Yoshio Masui and cell cycle control, past, present and future

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Biology of the Cell (1998) 90, 447452 o Elsevier, Paris

Yoshio Masui and cell cycle control, past, present and future Paul Nurse Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK

Yoshio Masui is a gentle, modest traditional biologist whose contributions have had a major influence on the recent advances that have taken place in understanding how the cell cycle is controlled. His work forms part of a long tradition of experimental zoologists beginning at the end of the last century, who have studied the process of cell division and its control. In this essay I want to place Yoshio Masui’s contributions in perspective, as the successor to these early experimentalists, and as the forerunner of the molecular biologists now working on cell cycle control. For a fuller treatment of much of the same historical material, I recommend Yoshio Masui’s own scholarly review (Masui (1992) Biochem Cell Bid 70, 920-945). After this I will briefly summarise the central role of cyclin dependent kinases (CDK’s) in controlling the cell cycle, following Yoshio Masui’s own example by emphasising their biological significance. Finally I will sketch out some of the unsolved problems of the future in this area of research as I see them. (0 Elsevier, Paris) Yoshio Masui / cell cycle / cyclin dependent kinases

SOME HISTORY The importance of the cell as the basic unit of life was first fully recognised by the mid-nineteenth century, following the identification of cells as common structural elements of both plants and animals (see review in Wilson, 1925). Initially there was some confusion as to how cells were formed, one favoured idea being that they arose within pre-existing cells by some process analogous to crystallisation. Virchow corrected this error with his recognition of the cell division process, and his proposal that all cells arise by pre-existing cells dividing into two (Wilson, 1925). His celebrated remark ‘omnis cellula ex cellula’ or, ‘all cells come from cells’, elegantly summarised his ideas. This placed cell division in a central position for the understanding of the growth and reproduction of all living organisms. Virchow was also a progenitor of pathology, and argued that disease had its origins in cellular malfunction, an insight which eventually would lead to the recognition of deranged cell division control as the basis of cancer. Rapidly improving microscopic techniques during the second half of the nineteenth century resulted in a more detailed and accurate descripYoshio Masui and cell cycle control

tion of the cell division process. The significance of centrosomes and chromosomes was recognised by Boveri and Flemming, who described the condensation and decondensation of chromosomes and the movement of the chromosomes by a spindle organised by the centrosomes (Flemming, 1879; Boveri, 1888). The name mitosis was given to these processes and events now also known as M-phase. Dividing cells are particularly evident in developing early embryos, and a combination of careful observation and inventive manipulation by experimental embryologists began to give insight into how cell division was controlled. Such studies led Hertwig to emphasise the importance of nuclear cytoplasmic relations for the control of division (Hertwig, 1903). The nucleus was identified as the seat of many of the changes that occur during mitosis and so it was natural to consider how the surrounding cytoplasm might control this nuclear behaviour. Study of embryos of differing ploidy led to the idea of the ‘karyoplasmic ratio’, meaning that there is a constancy in the proportion between nuclear and cytoplasmic masses. He proposed that increase in cytoplasmic mass unbalances the relationship between the nucleus and cytoplasm leading to a karyoplasmic tension which brings about Nurse

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cell division to restore the proper nuclear and cytoplasmic relationship. This idea is an important principle for understanding the control of cell division; it proposes that cell division can be regulated, at least on some occasions, by increase in cytoplasmic mass. Further support for this idea came from some dramatic experiments first performed by Hartmann and then repeated by Prescott (Hartmann, 1928; Prescott, 1955, 1956). Both these zoologists repeatedly microdissected growing amoebae, removing part of the cytoplasm leaving a smaller cytoplasmic mass which still contained the nucleus. Division was always delayed in these cells because the amoebae were never allowed to attain the cytoplasmic mass required for division to take place. Total growth of the cytoplasm was insufficient to induce nuclear division; what was required was growth to a critical mass. This suggested to some workers that a factor was accumulated proportional to cytoplasmic mass which acted to induce nuclear division. Work with the ciliate Tefrahymena by Zeuthen led to the proposal that this factor was a division protein, the accumulation of which could be influenced by stress conditions such as high temperature or pressure (Zeuthen, 1964). A transition point was identified after which ceils could divide after treatment with heat or pressure, whilst prior to this point cell division was delayed by the treatment. The cells which were delayed were set back to the same time in the cell cycle, and so became synchronised by the treatment. These cells took a fixed time interval to recover and go on to division as if they all had to repeat a particular process which required this fixed time to be completed. Similar delay kinetics were obtained with pulses of the protein synthesis inhibitor cyclohexamide which led Zeuthen to suggest that during this fixed time interval cells accu mulate proteins required to build unstable structures necessary for cell division to take place. The structures were sensitive to heat, pressure or to inhibition of protein synthesis, and so these treatments would destroy the structures setting the cell back in the cell cycle. Once past the transition point the structures became stabilised and the cell became resistant to these treatments. So by the 1960s it was generally thought that cell division was controlled by a division protein or set of division proteins which built up to a level necessary to generate a structure required for the onset of cell division. These proteins were likely to be accumulated at a rate proportional to cytoplasmic mass. The constancy of the karyoplasmic ratio sug-, gested that there was a fixed relation between DNA content within the nucleus and cytoplasmic mass, and so the division proteins.could well act on nuclear components. The division proteins would Yoshio fvlasuiand cell cycle control

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bring about changes on nuclear components which then promoted mitosis and cell division. The events of the cell cycle had been usefully categorised by Mitch&on as those which formed the DNA division cycle, mainly S-phase and mitosis, and those which formed the growth cycle which comprised most of the activities in the cytoplasm (Mitchison, 1971). Division proteins controlled by growth of the cytoplasm would drive S-phase and mitosis in the nucleus. However, the nature of these division proteins and the structures they formed let along how they influenced S-phase and mitosis remained corn-pletely unknown. So the important questions were, what were the division proteins and how could they be investigated? 8 The stage was now set for a series of important contributions from Yoshio Masui dealing with these questions. Initially, he recognised that amphibian oocytes and eggs.provided a powerful experimental system for investigating cell cycle control. Her&&g had realised that oocytes were unusual cells in that cell division was blocked but cell growth continued, producing giant cells that were easily manipulated experimentally (Hertwig, 1903). Several investigators including Van Beneden realised that some oocytes were arrested just prior to meiotic M-phase (Van Beneden, 1883). Further work by Heilbrunn showed that Rana oocytes could be matured by the addition of pituitary extracts and that the maturing oocytes underwent entry into M-phase (Heilbrunn rt al, 1939). Thus factors inducing M-phase were either activated or released from inhibition during oocyte maturation and so their study provided a means to investigate the division proteins controlling nuclear and cellular division. Masui and his collaborators made many contributions to this experimental system but I want to focus on three important advances crucial to the developing field of cell cycle control. The first was the optimisation of the maturing oocyte as an experimental system. He showed that Rana oocytes-prepared free of surrounding follicular cells underwent maturation by the addition of a steroid, probably progesterone (Masui, 1967). He concluded that during the maturation process, a progesterone like steroid was secreted by follicle cells surrounding the oocyte in response to the hormone gonadatrophin. Oocytes removed from the follicular cells could be induced to enter meiotic M-phase by the addition of progesterone. This provided a convenient experimental system to investigate factors inducing M-phase. The second advance was Masui and Markert’s demonstration that cytoplasm prepared from proNurse

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gesterone-treated Rana oocytes could induce maturation when injected into untreated oocytes (Masui and Markert, 1971). Because the oocytes underwent M-phase during maturation, this meant that the maturation promoting factors or MPF as they were to be called, could induce onset of M-phase. Cytoplasm containing MPF induced maturation in a dose dependent manner, and even cytoplasm completely lacking a nucleus could induce M-phase. This seminal paper identified MPF, albeit at this stage just a cytoplasmic extract, as the factor which controlled the onset of M-phase. Subsequent work by others (Schorderet-Slatkine and Drury, 1973; Reynhout and Smith, 1974) confirmed the existence of MPF in Xerzopus oocytes, the experimental system more widely used today. Thus MPF was identified as an excellent candidate for the division proteins bringing about nuclear and cellular division. The third advance was Lohka and Masui’s development of an in vitro system for studying MPF derived from amphibian eggs (Lohka and Masui, 1983). When sperm nuclei were added to a low speed supernatant derived from Rana activated eggs, they first formed a pro-nucleus and then proceeded through the cell cycle undergoing mitosis. Formation of a pro-nucleus and subsequent cell cycle progression did not occur in a high speed supernatant which lacked membrane vesicles. This procedure was extended to Xenopus egg extracts by Lohka, Hayes and Maller, and provided a good assay system for biochemical purification of MPF. Within several years MPF had been purified 3000fold and found to consist of two proteins with molecular masses of 32 kDa and 45 kDa (Lohka et al, 1988). These were the catalytic and cyclin subunits of the cyclin dependent protein kinase p34cdc* and cyclin B. MPF purified from starfish (Labbe et al, 1989) and clam (Draetta et al, 1989) oocytes were also found to contain the same p34cdQ and cyclin B sub-units. Thus the division proteins inducing Mphase were identified as the CDK p34cdc* /cyclin B.

A GENETIC DIVERSION Parallel work based on yeast genetics had also led to the same conclusion. Cell cycle mutants had been isolated in bacteria during the 1960s and these were used to study DNA replication. However, the first really comprehensive collection of cell cycle mutants were isolated by Hartwell in the budding yeast (Hartwell et al, 1970). Amongst these was the CDC28 gene which was implicated in the control of S-phase. A similar genetic approach in fission yeast identified the cdc2 gene and regulators of cdc2 as being important for controlling the onset of mitosis. The cdc2 gene was also required for S-phase onset and its cloning revealed that it was a homologue of Yoshio Masui and cell cycle control

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CJIC28 (see review, Nurse, 1990). The cdc2 gene encoded the protein kinase p34cdc2 which was shown to be regulated by tyrosine phosphorylation and cyclin association. Complementation using a human cDNA library identified the human CDC2 homologue (Lee and Nurse, 1987), and antibodies raised against fission yeast p34cdc* reacted with the Xerzopus (Gautier et al, 1988) and starfish (Labbe et al, 1989) 32 kDa components of MPF, thus linking MPF to the genetic studies in yeast.

CELL CYCLE CONTROL MECHANISMS The identification of MPF as a CDK promoting onset of M-phase derived directly from Yoshio Masui’s biological investigations of oocytes and eggs. Subsequent work has shown that CDKs are the core cell cycle regulators driving cells into Sphase and mitosis in a controlled orderly manner. I will now briefly summarise these mechanisms using the fission yeast with which I am most familiar as a simple model system to illustrate how the controls work. Onset of both S-phase and mitosis in fission yeast is brought about by p34cdc* being complexed with Gl/S cyclins at S-phase, and by p34cdc2 being complexed with G2/M cyclins at mitosis. However, the G2/M cyclin can substitute for the Gl/S cyclins, and so yeast strains can be built in which both S-phase and mitosis are apparently driven by a single p34cdc*/B cyclin complex (Fisher and Nurse, 1996). The total p34cdc2 catalytic activity assayed using Hl histone as a substrate increases as cells proceed through the cell cycle. At relatively low levels of protein kinase activity S-phase is initiated and at high levels mitosis is initiated. The changes in protein kinase activity are regulated by a variety of mechanisms. During early Gl when activity should be low, a CDK inhibitor ~25-1 is present which inhibits p34cdc2/B-cyclin (CorreaBordes and Nurse, 1995) and promotes mitotic Bcyclin turnover (Correa-Bordes et al, 1997). Later in the cell cycle, Cl/S cyclins are made which are required for the CDK activity in late Gl which brings about S-phase onset. After S-phase, G2/M cyclin accumulates in preparation for mitosis. However, during G2 CDK activity is kept at about one third of the maximum reached in mitosis by phosphorylation of a tyrosine residue in the catalytic site of p34cda (Moreno et al, 1989). At the onset of mitosis this tyrosine becomes dephosphorylated leading to maximal p34cdc* activation bringing about mitosis. To exit mitosis B-cyclin degradation occurs resulting in a dramatic fall in CDK activity. As a consequence cells enter the Gl of the next cell cycle with low p34cdc* activity ready for the next cycle of increasing p34cdc2 activity. Nurse

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This monotonic rise and fall of p34cdc2 CDK activity with S-phase being initiated at low activity and mitosis at high activity, automatically explains tvhy S-phase usually proceeds mitosis during the cell cycle. A second role for p34ek2- also explains why there is only one S-phase per normal cell cycle. During G2 the low CDK activity associated with tyrosine phosphorylated p34cdc2/G2/h4 B-cyclin blocks initiation of another round of DNA replication. If this activity is destroyed then cells can undergo multiple rounds of DNA replication (Hayles et aE, 1994). Thus the ~34~d~~/G2/M Bcyclin present in G2 prepares cells for M-phase and simultaneously inhibits onset of another S-phase. Only on mitotic exit when this CDK activity falls to a low level is this inhibition relieved allowing the Sphase of the next cell cycle to take place. This explains why there is only one S-phase per cell cycle. It may also explain why there is no intervening S-phase between the two M-phases which occur during meiosis. The p34~dcz/G2/M B-cyclin CDK activity does not fall to a low level between meiosis 1 and meiosis 11, and this level may be sufficient to prevent S-phase from taking place. Similarly, in endoreduplicating cells found in certain Drosophila tissues, p34 cdc*[G2/IM B-cycfin CDK activity is very low and this may allow multiple rounds of DNA replication. Therefore, changes in CDK activity may generate the basic variants seen in the cell cycle which can lead to M-phase without S-phase as in meiosis, and to S-phase without Mphase as in endoreduplicating cells. There is an apparent paradox in the above model because p34cdc2 activity both initiates S-phase onset and blocks another initiation of S-phase. This can be understood by a two step model for the initiation of DNA replication (Stern and Nurse, 1994). The idea is that step one requires the absence of p34dc2 activity whilst step 2 requires the presence of p34cdc* activity. On entering Gl, very low p34cdc2 CDK activity allows step one to be completed. Subsequent increase in activity as Gl proceeds allows step two to occur leading to S-phase. At the same time step one is prevented from taking place again. In budding yeast it has been proposed that this CDK activity might regulate the formation and activation of replicative complexes on the DNA. At step one very low CDK activity allows pre-replicative complexes to form in early Gl which bind to origin regions. At step two these complexes are activated by increasing CDK activity which initiates DNA replication, but no further pre-replicative complexes can form because of the CDK activity. This ensures that new complexes will not form on DNA molecules that have already been replicated, ensuring that any particular region of the DNA is only replicated once in any particular S-phase. Such Yoshio Masui and cell cycle control

a two step model explains the paradox and provides a very satisfactory solution to the problem of ensuring that all regions of the chromosome are only replicated once in each S-phase. This is a problem for all eukaryotic cells because they have multiple origins. A final role for the CDK p34cdcz in cell cycle control is to inhibit mitosis when S-phase is not completed. Blocks over S-phase usually prevent the subsequent mitosis until that block is relieved. In fission yeast this appears to operate directly through the p34cdc*/G2/M B-cyclin. Mutants which cannot maintain the inhibitory tyrosine phosphorylation proceed to mitosis even though S-phase is incomplete, suggesting that the failure to complete S-phase maintains the regulatory tyrosine af p34cW in a phosphorylated state (Enoch and Nurse, 1991; Enoch et al, 1991). The above summary shows that CDKs play a core role in bringing about an orderly progression through the cell cycle. They are necessary to bring about S-phase and M-phase, in maintaining only one S-phase per cell cycle, and in ensuring that Mphase is blocked until S-phase is completed. Also changes in CDK regulation can explain the cell cycle variations seen during meiosis and in endoreduplicating cells. It could be imagined that in the primitive progenitor eukaryotic cell there was a single CDK which increased in level as cells proceeded through the cell cycle. This was necessary to cope with the multiple origins required in eukaryotic cells because of their increased DNA content and also with ensuring that S-phase proceeds Mphase. These problems are not found in bacterial cells with a single origin of DNA replication, perhaps explaining why CDKs are not found in prokaryotes.

Although much has been achieved in understanding the molecular mechanisms which form the basis of cell cycle control, there remain a number of important issues which require attention. Four of the more interesting outstanding problems involving CDKs are: I) The precise quantitative in vtio regulation of CDK activity; 2) The effects of CDKs on the events of the cell cycle; 3) The control of CDKs by checkpoints; 4) The monitoring of cell mass increase and CDK regulation. The level of CDK activity is clearly important for regulating orderly progression through the cell cycle. There is a need both to undertake careful measurements of different CDKs throughout the Nurse

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cell cycle, and to precisely manipulate their level of activity in viva to determine their influence on cell cycle progression. For example, how much CDK activity is required in G2 to block a further round of S-phase? The relative contributions of CDK inhibitors, cyclin availability and phosphorylation state on CDK activity need to be evaluated. The activity which matters is that which pertains within the cell itself which may vary in different cellular locations. To address this problem requires the development of new methodologies which will allow the monitoring of CDK activity in viva preferably in different places within a cell. Fluorescent resonant electron transfer (FRET) may provide novel opportunities for such assays. The manner by which CDKs bring about S-phase and mitosis, and block S-phase during G2 is still very uncertain. What is required is an identification of the CDK substrates acting at different stages of the cell cycle and an assessment of how phosphorylation or dephosphorylation of these substrates affects the processes of S-phase and mitosis. Identifying in viva protein kinase substrates is very difficult and again new methods need to be developed. Genetic approaches stabilising CDK and substrate interactions and the use of FRET to measure close protein interactions may be useful. During mitosis we need to know better how CDKs bring about nuclear envelope break down, chromosome condensation and decondensation, spindle formation and chromosome movement. During S-phase the equivalent problems are how the DNA is opened up to become available for replication, and how DNA polymerisation is initiated. These are major problems of cell and molecular biology which the CDKs play an important role in regulating. Onset of mitosis is blocked when DNA replication is incomplete or damaged DNA remains unrepaired by the operation of checkpoint controls. It is likely that these blocks are brought about by manipulating CDK activity. Such controls may be particularly important for cancer, because mitosis and cell division can occur with damaged or partially replicated DNA when the checkpoints are defective. This will contribute to genomic instability allowing the genetic changes required for cancer to be accumulated. Therefore, it is important to understand the way in which signals are generated by damaged or partially replicated DNA and are then communicated to the CDKs. The final problem returns to the questions posed by the karyoplasmic ratio. How does a cell measure its mass, or rather its mass to DNA content ratio, and then feed this information into regulating CDK activity? This is a difficult problem because the regulatory circuits involved may be rather distant from the CDK. Several models have been develoYoshio Masui and cell cycle control

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ped mainly derived from studies on the control of DNA replication in bacteria, but there has been surprisingly little progress in recent years of cell cycle research. A central element of these models is to have a counting mechanism for molecules. Concentration of a substance in a cell is given by the numbers of molecules of that substance divided by the volume of the cell. If the volume is proportional to cell mass, then if the numbers of molecules could be counted as well as their concentration measured, cell mass could be computed from the cell volwne. Counting can be carried out by titrating the key molecule, either onto DNA sites or onto sites proportional to DNA content such as chromatin or some other nuclear structure. Such ideas provide a way to think about this problem, but they need to be investigated to see if they provide any new insight into how CDK activity is regulated. The great advances of molecular biology and genetics are providing the tools to address these problems. But in addition to using these tools, really clear thinking together with an appreciation of the relevant biology are also required. It is these qualities which are the trademark of Yoshio Masui’s work, and following his example will help us to address the outstanding problems of cell cycle control in the future.

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Hayles J, Fisher D, Wollard

A and Nurse P (1994) Temporal

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order of S-phase and mitosis in fission yeast is determined by the state of the p34cdc2-mitotic 6 cyclin complex. Cell 78, 813-822 Heilbrunn LV, Daugherty K and Wilbur KM (1939) Initiation of maturation in the frog egg. Pkysiol Zooll2,97-100 Hertwig R (1903) Ueber Korrelation von ZeM und Kerngrosse und ihre Bedeutung fti die geschlechtlich Differenzienmg und die Teilung der Zelle. BiuZ Z&r&I 33,4-62 Labbe JC, Picard A, Peaucellier G, Cavadore JC, Nurse P and Do&e M (1989) Puriiication of MPF from starfish: identification as the Hl histone kinase p34da and a possible mechanism for its periodic activation. Cell 57,253-263 Lee MG and Nurse P, (1987) Compfementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature 327‘31-35 Lohka MJ and Masui Y (1983) Formation in vitro of sperm pronuclei and mitotic chromosomes induced by ooplasmic components. Science (Wash DC) 220,7l9-721 Lohka MJ, Hayes MK and MaHer JM (1988) Purification of maturation-promoting factor, an intracellular regulator of early mitotic events. Pror Nail Acad Sci USA 85,3009-3013 Masui Y (1967) Relative roles of the pituitary, follicle cells and progesterone in the induction of oocyte maturation in Rana pipiens. ] Exp Zoo1 166,365-376 Masui Y (1992) Towards understanding the control of the division cycle in animal cells. Biochem Cell Biol70,920-945 Masui Y and Markert CL (1971) Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. I Exp zufd 177,X+145 Mitch&on JM (1971) The biology of Ihe ceil cycle. Cambridge Uni. versity Press, Cambridge

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15 April

1998

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