nägeli and Remak who correctly described the division of plant and animal cells, and with Virchow who promoted the idea that all cells were produced by the fission of preexisting cells (for a pithy, historical account of this period, see 12Harris H The Birth of the Cell. Yale University Press, New Haven1999Google Scholar). At around the same time, Kolliker realized that early embryonic cleavage represented a series of cell divisions producing cells that eventually became differentiated into various tissues and organs (for a great near contemporary account of cell work at this time, see 50Wilson E The Cell in Development and Heredity. Macmillan, New York1925Google Scholar). This idea was extended further during the 1870s and 1880s by Pringsheim, Strasburger, and Hertwig (44Sharp L.W An Introduction to Cytology. McGraw Hill Book Company, New York1921Google Scholar), who recognized that eggs and sperm were single cells which became joined together at fertilization, so even the most complex multicellular organisms passed through a single celled stage. Thus, cell division was established as the basis of growth and development of both animals and plants. Improvements in microscopes and microscopic techniques led to a detailed description of the changes occurring to the chromosomes during mitosis, the most conspicuous event of cell division. A critical feature observed by Flemming and Strasburger during the 1880s was the appearance of elongate chromosomal threads formed from the nucleus, which then split lengthways before shortening and thickening later in mitosis (50Wilson E The Cell in Development and Heredity. Macmillan, New York1925Google Scholar, 10Flemming W Contributions to the knowledge of the cell and its vital processes. Part II.J. Cell Biol. 1965; 25: 3-69PubMed Google Scholar). Van Beneden later showed that the longitudinal halves of each split chromosome separated apart into the two daughter nuclei, and that the chromosomes of a fertilized nematode egg were derived in equal numbers from the egg and sperm. With this discovery, Weissman came to the important conclusion that the chromosomes were the basis of heredity, and that germ cells formed a continuous line of heredity between the generations (44Sharp L.W An Introduction to Cytology. McGraw Hill Book Company, New York1921Google Scholar, 50Wilson E The Cell in Development and Heredity. Macmillan, New York1925Google Scholar). The link between the cell cycle and genetics was further strengthened by the rediscovery of Mendel's Laws of Inheritance by DeVries, Correns, and Tschermak at the turn of the century. Mendel's postulate that a zygote contains two sets of “qualities” whilst the maternal and paternal gametes have a single set, paralleled the generation of haploid and diploid sets of chromosomes during meiosis, fertilization, and mitosis. Mendel's abstract laws could thus be explained by the concrete behavior of the chromosomes during the cell cycle. This pioneering work was confirmed during the first two decades of the twentieth century, placing the cell cycle firmly at the centre of the growth, development and heredity of all living organisms (50Wilson E The Cell in Development and Heredity. Macmillan, New York1925Google Scholar). At this time there were also speculations relevant to the control of the cell cycle. Hertwig proposed the concept of the karyoplasmic ratio (50Wilson E The Cell in Development and Heredity. Macmillan, New York1925Google Scholar), arguing that there is a constancy between nuclear and cytoplasmic volume, and that cell cleavage takes place when this ratio becomes unbalanced. Implicit in this concept is the idea that progression through the cell cycle is coordinated with cellular growth, an idea which was only developed further later in the century. The period between 1920 and 1950 was somewhat of a Dark Ages for the cell cycle. The most interesting work dealt with the various chromosomal changes that can occur during mitosis and meiosis, and their significance for genetic transmission (6Darlington C.D The Evolution of Genetic Systems. Cambridge University Press, London1958Google Scholar). However, for new insights into the events and controls of the cell cycle, we have to move on three decades to work started in the 1950s. The events concerned with the replication and partition of the chromosomes are common to all cell cycles, because with few exceptions, a newly divided cell needs to receive a full genome complement to survive. Chromosomes are present in low copy number, and so special mechanisms are required to ensure their precise replication and partition. The double-helical base-paired structure of DNA provided a deeply satisfying solution to the problem of how replication can be so precise (49Watson J.D Crick F.H Molecular structure of nucleic acids a structure for deoxyribose nucleic acid.Nature. 1953; 171: 737-738Crossref PubMed Scopus (7606) Google Scholar). Also at this time, microspectrophotometric (46Swift H The constancy of deoxyribonucleic acid in plant nuclei.Proc. Natl. Acad. Sci. USA. 1950; 36: 643-653Crossref PubMed Scopus (217) Google Scholar) and autoradiographic studies (17Howard A Pelc S Synthesis of deoxyribonucleic acid in normal and irradiated cells and its relation to chromosome breakage.Heredity. 1953; 6: 261-273Google Scholar) in eukaryotic cells showed that DNA replication occurs during a restricted part of interphase called S phase. This work led to the eukaryotic cell cycle being divided into S phase and M phase or mitosis, with the gap before S phase being called G1 and after S phase G2 (32Mitchison J.M The Biology of the Cell Cycle. Cambridge University Press, Cambridge1971Google Scholar). In prokaryotes, chromosome replication and partition need not be temporally separated, and can overlap during the cell cycle of rapidly growing bacteria. It seems likely that the separation of these processes into S phase and M phase and the controls which regulate their onset both evolved as DNA content increased during the emergence of eukaryotic cells. The discovery of S phase also identified two key problems still important today: how does the machinery of DNA replication work, and what determines the onset of S phase during the cell cycle? The first of these problems has been worked on for the past 40 years and has resulted in the gradual unravelling of the molecular mechanisms and enzymology of the process of DNA replication, starting with the pioneering discovery of a DNA polymerase (23Kornberg A Lehman I Bessman M Simms E Enzymic synthesis of deoxyribonucleic acid.Biochim. Biophys. Acta. 1956; 21: 197-198Crossref PubMed Scopus (97) Google Scholar). Important advances were the development of the T4 (35Morris C.F Sinha N.K Alberts B.M Reconstruction of bacteriophage T4 DNA replication apparatus from purified components rolling circle replication following de novo chain initiation on a single-stranded circular DNA template.Proc. Natl. Acad. 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Initiation of DNA replication was found to occur at specific origin regions defined by distinct DNA sequence motifs in prokaryotic and viral systems, and in the budding yeast (45Stinchcomb D.T Struhl K Davis R.W Isolation and characterisation of a yeast chromosomal replicator.Nature. 1979; 282: 39-43Crossref PubMed Scopus (469) Google Scholar). However, such unique DNA sequence specificity is not found in most eukaryotes studied, an example being Xenopus eggs (31Mechali M Kearsey S Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast.Cell. 1984; 38: 55-64Abstract Full Text PDF PubMed Scopus (116) Google Scholar). Higher eukaryotic origins appear to have a more extended structure probably reflecting a degenerate organization and possibly a role for higher order nuclear structure. The end of the century has seen increased attention directed toward the molecular mechanisms by which replication complexes are built at origins. Chromatin associated origin recognition complexes (ORCs), identified because they bind yeast origins, are thought to act as “landing pads” for the replication complexes (1Bell S.P Stillman B Nucleotide dependent recognition of chromosomal origins of DNA replication by a multi-protein complex.Nature. 1992; 357: 128-134Crossref PubMed Scopus (956) Google Scholar). A key step for this is carried out by the initiator protein Cdc6p/Cdc18p, which loads Mcm proteins onto chromatin to “license” DNA for replication (2Blow J.J Laskey R.A A role for the nuclear envelope in controlling DNA replication within the cell cycle.Nature. 1988; 332: 546-548Crossref PubMed Scopus (455) Google Scholar, 7Diffley J.F.X Once and only once upon a time specifying and regulating origins of DNA replication in eukaryotic cells.Genes Dev. 1996; 10: 2819-2830Crossref PubMed Scopus (209) Google Scholar). Licensing ensures that no DNA is replicated twice during an S phase, and that there is only one S phase each cell cycle. Once replication complexes are built and activated, a series of replicating forks are set up along the chromosomes generating bubbles that eventually fuse together to complete DNA replication. The next major event of the cell cycle is mitosis (see Figure 1 and Figure 2). Early work up to the 1960s focused on descriptive and structural studies, moving from light microscopy to electron microscopy and biochemical analysis. The mitotic spindle was first described by Boveri at the turn of the century as a system of astral rays extending between the centrosomes (50Wilson E The Cell in Development and Heredity. Macmillan, New York1925Google Scholar). Improvements in fixation and electron microscopy demonstrated that the spindle was made up of microtubules (11Harris P Electron microscope studies of mitosis in sea urchin blastomeres.J. Cell Biol. 1961; 11: 419Crossref Scopus (35) Google Scholar), and isolation of the mitotic apparatus and the identification of a colchicine binding protein (30Mazia D Mitosis and the physiology of cell division.in: Brachet J Mirsky A.E The Cell, Vol. 3. Academic Press, Inc, New York1961Google Scholar) eventually led to the discovery that microtubules were composed of tubulin polymers (21Kiefer B Sakai H Solari A.J Mazia D The molecular unit of the microtubules of the mitotic apparatus.J. Mol. Biol. 1966; 20: 75-79Crossref PubMed Scopus (39) Google Scholar). Fluorescence imaging and in vitro assays revealed that microtubules oscillate between growing and shrinking states, a process called dynamic instability (33Mitchison T Kirschner M Dynamic instability of microtubule growth.Nature. 1984; 312: 237-242Crossref PubMed Scopus (2166) Google Scholar). Microtubular organizing centres (MTOCs) were found to be located at centrosomes and at kinetechores, and shown to seed new microtubular growth, and to capture and stabilize preexisting microtubules preventing their shrinkage (40Nicklas R.B How cells get the right chromosomes.Science. 1997; 275: 632-637Crossref PubMed Scopus (499) Google Scholar). Tubulin subunit turnover and microtubular motors can move chromosomes (19Inoue S Salmon E.D Force generation by microtubule assembly/disassembly in mitosis and related movements.Mol. Biol. Cell. 1995; 6: 1619-1640Crossref PubMed Scopus (430) Google Scholar), and motors may also contribute to the building of mitotic spindles by organizing and bundling microtubules (48Vernos I Karsenti E Motors involved in spindle assembly and chromosome segregation.Curr. Opin. Cell Biol. 1996; 8: 4-9Crossref PubMed Scopus (77) Google Scholar).Figure 2Imaging Mitosis at the Turn of the MillenniumShow full captionCurrently available light microscope technology allows detailed and spectacular imaging of spindle organization in metaphase cells. This shows a salamander (newt) lung cell, fixed in metaphase, and photographed by epifluorescence after labeling the chromosomes (blue) and DAPI, and staining the microtubules (green) and keratin filaments (red) by indirect immunofluorescence methods. This cell contains a single monooriented chromosome that will delay anaphase onset until it becomes properly bioriented and positioned near the spindle equator. In epithelia, the spindle is often surrounded by a cage of intermediate filaments that formerly surrounded the nucleus. (Figure kindly provided by Conly L. Rieder, Division of Molecular Medicine, Wadsworth Center, N. Y. State Department of Health, Albany, New York.)View Large Image Figure ViewerDownload Hi-res image Download (PPT) Currently available light microscope technology allows detailed and spectacular imaging of spindle organization in metaphase cells. This shows a salamander (newt) lung cell, fixed in metaphase, and photographed by epifluorescence after labeling the chromosomes (blue) and DAPI, and staining the microtubules (green) and keratin filaments (red) by indirect immunofluorescence methods. This cell contains a single monooriented chromosome that will delay anaphase onset until it becomes properly bioriented and positioned near the spindle equator. In epithelia, the spindle is often surrounded by a cage of intermediate filaments that formerly surrounded the nucleus. (Figure kindly provided by Conly L. Rieder, Division of Molecular Medicine, Wadsworth Center, N. Y. State Department of Health, Albany, New York.) These properties provide a satisfying although still incomplete explanation for the events of mitosis. Initially the centrosomes duplicate, separate, and then generate a microtubular spindle between them. This establishes a bipolarized cellular state that is an essential early step of mitosis. The chromosomes, composed of two sister chromatids, become condensed, and each chromatid has a kinetochore located at its centromere, which is able to capture spindle microtubules. A stable configuration of chromosomes on the spindle is only achieved when one sister chromatid kinetochore becomes attached to microtubules emanating from one pole of the spindle, and the other sister chromatid kinetochore becomes similarly attached to the other pole (40Nicklas R.B How cells get the right chromosomes.Science. 1997; 275: 632-637Crossref PubMed Scopus (499) Google Scholar). This is the crucial step of mitosis because when this is achieved, the replicated DNA molecules of each chromosome become separately oriented toward opposite poles of the cell. At this point cohesion between sister chromatids is lost (38Nasmyth K Separating sister chromatids.Trends. Biochem. Sci. 1999; 24: 98-104Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), and the chromatids move apart to form two nuclei that become separated by cytokinesis. Controls of the cell cycle regulate the onset of events such as S phase and mitosis, and ensure that these events occur in the correct sequence, are coordinated with cellular growth, and are corrected for errors in their execution. Early speculations about controls included roles for such diverse processes and components as energy reservoirs, heat labile division structures, and limit cycle oscillators (for a fine review, see 32Mitchison J.M The Biology of the Cell Cycle. Cambridge University Press, Cambridge1971Google Scholar). In the last 25 years that there has been more consistent progress in understanding these controls, largely as a consequence of better conceptualization of the problems combined with more effective methodologies. An important conceptual advance was the idea that the cell cycle should be considered as a temporally organized sequence of events analogous to simple developmental systems such as phage morphogenesis (13Hartwell L Saccharomyces cerevisiae cell cycle.Bacteriol. Rev. 1974; 38: 164-198Crossref PubMed Google Scholar). This thinking focused attention on the way in which different cell cycle events were linked together in an orderly sequence. Later events were often found to be dependent upon the successful completion of earlier events, and it was reasoned that these dependencies could be of two types, either directly coupled or based on a linking signaling control. Direct coupling is hard-wired, like sequential substrate–product relationships in a metabolic pathway, and is most relevant for sequential events in processes involving direct molecular interactions like building DNA replication complexes. Failure of an early step in this type of process fails to generate the correct “product” required as the “substrate” for the next step, making the later step dependent on the earlier one. By contrast, dependencies operating through a signaling control can link more distant events separated either in space within the cell or in time between different phases of the cell cycle. For example inhibiting S phase by reducing the supply of deoxyribonucleotides blocks the temporally distant and unrelated event of mitosis. This block requires a set of proteins acting in a signal transduction pathway, which communicate the fact that S phase is incomplete to the effectors of mitosis. This idea was developed further to generate the concept of the checkpoint control (14Hartwell L Weinert T Checkpoints controls that ensure the order of cell cycle events.Science. 1989; 246: 629-634Crossref PubMed Scopus (2341) Google Scholar). At different points in the cell cycle, the cell “checks” if an earlier event, such as S phase, has been properly executed before proceeding to a later event, such as mitosis. The checkpoint concept also covers other situations such as blocking mitosis after DNA damage until the damage is repaired, a mechanism which helps ensure faithful genomic transmission. A second important conceptual advance was the idea that certain cell cycle events acted as major rate-limiting steps for cell cycle progression. Extending earlier ideas about trigger points and division proteins, an analogy was made between cell cycle control and the control of flux through a metabolic pathway (for a relevant review of flux control, see 20Kacser H Porteous J Control of metabolism what do we have to measure?.Trends Biochem. 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Sci. 1987; 12: 5-14Abstract Full Text PDF Scopus (134) Google Scholar). These conceptual advances were complemented by development of powerful new experimental approaches. One was the application of genetics coupled with molecular biology, which was particularly effective for cell cycle studies with the yeasts (15Hartwell L.H Mortimer R.K Culotti J Culotti M Genetic control of the cell division cycle in yeast genetic analysis of cdc mutants.Genetics. 1973; 74: 267-286Crossref PubMed Google Scholar, 39Nasmyth K Reed S Isolation of genes by complementation in yeast molecular cloning on a cell cycle gene.Proc. Natl. Acad. Sci. USA. 1980; 77: 2119-2123Crossref PubMed Scopus (302) Google Scholar). Cell cycle mutants were isolated, the genes defined by these mutants identified, physiologically characterized, cloned by complementation, and the cloned gene used as the starting point for subsequent biochemical analysis. A second experimental approach was the use of complex cell extracts derived from amphibian or marine invertebrate oocytes or eggs to generate in vitro systems able to carry out steps of the cell cycle in vitro (27Lohka M Masui Y Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic contents.Science. 1983; 220: 719-721Crossref PubMed Scopus (448) Google Scholar). The ability to deplete and purify certain components from these complex cell-free extracts also allowed a biochemical analysis of these steps. This allowed the purification of maturation-promoting factor (MPF), a factor that promotes the onset of M phase (28Lohka M.J Hayes M.K Maller J.L Purification of maturation-promoting factor, an intracellular regulator of early mitotic events.Proc. Natl. Acad. Sci. USA. 1988; 85: 3009-3013Crossref PubMed Scopus (444) Google Scholar). Both approaches complemented each other, and the fact that the cell cycle and its control turned out to be highly conserved meant that studies could be made and compared in a variety of biological systems, each with its own advantages. Factors that could advance cell cycle progression were good candidates for components which act as rate-limiting steps in the cell cycle, and were identified genetically in fission yeast by mutants which accelerated cell division, and in amphibian eggs by the purification of MPF. A network of genes was characterized in yeast that regulated the onset of mitosis; core to this network was the Cdc2p protein kinase activated by the Cdc25p protein phosphatase and inhibited by the Wee1p protein kinase (42Nurse P Universal control mechanism regulating onset of M-phase.Nature. 1990; 344: 503-508Crossref PubMed Scopus (2182) Google Scholar) MPF was identified in Rana oocytes induced to enter M phase as part of the egg maturation process by injection with cytoplasm derived from eggs in M phase (29Masui Y Markert C Cytoplasmic control of nuclear behaviour during meiotic maturation of frog oocytes.J. Exp. Zool. 1971; 177: 129-146Crossref PubMed Scopus (1127) Google Scholar). MPF purified from Xenopus (28Lohka M.J Hayes M.K Maller J.L Purification of maturation-promoting factor, an intracellular regulator of early mitotic events.Proc. Natl. Acad. Sci. 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They were shown to act as universal cell cycle regulators from yeast to mammals by the cloning of the human CDC2 gene by complementation of a cdc2 mutant in fission yeast (24Lee M.G Nurse P Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2.Nature. 1987; 327: 31-35Crossref PubMed Scopus (737) Google Scholar). Interestingly, a cell cycle periodic CDK-like activity had been proposed as a mitosis regulator in the slime mold Physarum a decade previously, but experimental limitations of the slime mold unfortunately prevented this initial work from being developed further (3Bradbury E.M Inglis R.J Matthews H.R Control of cell division by very lysine rich histone (f1) phosphorylation.Nature. 1974; 247: 257-261Crossref PubMed Scopus (278) Google Scholar). It has been proposed that CDKs act as a “cell cycle engine” (37Murray A Hunt T The Cell Cycle. W. H. Freeman & Co, New York1993Google Scholar), driving cells through the cell cycle. Different CDKs control the onset of S phase and M phase (47van den Heuvel S Harlow E Distinct roles for cyclin-dependent kinases in cell cycle control.Science. 1993; 262: 2050-2054Crossref PubMed Scopus (958) Google Scholar), and increasing the activity of these CDKs can advance both events. CDKs are regulated by the availability of the cyclin subunit, by changes in phosphorylation of a catalytic site tyrosine residue controlled by Cdc25p and Wee1p, and by association with CKI inhibitors (42Nurse P Universal control mechanism regulating onset of M-phase.Nature. 1990; 344: 503-508Crossref PubMed Scopus (2182) Google Scholar, 34Morgan D.O Principles of CDK regulation.Nature. 1995; 374: 131-134Crossref PubMed Scopus (2859) Google Scholar). In metazoan cells CDKs act in early G1 to activate E2F-dependent transcription of genes required for S phase, in late G1 to initiate S phase, and finally in G2 to initiate mitosis. In the yeasts the range of CDKs is more limited and in certain circumstances the same CDK appears to initiate S phase at a low activity and mitosis at a high activity (9Fisher D.L Nurse P A single fission yeast mitotic cyclin B-p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins.EMBO J. 1996; 15: 850-860Crossref PubMed Scopus (226) Google Scholar). The fact that a gradual increase in a single CDK activity can drive cells through the whole sequence of cell cycle events might indicate that a similar regulatory situation operated in the primeval eukaryotic cell. The higher levels of CDK activity present during G2 have also been shown to block initiation of a further S phase (16Hayles J Fisher D Woollard A Nurse P Temporal order of S-phase and mitosis in fission yeast is determined by the state of the p34cdc2/mitotic B cyclin complex.Cell. 1994; 78: 813-822Abstract Full Text PDF PubMed Scopus (323) Google Scholar), probably by regulating activity of the Cdc6p/Cdc18p ini