Views Often Encounter One of the Last Big Unsolved Problems Coveries That Revealed the Central Resistance from Others in the Field

Journal of Biology BioMed Central

Research news Growing without a size checkpoint Jonathan B Weitzman

Mammalian cells, unlike yeast, seem to maintain a constant distribution of cell sizes without a cell-size checkpoint.

Some scientific concepts are so taken Yeast cell biologist Doug Kellogg fission yeast Schizosaccharomyces pombe for granted that they are no longer (University of California Santa Cruz, that produce abnormally small cells challenged, despite a lack of experi- USA) shares Raff’s amazement. “We [2,3]. These famous wee mutants mental data to support them. really don’t know very much about this (named after the Scottish term for Researchers who bravely challenge such fundamental issue,” says Kellogg. “It’s small) gave birth to an explosion of dis- widely accepted views often encounter one of the last big unsolved problems coveries that revealed the central resistance from others in the field. In in cell biology.” machinery underlying cell-cycle progres- this issue of the Journal of Biology [1], sion. Equivalent whi mutants (named Ian Conlon and Martin Raff describe a The history from yeast after the bottle of whisky opened to cele- series of experiments that questions a Much of the current understanding of brate their discovery) were subse- basic assumption about the way that cell growth comes from experiments quently isolated from the budding mammalian cell size is maintained performed in yeast, which showed that yeast Saccharomyces cerevisiae. during proliferation, and they demol- yeast cells must attain a critical size in “I did lots of experiments in those ish widely accepted doctrine (see ‘The order to progress through the cell days, together with Murdoch Mitchison bottom line’ box for a summary of cycle and divide (see the ‘Background’ and Peter Fantes, looking at cell size. their work). box). In the 1970s Paul Nurse and But the world wasn’t interested,” recalls Most proliferating cells in culture colleagues identified mutants of the Nurse (now at Cancer Research UK, maintain a constant distribution of sizes and a constant average size, presumably by coordinating cell growth with progression through the cell division cycle. The bottom line But it is far from clear how this coordi- • The growth rate of primary rat Schwann cells is linear and indepen- nation is orchestrated at the molecular dent of cell size, unlike the situation for yeast, in which bigger cells level. Raff was perplexed that such a grow faster. fundamental issue has attracted relatively little attention over the last couple • Mammalian cell growth and the cell division cycle are regulated by of decades: the impressive advances in extracellular factors, and it takes several cell division cycles for mam- understanding the mechanisms of cell- malian cells to adapt to a new growth environment, whereas yeast cycle progression have vastly overshad- cells adapt within a single cell cycle. owed any insights gained into how cells coordinate their growth with their size. • Unlike yeast cells, mammalian cells seem to coordinate growth with “It’s quite inexplicable,” says Raff, “why cell-cycle progression without operating a cell-size checkpoint. this question has been so neglected.”

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cells without affecting cell growth, Background whereas insulin-like growth factor-1 • In yeast cells a cell-size checkpoint operates to ensure that cells, (IGF-1) promoted both cell growth which grow exponentially, divide when they have completed a critical and cell division [4]. These experi- amount of cell growth (increase in cell mass), and that they reach a ments implied that extracellular specific size before initiating cell-cycle progression and cell division. signals are important for controlling Schwann cell size at division, and • It has been widely assumed that there is a similar cell-size check- raised the intriguing possibility that point in animal cells. mammalian cells do not require a size checkpoint. “So, we decided to • In cultured primary rat Schwann cells, isolated by immunopanning do similar experiments to those done from the early post-natal sciatic nerve, extracellular ‘growth factors’ by Nurse and colleagues in yeast,” differentially influence cell-cycle progression and cell growth. Glial says Conlon. “We arrested cells at a growth factor (GGF) stimulates cell division but not cell growth, certain point within the cell cycle and whereas insulin-like growth factor-1 (IGF-1) stimulates both cell measured their size, to see whether growth and division. So, Schwann cells might be able to maintain a con- they grew exponentially.” (See the stant size without operating a checkpoint. ‘Behind the scenes’ box for more of the background to the work.) • The Coulter Counter can accurately measure mammalian cell size The mode of cell growth is critical, by monitoring changes in the current flow caused by volume displace- explains Robert Brooks (King’s College ment of the electrolyte conducting liquid. Accurate measurements are London, UK). “If growth is exponen- essential if conclusions are to be drawn about the rate of cell growth. tial, then cells must have a size control over division, since otherwise random differences in size at division would increase continuously from generation London). “Then, when things went bigger, there is presumably another to generation. This does not happen. molecular, research on cell size and cell checkpoint that prevents division from Conversely, if growth is not exponential, growth sort of stopped”. Instead, Nurse occurring before the cells have reached then such a size control is not neces- and others focussed on the cell division an adequate size, to prevent them sary,” says Brooks. “Given that cells only cycle, and the intricate ballet performed getting progressively smaller. double in size (on average) from one by cyclins and cyclin-dependent kinases Further evidence to support the idea division to the next, it is actually very that coordinate its different phases. The of a cell-size checkpoint came from difficult experimentally to distinguish remarkable conservation of the molecu- observations of cell size when yeast between linear and exponential growth lar machinery controlling the eukaryotic were switched from growing in one over such a narrow (two-fold) range,” cell cycle meant that discoveries in yeast type of medium to growing in another says Brooks. Nurse agrees and stresses were quickly extended to mammals. But [2,3]. In nutrient-rich medium the cells the need for very precise measurements. as Conlon and Raff now reveal [1], the grow faster and are bigger than cells Conlon and Raff overcame these mechanisms that coordinate cell growth growing in nutrient-poor medium. technical difficulties by measuring size with cell division do not seem to be When cells are switched from nutrient- over a longer time period than one cell similarly conserved between yeast and poor to nutrient-rich conditions they cycle. They blocked cell division using mammalian cells. rapidly adjust their size before they an inhibitor of DNA polymerase ␣ and When cell-cycle progression is divide. “These experiments showed monitored cell size using a Coulter blocked in yeast the cells continue to categorically that there is a cell-size Counter. “This machine measures grow. In fact big cells grow faster than checkpoint in yeast,” says Conlon. liquid displacement, allowing very small cells, so cell growth is exponential “Everyone assumed the same applied accurate measurement of real cell [2,3]. This finding suggests that normal in animal cells, but the question had volume,” says Conlon. He and Raff yeast cells must operate a ‘checkpoint’ not really been tested.” found that the Schwann cells grew in a in order to maintain a constant average linear fashion, adding a constant cell size: the checkpoint ensures that Schwann cells do it differently amount of volume each day, indepen- cells do not continue growing after a During work for his PhD thesis, dent of their size. The linear growth certain size (until they have divided). Conlon discovered that glial growth continued until cells reached over eight While this cell-size checkpoint operates factor (GGF) could stimulate cell-cycle times their normal size. “This is a to prevent cells from getting bigger and progression in primary rat Schwann remarkable feat for the cells – they

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But when Schwann cells were shifted Behind the scenes from serum-free medium to serum- Journal of Biology asked Martin Raff about the experiments that led him to containing medium it took them conclude that mammalian cells lack a cell-size checkpoint. around six cell divisions to attain the average size of cells grown in nutrient- What prompted you to measure the size of proliferating rich medium. These results are consis- Schwann cells? tent with the lack of a cell-size This work came out of Ian Conlon’s PhD thesis project. Ian had found that checkpoint that prevents division at an two different growth factors had distinct effects on cell-cycle progression inappropriate size. and cell growth. Glial growth factor (GGF) stimulated the cell cycle Conlon and Raff suggest that extra- without affecting growth, so that cells divided at different sizes. We went cellular signals are largely responsible to speak to Paul Nurse, who had done some of the early experiments on for regulating cell size in Schwann the size checkpoint in yeast, and that got us thinking about the possibility cells. “Animal cells hardly do anything of mammalian cell growth without cell-size checkpoints. without signals from other cells,” says Raff, implying that it is differences in How long did it take to do the experiments and what were the the lifestyles of yeast and animal cells steps that ensured success? that account for their different modes Our earlier study of extracellular mitogens took about two years, and it of regulation. Nurse emphasizes that took another year to do the experiments in the Journal of Biology article. microbes such as yeast are under a Initially it took time to get the system running and to perfect the Schwann strong selective pressure to respond to cell culture conditions. It was important to use ‘normal’ primary cells, as their environmental conditions, whereas most cell lines have screwed up regulation. But the experiments are rela- metazoan cells are not. Kellogg agrees tively straightforward technically - they could have been done years ago. that the yeast lifestyle makes yeast cells quite different, noting that “yeast What was your initial reaction to the results and how were they growth is determined from within, received by others? whereas animal cells live as a commu- We were very surprised that mammalian cells don’t have a cell-size check- nity of cells.” Raff wonders how the point. Most people believed that the checkpoint existed - I am even asso- metazoan extracellular signals are ciated with two textbooks that talk about the checkpoint. It was being regulated and how they are surprising that cells grow at a constant rate over such a wide size range. I linked to the coordination of protein find that quite remarkable. But everyone had swallowed the yeast dogma, synthesis and degradation. “They must and changing perspective is always harder. be tightly coupled,“ says Raff, citing work from James Franklin and Eugene What are the next steps? Johnson who demonstrated that neuro- I am retired and closing my lab. So, we hope our results will get other trophic factors regulate the rates of people interested enough to follow up. There are a number of obvious protein synthesis and degradation in questions. First, these experiments should be repeated in a few more cell sympathetic neurons [5]. types to confirm that this is a general phenomenon. To me the most inter- esting question is how protein synthesis and degradation are coupled, to A growth industry maintain strict size control. It is also important to figure out how the The community of researchers investi- extracellular growth signals are regulated. gating cell size is still relatively small, but it is clearly growing. “More and more people are coming back to look at this problem,” says Kellogg, who must really care about this,” says Raff. degradation, says Brooks. “It would be welcomes the renewed interest. Neither The experiments also revealed that the nice to know how this works.” Conlon nor Raff, at opposite ends of rates of both protein synthesis and The finding that growth is not expo- the career spectrum, will be pursuing protein degradation increased with cell nential (unlike in yeast) suggested that the problem further, and they hope size, such that the net accumulation of Schwann cells do not need a check- that their study will inspire others to protein is independent of size. “This point. To further challenge the check- tackle this fundamental question of suggests that big cells know they are big point notion Conlon and Raff tried the how cells acquire their required size in and slow down their net rate of growth same type of medium-switching experi- multicellular organisms. Kellogg expects accordingly,” by increasing their rate of ments as had been carried out in yeast. that there are many layers of redundant

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:mechanisms, making it difficult to phosphoinositide 3؅-kinase and ribo- 2. Nurse P, Thuriaux P, Nasmyth K tackle genetically and to distinguish somal S6 kinase, that have recently Genetic control of the cell division cycle in the fission yeast Schizosac- between primary and secondary effects. been linked to cell-size control in flies charomyces pombe. Mol Gen Genet “It’s a real struggle but we are going to and mice [7]. 1976, 146:167-178. get around it,” he says. He cites a recent After almost 25 years of relative 3. Fantes P, Nurse P: Control of cell size at division in fission yeast by a study from Mike Tyers’ laboratory neglect, it looks as though the field of growth modulated size control over (University of Toronto, Canada) that cell-size control is finally growing up. nuclear division. Exp Cell Res 1977, used a systematic approach to assess The next decade seems likely to 107:377-386. 4. Conlon IJ, Dunn GA, Mudge AW, Raff the effects on cell growth of mutation produce results as remarkable as the MC: Extracellular control of cell of each of the 6,000 genes in budding discoveries of the previous decades size. Nat Cell Biol 2001, 3:918-921. yeast [6]; this study highlighted the about the molecular mechanisms regu- 5. Franklin JL, Johnson EM. Control of neuronal size homeostasis by importance of ribosome biogenesis in lating the cell cycle. Now that the trophic factor-mediated coupling of regulating growth. misapprehension of a mammalian protein degradation to protein syn- Nurse is hopeful that such genome- cell-size checkpoint has been wiped thesis. J Cell Biol 1998, 142:1313-1324. 6. Jorgensen P, Nishikawa JL, Breitkreutz BJ, wide approaches will one day provide away, there should be a renewed inter- Tyers M: Systematic identification of the answer to what limits growth, be it ested in determining how cells grow pathways that couple cell growth ribosomes, energy metabolism, protein and maintain a constant size, and what and division in yeast. Science 2002, 297:395-400. production or gene activation. He wants the fundamental differences might be 7. Neufeld TP, Edgar BA: Connections to see more experiments with different between the regulation of growth in between growth and the cell cycle. cell types, different patterns of growth yeast and mammalian cells. Curr Opin Cell Biol 1998, 10:784-790. and different growth-perturbation conditions. Conlon adds that it will be References important to link the extracellular 1. Conlon I, Raff M: Differences in the Jonathan B Weitzman is a scientist and way a mammalian cell and yeast growth signals to intracellular signaling cells co-ordinate cell growth and science writer based in Paris, France. pathways, such as those involving cell-cycle progression. J Biol 2003, 2:7. E-mail: [email protected]

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