Environmental control of cell size at division

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Environmental control of cell size at division Elizabeth Davie and Janni Petersen Tight coupling between cell growth and cell cycle progression allows cells to adjust their size to the demands of proliferation in varying nutrient environments. Target of rapamycin (TOR) signalling pathways co-ordinate cell growth with cell cycle progression in response to altered nutritional availability. To increase cell size the active TOR Complex 1 (TORC1) promotes cell growth to delay mitosis and cell division, whereas under limited nutrients TORC1 activity is decreased to reduce cell size. It remains unclear why cell size is subject to such tight control. Recent evidence suggests that in addition to modulating cell size, changes in nutrient availability also alter nuclear:cytoplasmic (N/C) ratios and may therefore compromise optimal cellular physiology. This could explain why cells increase their size when conditions are favourable, despite being competent to survive at a smaller size if necessary. Address University of Manchester, C.4255 Michael Smith Building, Faculty of Life Sciences, Oxford Road, Manchester M13 9PT, UK Corresponding author: Petersen, Janni ([email protected]) Current Opinion in Cell Biology 2012, 24:838–844 This review comes from a themed issue on Cell division, growth and death Edited by Julia Promisel Cooper and Richard J Youle For a complete overview see the Issue and the Editorial Available online 2nd September 2012 0955-0674/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ceb.2012.08.003

Introduction Eukaryotic cell size is an important characteristic for adaptation and survival. Because of the importance of cell size in influencing cellular physiology, organ and organism size, cells have evolved a variety of mechanisms to ensure that it is appropriately controlled. Hence, there is a need for a tight co-ordination between cell growth and cell division. Given that size is an adaptive trait, it is unsurprising that it can be dictated by several environmental factors, one of the most important being nutrient availability. In the presence of rich nutrients, cells maintain high levels of macromolecular synthesis to promote growth and increase size. Conversely, limitations in nutritional environment restrain protein synthesis to conserve crucial metabolites and consequently, reduce size. This has been demonstrated in both uni-cellular and multicellular organisms. One striking example is that wild type flies in nutrient-limiting conditions develop to less than Current Opinion in Cell Biology 2012, 24:838–844

half the size of their well-fed counterparts [1]. Importantly, this reduction in body size was found to result from a reduction in cell size rather than in cell number. Thus, under conditions of poor nutrition, cells can adapt to maintain proliferation and continue to survive, albeit at a smaller size. This concept is supported by the advancement of cell division at a reduced size following moderate starvation of HEK293 cells or following nutrient downshift of fission yeast cultures [2,3]. Evidently, to increase or decrease growth rate and adjust the timing of division accordingly, cells must continuously monitor their nutritional status. The inability to do so is a recognised cancer hallmark. In all eukaryotes, the target of rapamycin (TOR) acts as a major nutrient sensor. How nutritional status is sensed to regulate TOR and cell size remains somewhat elusive, in part due to challenges presented by the complex nature of mammalian signalling networks. Many aspects of TOR signalling are conserved in simple eukaryotes such as fission yeast S. pombe (schizosaccharomyces pombe) and budding yeast S. cerevisiae (saccharomyces cerevisiae). The mode of growth of fission yeast lends itself to studying the coupling between cell growth and division size and so is the major focus of this review. The role played by TOR signalling in budding yeast and higher eukaryotes is the focus of some excellent reviews [4,5]. Rod-shaped fission yeast cells are ideal for studying cell size control because they maintain a constant diameter and grow by tip elongation (Figure 1a). Therefore, because cell growth is two-dimensional in this organism, simple measurements of cell length at division give an accurate assessment of cell size. Under steady-state conditions, tip growth terminates at a defined cell length when cells commit to mitosis and cell division. Fission yeast cells are ‘born’ in the G2 phase of the cell cycle, thus cells only grow in G2. Therefore, fission yeast cells either advance or delay mitotic commitment to control their size in response to altered nutrient environment (Figure 1a). Consequently, fission yeast provides a powerful system in which to study nutrient sensing and regulation of size. In this review we discuss the role of TOR in the control of cell size. We focus on nutritional-regulation of TOR, with the intention of integrating findings from both fission yeast and higher eukaryotes.

Nutrient availability impacts on TOR Complex 1 (TORC1) signalling to modulate cell size The role of TOR signalling in regulating nutrientmediated cell growth is well described in diverse eukaryotic species [6]. The link between nutrient availability www.sciencedirect.com

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Figure 1

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(a) Schematic of cell cycle progression under different environmental conditions in S. pombe. Under steady-state conditions cells terminate tip growth and commit to mitosis (M) at a defined size. In poor nutrient environments, TOR activity is low and cells advance into mitosis at a smaller size. In rich nutrient environments TOR is more active to prolong growth and delay mitotic onset. (b) Shifting S. pombe cells from media containing glutamate (nutrient-rich) into a range of different nutrient environments, alters cell size at division to match the quality of the new environment. A shift from glutamate to proline advances mitosis to reduce cell size.

and TOR evolved from studies in S. cerevisiae, that found phenotypic similarities between cells inactivated for tor and those deprived of nutrients [7]. This association has since been reproduced in fission yeast, flies, mice and several mammalian cell lines [8,9]. TOR is a highly conserved protein kinase that nucleates two structurally and functionally distinct complexes, TORC1 and TORC2. Unlike mammals, which contain a single TOR gene, S. cerevisiae and S. pombe contain two tor genes, tor1 and tor2. In S. pombe, the shift from rich to poor nitrogen source (nutrient stress), to advance mitosis at a reduced size (Figure 1), is dependent on Tor1 [10]. Furthermore, inhibition of TOR signalling with rapamycin mimics the effect of nutrient stress. Tor1 has also recently been implicated in the reduction of cell size in response to glucose limitation [11], indicating that Tor1 is regulated by various nutritional inputs. The majority of the literature places Tor1 in TORC2 and Tor2 in TORC1 [12–14], thereby suggesting TORC2 to www.sciencedirect.com

be responsible for size control under conditions of nutrient stress. However, increasing evidence suggests that this may not be the case. Deletion of the TORC2-specific subunits Sin1 and Ste20 (rictor in mammals) has no impact on the response to nutrient stress, whereas deletion of the TORC1-specific subunit Mip1 does compromise this response [15]. Furthermore, Mip1 and Tor1 physically interact in minimal media. More recently, we have observed a specific downregulation of TORC1 but not TORC2 in response to nutrient stress. We find phosphorylation of a TORC1-specific substrate Maf1 [16,17] to be reduced after nutrient stress, whereas phosphorylation on Gad8 Ser546, a TORC2-specific site [18], appears to remain unchanged (our unpublished findings). Given that rapamycin is thought to predominantly inhibit TORC1 across all eukaryotes, and that rapamycin treatment mimics nutrient stress-induced advanced mitotic onset, not only in S. pombe but also in mammalian cell lines [19], we would argue that TORC1 regulates the environmental modulation of cell size. Consistently, Current Opinion in Cell Biology 2012, 24:838–844

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there is evidence that both upstream and downstream components of the TORC1 pathway affect cell size. Knockout of the TORC1 activators Rheb and MAP4K3 reduces cell size in MEFs and Drosophila respectively [20,21]. Similarly, knockout of the TORC1 substrate S6K in both Drosophila and in mice results in the same small cell phenotype [1,22], whereas mutation of the TORC1 negative regulator TSC1 in Drosophila significantly increases cell size [23]. Reduced TORC1 activity in nutrient-poor S. pombe cultures modulates cell size by boosting MAP kinase (Sty1) activity. Active Sty1 promotes phosphorylation of Pololike kinase (Plo1), leading to the promotion of mitosis [10,24]. Since the mechanisms downstream of TOR signalling that regulate growth and mitotic commitment have been reviewed previously [9], we will not discuss these here. Although, it is worth pointing out that TORdependent regulation of MAPK signalling is likely to be conserved. The mTOR pathway has been found to regulate mammalian ERK activity, which is important for controlling mitotic onset in mammals [25,26]. There is also an emerging, stress-dependent crosstalk between the Sty1 homologue, p38 MAPK and mTOR pathways. Cells fail to reduce in size following glucose deprivation when p38b is deleted [27], suggesting an importance for Stress MAP kinase signalling in mammalian nutrient-mediated size control.

Nutrient sensing upstream of TORC1 The nutrient sensing ability of TORC1 remains poorly understood and the mechanisms of TORC1 modulation by amino acid, glucose and nitrogen availability are not yet clear. Reduced carbon levels that alter ATP energy levels or changes in the concentrations of amino acids are both sensed by the cells [5,28]. Fission yeast cells can also specifically sense a poor nitrogen source. As mentioned above, when the nitrogen source is changed from glutamate (good nitrogen source) to proline (poor nitrogen source) cells reduce their cell size at division [3,10]. Importantly, cell size is reduced even in the presence of twice the amount of the initial glucose concentration (Figure 1b). Furthermore, if cells are transferred into another poor nitrogen source Uracil, they also reduce cell size at division. Therefore, because these nutrient-shifted cells are prototroph, it is not a change in amino acids or carbon that the cells are sensing, but specifically the ‘quality’ of the nitrogen source. Initially, it was thought that nutrient-mediated TORC1 activation was achieved in a similar manner to growth factor stimulation, through a PI3K/TSC/Rheb signalling module. Phosphorylation of TSC2 causes inactivation of the Tsc1/2 complex, which in its active state functions as a negative regulator of Rheb and consequently, TORC1 (Figure 2a) [29–32]. Earlier studies found amino acid depletion of mammalian cells to impair Rheb–TOR Current Opinion in Cell Biology 2012, 24:838–844

binding and result in rapid dephosphorylation of the TORC1 effector S6K [33]. Rheb and its upstream regulators were quickly implicated as nutrient-dependent regulators of TOR. However, in some studies TORC1 was still found to be regulated in the absence of TSC2 [34,35]. This opened up the field to the existence of TSCindependent, and potentially Rheb-independent mechanisms of nutrient-mediated TORC1 regulation. The current outlook considers RagGTPases and the class III PI3K hVps34 (reviewed in [36]) to play key roles in amino acid sensing to TORC1 (Figure 2a). The Rag proteins are a unique family of Ras-related GTPases, homologous to Gtr proteins in yeast [37]. In yeast, Drosophila and human cells Gtr/Rag proteins have been found to act as heterodimers to modulate TORC1 activity in response to changes in amino acid levels [38,39,40,41]. It was noticed by Sancak et al., that the presence of amino acids induced TORC1 to relocate to lysosomal membranes [39,42]. This change in localisation was ablated by Rag knockdown and promoted by Rag overexpression even in the absence of amino acids. Rag heterodimers were later discovered to be anchored to the lysosome membranes via a ‘Ragulator’ complex [42]. Sabatini and colleagues therefore proposed that in response to fluctuations in amino acid levels, Rag proteins induce the relocation of TORC1 to Rheb-containing vesicles, where it can become activated by interaction with Rheb. In more recent years, additional Rag interacting proteins have been identified, including the adaptor protein p62, which binds to Rag proteins to stabilise the formation of the active Rag heterodimer and induce TORC1 activation [43]. How Rags sense nutrient status remains unclear, although findings from both S. cerevisiae and S. pombe propose the GTP exchange factor Vam6 to be required for activation of Gtr1/2 upstream of TORC1 [40,41] (Figure 2a). The model for Rag-dependent activation of TORC1 on the lysosome could explain why MEFs null for TSC2 remain sensitive to amino acid deprivation despite having constitutively active Rheb; without amino acid stimulation TORC1 may not localise to Rheb-containing cellular compartments. Duran and Hall provide a current in depth view of Gtr/Rag-TOR regulation [44]. In response to glucose, rather than amino acid, deprivation TORC1 downregulation is thought to occur through activation of the AMP-activated protein kinase (AMPK). Depletion of glucose compromises the cellular energy status, resulting in activation of AMPK, which in turn can activate Tsc1/2 or directly phosphorylate raptor to inhibit TORC1 [45,46]. Recent data from glucose limitation studies in S. pombe show that under low glucose, the calcium-dependent and calmodulin-dependent protein kinase kinase (CaMKK) Ssp1 is required to accelerate progression through G2 phase [47]. Further www.sciencedirect.com

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Figure 2

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(a) Under nutrient-rich conditions TORC1 is active to promote cell growth and increase cell size at division. Nutrient sensing upstream of TORC1 is unclear. Nutrients may be sensed by Vam6, which may activate Gtr1/2 to promote TORC1 activity. In mammals Rag proteins (Gtr homologues) are localised to the lysosome by a ragulator complex. Rheb is similarly localised, and so active Rags may localise TORC1 to its upstream activator. Gtrs in yeast are localised to the vacuole and so this mechanism could be conserved. In rich environments, the negative regulators of TORC1, Tsc1/2 and AMPK are inactive. (b) When nutrient conditions are poor TORC1 is inhibited to reduce cell size at division. Nuclear size does not appear to decrease, therefore altering the N/C ratio. Poor nutrients could inhibit TORC1 through inactivation of Gtr1/2. Alternatively, activation of kinases such as Ssp1 and Ssp2 could result in TORC1 inhibition, potentially through activation of Tsc1/2, which is a known AMPK substrate in response to glucose deprivation in mammals.

evidence suggests Ssp1 to directly phosphorylate the AMPKa homologue Ssp2 to promote the nutritional stress response [48], although it remains unclear at this stage whether AMPK signals through Tsc proteins to modulate TORC1 in these circumstances. Interestingly, Ssp1 and Ssp2 have also been implicated in the response to complete nitrogen starvation, which induces G1 arrest, cell cycle exit and sexual differentiation [48]. Given that disruption of Tsc1/2 can partially rescue the reduced TORC1 activity in nitrogen-depleted cells [49], it could be predicted that Ssp1, Ssp2 and the Tsc1/2 complex could sense nitrogen starvation.

Why is cell size under tight control? Having discussed the presence of, and potential mechanisms for, cell size control in response to changes in nutrient availability, the following question is intriguing: If cells can still proliferate, and whole organisms survive at a small size under poor nutrient conditions, then why do cells make the extra effort to increase their size in rich www.sciencedirect.com

nutrient environments? Interestingly, increased cell size leads to an altered nuclear to cytoplasmic (N/C) ratio. We find that despite being small in overall size, S. pombe cells grown in a poor nutrient environment have nuclei of similar volume to those of larger cells grown in good nutrient environmental controls, that is, cells in proline have a larger N/C ratio (our unpublished work) (Figure 3). From our preliminary studies it appears that the N/C ratio of cells in either nutrient environment remains relatively constant over time, but is significantly different between the two conditions. This expands on the work of Neumann and Nurse, who determined that N/C ratios remain constant over a range of different cell sizes [50]. Our data support the view that N/C ratios remain constant in individual cells as the cell cycle progresses. However, where Neumann and Nurse found ratios to be constant even between cells grown in a rich environment and nitrogen-starved cells, our data suggest that the N/C ratio changes when cells are subjected to moderate nitrogen stress. Importantly, when cells are nitrogen starved they Current Opinion in Cell Biology 2012, 24:838–844

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(a) S. pombe cultures, containing the cut11.GFP- nuclear envelope marker [51] were grown in media containing either glutamate or proline. (b) The nuclear to cytoplasmic (N/C) ratio was calculated for over 250 cells and plotted against cell length. Cells in either environment maintain fairly constant N/C ratios as they increase in length throughout the cell cycle. Cells grown with proline as a nitrogen source have increased N/C ratios compared with cells in the rich nitrogen source (b: compare blue and red).

arrest in G1 with a haploid complement of DNA, whereas under our nitrogen stress cells continue to proliferate. Because spatial aspects of cell physiology, such as the positioning of organelles and of cytoplasmic signalling modules, are important for cell function, we postulate that the altered N/C ratio in small proline-grown cells may Current Opinion in Cell Biology 2012, 24:838–844

prevent optimal positioning or signalling, providing reason for cells to grow bigger when conditions permit. Interestingly, Nurse and colleagues have shown that intracellular protein concentrations remain constant in media containing either glutamate or proline [3], implying that in smaller proline cells with bigger N/C ratios, the www.sciencedirect.com

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cytoplasmic content may be more ‘compact’. Perhaps this is less favourable for the appropriate function of cytoplasmic signalling modules. Further investigation is needed to ascertain why nuclear size and cell size do not follow a linear relationship across different qualities of nutrient environment, and therefore, whether nutrient stress could instigate a novel control over N/C ratio.

Conclusions It is clear that nutrient availability impacts on the control of cell size through regulation of the TORC1 signalling pathway. In recent years, there has been a substantial increase in our knowledge of the mechanisms of nutritional input to TORC1. Yet, many details of the molecular basis for such mechanisms remain elusive, as do the mechanisms linking TORC1 to mitotic regulators in mammalian systems. Because mammalian cells also get smaller when TOR is inhibited, by definition they also undergo mitosis at a reduced cell size. Our recent data suggest that, as well as altering overall cell size; changes in nutrient environment may also alter N/C ratios. It seems that key signalling components involved in controlling cell growth and division are conserved, and so by continuing to study nutrient-dependent regulation of TORC1 in yeast we hope to further expand upon our knowledge in this key area.

Acknowledgements We thank Iain Hagan for helpful comments and discussion on the manuscript. Work in the Petersen lab is supported by Cancer Research UK and by the Wellcome Trust. We apologise to authors whose work could not be discussed and cited due to space constraints.

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46. Inoki K, Zhu T, Guan KL: TSC2 mediates cellular energy response to control cell growth and survival. Cell 2003, 115:577-590. 47. Hanyu Y, Imai KK, Kawasaki Y, Nakamura T, Nakaseko Y, Nagao K, Kokubu A, Ebe M, Fujisawa A, Hayashi T et al.:  Schizosaccharomyces pombe cell division cycle under limited glucose requires Ssp1 kinase, the putative CaMKK, and Sds23, a PP2A-related phosphatase inhibitor. Genes Cells 2009, 14:539-554. This study together with Ref. [48] provides evidence for the role of CaMKK/Ssp1 in the response to nitrogen starvation and to glucose limitation. It is shown that Ssp1 is required for cells to progress through G2 under low glucose. 48. Valbuena N, Moreno S: AMPK phosphorylation by Ssp1 is  required for proper sexual differentiation in fission yeast. J Cell Sci 2012, 125:2655-2664. This study together with Ref. [47] provides evidence for the role of CaMKK/Ssp1 in the response to nitrogen starvation and to glucose limitation. It is suggested that Ssp1 phosphorylates the AMPKa homologue Ssp2 following nutrient depletion, which could modulate TOR signalling to regulate cell size at division. 49. Nakashima A, Sato T, Tamanoi F: Fission yeast TORC1 regulates phosphorylation of ribosomal S6 proteins in response to nutrients and its activity is inhibited by rapamycin. J Cell Sci 2010, 123:777-786. 50. Neumann FR, Nurse P: Nuclear size control in fission yeast.  J Cell Biol 2007, 179:593-600. An interesting paper looking at the control of nuclear size and therefore nuclear:cytoplamic ratios in fission yeast. The authors find that N/C ratios remain constant over a range of cell sizes and when cells are grown in rich media or nitrogen starved. 51. West RR, Vaisberg EV, Ding R, Nurse P, McIntosh JR: cut11(+): a gene required for cell cycle-dependent spindle pole body anchoring in the nuclear envelope and bipolar spindle formation in Schizosaccharomyces pombe. Mol Biol Cell 1998, 9:2839-2855.

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