Control of the Cell Cycle

Restriction point control of the mammalian cell cycle via the cyclin E/Cdk2:p27 complex Riaan Conradie 1 , Frank J. Bruggeman 2 , Andrea Ciliberto 3 , Attila Csika ´ sz-Nagy 4 , Bela Nova ´ k 5 , Hans V. Westerhoff 2,6 and Jacky L. Snoep 1,2,6 1 Triple J Group for Molecular Cell Physiology, Department of Biochemistry, Stellenbosch University, Matieland, South Africa 2 Molecular Cell Physiology & Netherlands Institute for Systems Biology, Vrije Universiteit, Amsterdam, The Netherlands 3 FIRC Institute of Molecular Oncology Foundation, Milan, Italy 4 University of Trento Centre for Computational and Systems Biology, Povo (Trento), Italy 5 Oxford Centre for Integrative Systems Biology, University of Oxford, UK 6 Manchester Centre for Integrative Systems Biology, Manchester Interdisciplinary Biocentre, The University of Manchester, UK Keywords cell cycle; kinetic modeling; metabolic control analysis; restriction point; systems biology Correspondence J. L. Snoep, Triple J Group for Molecular Cell Physiology, Department of Biochemistry, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa Fax: +272 1808 5863 Tel: +272 1808 5844 E-mail: jls@sun.ac.za Note The mathematical model described here has been submitted to the JWS Online Cellular Systems Modelling Database and can be accessed at http://jjj.biochem.sun.ac.za/ database/conradie/index.html free of charge (Received 14 May 2009, revised 21 October 2009, accepted 30 October 2009) doi:10.1111/j.1742-4658.2009.07473.x Numerous top-down kinetic models have been constructed to describe the cell cycle. These models have typically been constructed, validated and ana- lyzed using model species (molecular intermediates and proteins) and phe- notypic observations, and therefore do not focus on the individual model processes (reaction steps). We have developed a method to: (a) quantify the importance of each of the reaction steps in a kinetic model for the position- ing of a switch point [i.e. the restriction point (RP)]; (b) relate this control of reaction steps to their effects on molecular species, using sensitivity and co-control analysis; and thereby (c) go beyond a correlation towards a cau- sal relationship between molecular species and effects. The method is gen- eric and can be applied to responses of any type, but is most useful for the analysis of dynamic and emergent responses such as switch points in the cell cycle. The strength of the analysis is illustrated for an existing mamma- lian cell cycle model focusing on the RP [Novak B, Tyson J (2004) J Theor Biol 230, 563–579]. The reactions in the model with the highest RP control were those involved in: (a) the interplay between retinoblastoma protein and E2F transcription factor; (b) those synthesizing the delayed response genes and cyclin D/Cdk4 in response to growth signals; (c) the E2F-depen- dent cyclin E/Cdk2 synthesis reaction; as well as (d) p27 formation reac- tions. Nine of the 23 intermediates were shown to have a good correlation between their concentration control and RP control. Sensitivity and co-control analysis indicated that the strongest control of the RP is medi- ated via the cyclin E/Cdk2:p27 complex concentration. Any perturbation of the RP could be related to a change in the concentration of this complex; apparent effects of other molecular species were indirect and always worked through cyclin E/Cdk2:p27, indicating a causal relationship between this complex and the positioning of the RP. Abbreviations DRG Control of the Cell Cycle Control of the Cell Cycle Bởi: OpenStaxCollege The length of the cell cycle is highly variable, even within the cells of a single organism In humans, the frequency of cell turnover ranges from a few hours in early embryonic development, to an average of two to five days for epithelial cells, and to an entire human lifetime spent in G0 by specialized cells, such as cortical neurons or cardiac muscle cells There is also variation in the time that a cell spends in each phase of the cell cycle When fast-dividing mammalian cells are grown in culture (outside the body under optimal growing conditions), the length of the cycle is about 24 hours In rapidly dividing human cells with a 24-hour cell cycle, the G1 phase lasts approximately nine hours, the S phase lasts 10 hours, the G2 phase lasts about four and one-half hours, and the M phase lasts approximately one-half hour In early embryos of fruit flies, the cell cycle is completed in about eight minutes The timing of events in the cell cycle is controlled by mechanisms that are both internal and external to the cell Regulation of the Cell Cycle by External Events Both the initiation and inhibition of cell division are triggered by events external to the cell when it is about to begin the replication process An event may be as simple as the death of a nearby cell or as sweeping as the release of growth-promoting hormones, such as human growth hormone (HGH) A lack of HGH can inhibit cell division, resulting in dwarfism, whereas too much HGH can result in gigantism Crowding of cells can also inhibit cell division Another factor that can initiate cell division is the size of the cell; as a cell grows, it becomes inefficient due to its decreasing surface-to-volume ratio The solution to this problem is to divide Whatever the source of the message, the cell receives the signal, and a series of events within the cell allows it to proceed into interphase Moving forward from this initiation point, every parameter required during each cell cycle phase must be met or the cycle cannot progress Regulation at Internal Checkpoints It is essential that the daughter cells produced be exact duplicates of the parent cell Mistakes in the duplication or distribution of the chromosomes lead to mutations that 1/10 Control of the Cell Cycle may be passed forward to every new cell produced from an abnormal cell To prevent a compromised cell from continuing to divide, there are internal control mechanisms that operate at three main cell cycle checkpoints A checkpoint is one of several points in the eukaryotic cell cycle at which the progression of a cell to the next stage in the cycle can be halted until conditions are favorable These checkpoints occur near the end of G1, at the G2/M transition, and during metaphase ([link]) The cell cycle is controlled at three checkpoints The integrity of the DNA is assessed at the G checkpoint Proper chromosome duplication is assessed at the G2 checkpoint Attachment of each kinetochore to a spindle fiber is assessed at the M checkpoint The G1 Checkpoint The G1 checkpoint determines whether all conditions are favorable for cell division to proceed The G1 checkpoint, also called the restriction point (in yeast), is a point at which the cell irreversibly commits to the cell division process External influences, such as growth factors, play a large role in carrying the cell past the G1 checkpoint In addition to adequate reserves and cell size, there is a check for genomic DNA damage at the G1 checkpoint A cell that does not meet all the requirements will not be allowed to progress into the S phase The cell can halt the cycle and attempt to remedy the problematic condition, or the cell can advance into G0 and await further signals when conditions improve 2/10 Control of the Cell Cycle The G2 Checkpoint The G2 checkpoint bars entry into the mitotic phase if certain conditions are not met As at the G1 checkpoint, cell size and protein reserves are assessed However, the most important role of the G2 checkpoint is to ensure that all of the chromosomes have been replicated and that the replicated DNA is not damaged If the checkpoint mechanisms detect problems with the DNA, the cell cycle is halted, and the cell attempts to either complete DNA replication or repair the damaged DNA The M Checkpoint The M checkpoint occurs near the end of the metaphase stage of karyokinesis The M checkpoint is also known as the spindle checkpoint, because it determines whether all the sister chromatids are correctly attached to the spindle microtubules Because the separation of the sister chromatids during anaphase is an irreversible step, the cycle will not proceed until the kinetochores of each pair of sister chromatids are firmly anchored to at least two spindle fibers arising from opposite poles of the cell Link to Learning Watch what occurs at the G1, G2, and M checkpoints by visiting this website to see an animation of the cell cycle Regulator ...Site-specific phosphorylation of MCM4 during the cell cycle in mammalian cells Yuki Komamura-Kohno 1 , Kumiko Karasawa-Shimizu 1 , Takako Saitoh 1 , Michio Sato 1 , Fumio Hanaoka 2 , Shoji Tanaka 1 and Yukio Ishimi 1,3 1 Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan 2 Graduate School of Frontier Biosciences, Osaka University, Japan 3 Faculty of Science, Ibaraki University, Mito, Japan MCM2–7 proteins are essential for eukaryotic DNA replication and are the most likely candidates for the replicative DNA helicase responsible for unwinding DNA at the replication forks [1–3]. Consistent with their primary amino acid sequences, a subcomplex of MCM4 ⁄ 6 ⁄ 7 functions as DNA helicase in vitro [4]. It has been suggested that MCM2, -3 and -5 play a regu- latory role in the function of MCM4 ⁄ 6 ⁄ 7 DNA heli- case, because addition of MCM2 or MCM3 ⁄ 5to MCM4 ⁄ 6 ⁄ 7 complex resulted in inhibition of the MCM4 ⁄ 6 ⁄ 7 DNA helicase [5,6]. Thus MCM2–7 com- plex, a major MCM complex on chromatin during the G 1 phase, has to be activated to show DNA helicase activity. It is possible that several proteins, including CDC7 kinase and CDC45 are involved in this activa- tion. Evidence suggests that MCM2–7 proteins may have additional functions during the cell cycle [3]. Cyclin-dependent kinases (CDK), which play a critical Keywords CDK; cell cycle; DNA replication; MCM proteins; phosphorylation Correspondence Y. Ishimi, Faculty of Science, Ibaraki University, 2-1-1 Bunkyo, Mito 310-8512, Ibaraki, Japan Fax: +81 29 228 8439 Tel: +81 29 228 8439 E-mail: ishimi@mx.ibaraki.ac.jp (Received 5 September 2005, revised 6 January 2006, accepted 18 January 2006) doi:10.1111/j.1742-4658.2006.05146.x MCM4, a subunit of a putative replicative helicase, is phosphorylated dur- ing the cell cycle, at least in part by cyclin-dependent kinases (CDK), which play a central role in the regulation of DNA replication. However, detailed characterization of the phosphorylation of MCM4 remains to be per- formed. We examined the phosphorylation of human MCM4 at Ser3, Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110 using anti-phosphoMCM4 sera. Western blot analysis of HeLa cells indicated that phosphorylation of MCM4 at these seven sites can be classified into two groups: (a) phos- phorylation that is greatly enhanced in the G 2 and M phases (Thr7, Thr19, Ser32, Ser54, Ser88 and Thr110), and (b) phosphorylation that is firmly detected during interphase (Ser3). We present data indicating that phos- phorylation at Thr7, Thr19, Ser32, Ser88 and Thr110 in the M phase requires CDK1, using a temperature-sensitive mutant of mouse CDK1, and phosphorylation at sites 3 and 32 during interphase requires CDK2, using a dominant-negative mutant of human CDK2. Based on these results and those from in vitro phosphorylation of MCM4 with CDK2 ⁄ cyclin A, we discuss the kinases responsible for MCM4 phosphorylation. Phosphor- ylated MCM4 detected using anti-phospho sera exhibited different affinities for chromatin. Studies on the nuclear localization of chromatin-bound MCM4 phosphorylated at sites 3 and 32 suggested that they are not gener- ally colocalized with replicating DNA. Unexpectedly, MCM4 phosphoryl- ated at site 32 was enriched in the nucleolus through the cell cycle. These results suggest that phosphorylation of MCM4 has several distinct and site-specific roles in the function of MCM during the mammalian cell Injection of poly(b- L -malate) into the plasmodium of Physarum polycephalum shortens the cell cycle and increases the growth rate Michael Karl 1 , Roger Anderson 2 and Eggehard Holler 1 1 Institut fu ¨ r Biophysik und Physikalische Biochemie der Universita ¨ t Regensburg, Germany; 2 Molecular Biology and Biotechnology, University of Sheffield, UK Poly(b- L -malate) (PMLA) has been reported as an uncon- ventional, physiologically important biopolymer in plasmo- dia of myxomycetes, and has been proposed to function in the s torage and transport of nuclear proteins by mimicking the phospho(deoxy)ribose backbone of nucleic acids. It is distributed in the cytoplasm a nd especially in the nuclei of these giant, multinucleate cells. We report here f or the first time an increase in growth rate and a shortening of the cell cycle after the injection of purified PMLA. By comparing two strains of Physarum polycephalum that differed in their production levels of PMLA, it was found that growth activation and cell cycle shortening correlated with the relative increases of PMLA levels in the cytoplasm or the nuclei. Growth rates of a low PMLA producer strain (LU897 · LU898) were increased by 40–50% while those of a high producer strain (M 3 CVIII) were increased by only 0–17% in comparison with controls. In both strains, shortening of the cell cycle occurred to a similar extent (7.2– 9.5%), and t his w as associat ed with similar increases in nuclear PMLA levels. The effects showed saturation de- pendences with regard to the amount of injected PMLA. A steep rise of i ntracellular PMLA shortly a fter injection was followed by the appearance of histone H1 in the cytoplasm. The increase i n growth rate, the shortening of the cell cycle duration and the appearance of H1 in the cytoplasm suggest that PMLA competes with nucleic acids in binding to pro- teins that control translation and/or transcription. Thus, PMLA could play an important role in the coordination of molecular p athways t hat a re responsible f or the synchronous functioning of the multinucleate plasmodium. Keywords 1 : cell cycle; g rowth rate; P hysarum polycephalum; plasmodium; polymalic acid. In the absence of cytokinesis, repeated nuclear divisions give rise to giant multinucleate cells (plasmodia) in Physarum polycephalum [1], a well studied representative of the myxomycete family. One of the notable features of plasmodia is the high synchrony of events during the cell cycle. The maintenance of this synchrony over large cellular distances must require an activity that accounts for the rapid and ubiquitous distribution and coordination of protein activities in the periodical cell cycle eve nts. W e h ave previously identified the unusual polyanion poly(b- L -ma- late) (PMLA) a s a specific component of the plasmodium that fulfils the requirements for such a Ôdistributing activityÕ [2,3]. Its level in the nuclei i s kept constant by constitutive synthesis and secretion of excess polymer from the cyto- plasm to the culture medium, and the levels in the nuclei for different strains are of the same magnitude [4]. PMLA binds reversibly to histones, DNA polymerases, a nd other DNA- interacting proteins, thus favouring the formation of large complexes consisting o f a variety of Genome Biology 2009, 10:R123 Open Access 2009Castellanoet al.Volume 10, Issue 11, Article R123 Research Serum-dependent transcriptional networks identify distinct functional roles for H-Ras and N-Ras during initial stages of the cell cycle Esther Castellano *† , Carmen Guerrero * , Alejandro Núñez * , Javier De Las Rivas * and Eugenio Santos * Addresses: * Centro de Investigación del Cáncer, IBMCC (CSIC-USAL), University of Salamanca, Campus Unamuno, 37007 Salamanca, Spain. † Current address: Signal Transduction Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK. Correspondence: Eugenio Santos. Email: esantos@usal.es © 2009 Castellano et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ras isoforms and the cell cycle<p>Transcriptional and functional analysis reveals that the H-Ras and N-Ras isoforms have different roles in the initial phases of the mouse cell cycle</p> Abstract Background: Using oligonucleotide microarrays, we compared transcriptional profiles corresponding to the initial cell cycle stages of mouse fibroblasts lacking the small GTPases H-Ras and/or N-Ras with those of matching, wild-type controls. Results: Serum-starved wild-type and knockout ras fibroblasts had very similar transcriptional profiles, indicating that H-Ras and N-Ras do not significantly control transcriptional responses to serum deprivation stress. In contrast, genomic disruption of H-ras or N-ras, individually or in combination, determined specific differential gene expression profiles in response to post- starvation stimulation with serum for 1 hour (G0/G1 transition) or 8 hours (mid-G1 progression). The absence of N-Ras caused significantly higher changes than the absence of H-Ras in the wave of transcriptional activation linked to G0/G1 transition. In contrast, the absence of H-Ras affected the profile of the transcriptional wave detected during G1 progression more strongly than did the absence of N-Ras. H-Ras was predominantly functionally associated with growth and proliferation, whereas N-Ras had a closer link to the regulation of development, the cell cycle, immunomodulation and apoptosis. Mechanistic analysis indicated that extracellular signal-regulated kinase (ERK)-dependent activation of signal transducer and activator of transcription 1 (Stat1) mediates the regulatory effect of N-Ras on defense and immunity, whereas the pro-apoptotic effects of N-Ras are mediated through ERK and p38 mitogen-activated protein kinase signaling. Conclusions: Our observations confirm the notion of an absolute requirement for different peaks of Ras activity during the initial stages of the cell cycle and document the functional specificity of H- Ras and N-Ras during those processes. Published: 6 November 2009 Genome Biology 2009, 10:R123 (doi:10.1186/gb-2009-10-11-r123) Received: 2 July 2009 Accepted: 6 November 2009 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2009/10/11/R123 http://genomebiology.com/2009/10/11/R123 Genome Biology 2009, Volume 10, Issue 11, Article R123 Castellano et al. R123.2 Genome Biology 2009, 10:R123 Background The mammalian H-Ras, N-Ras and K-Ras proteins are highly related small GTPases functioning as critical components of cellular signaling pathways controlling proliferation, differ- entiation or survival. They act as molecular switches cycling between inactive (GDP-bound) and active (GTP-bound) states in a process modulated under physiological conditions by a variety of specific regulatory proteins, including GAPs (GTPase activating proteins) and GEFs (guanine nucleotide exchange factors) [1-3]. Hyperactivating point mutations BioMed Central Page 1 of 5 (page number not for citation purposes) Theoretical Biology and Medical Modelling Open Access Research Single cell studies of the cell cycle and some models JM Mitchison* Address: Institute for Cell, Animal and Population Biology, University of Edinburgh, Edinburgh EH9 3JT, UK Email: JM Mitchison* - j.m.mitchison@ed.ac.uk * Corresponding author Abstract Analysis of growth and division often involves measurements made on cell populations, which tend to average data. The value of single cell analysis needs to be appreciated, and models based on findings from single cells should be taken into greater consideration in our understanding of the way in which cell size and division are co-ordinated. Examples are given of some single cell analyses in mammalian cells, yeast and other microorganisms. There is also a short discussion on how far the results are in accord with simple models. Introduction What is the point of single cell studies of the cell cycle? The simple answer is that they provide extra information that is not available from studies of cell populations. Without them a cell biologist can be misled. It is easiest for me to start with the theme of the extensive results on single cells of the fission yeast Schizosaccharomy- ces pombe with which I have worked since the mid-1950s. It was then a fairly obscure organism for physiological studies though it had a good genetic background found by U. Leupold in Bern [1]. Since then it has flourished and quite large international meetings are now devoted entirely to it. For those unfamiliar with it, it is like a scaled-up bacterial rod with division at a medial septum, unlike budding yeasts. One the early results on its growth came from a single cell study by Bayne-Jones and Adolph [2]. Here I need to make a small digression about references. They will be given in this article but there are much longer accounts of nearly all the topics in my recent 100-page review [3]. When I took up fission yeast in the mid-fifties, I used a new micro- scopic technique, which gave by optical interferometry the total dry mass of single growing cells as well as their volume [4]. Volume increased, approximately in an expo- nential curve, through the first three quarters of the cycle but then stayed constant for the last quarter between mitosis and division. But total dry mass increased approx- imately linearly through the whole cycle. This was the first demonstration of linear growth, and I was surprised. Early synchrony techniques by induction This period of the fifties was when attention in this field was largely focused on the successful synchronisation of Tetrahymena and Chlorella by periodic changes in their environment. Good synchronous cultures would mean that powerful biochemical techniques, often enzyme activity assays at that time, could be applied in a cell cycle context. In the next 15 years, induction synchrony was somewhat improved but the cell cycles were always and inevitably distorted. Methods were also developed to select out a fraction of an asynchronous culture in one stage of the cycle and grow it up separately (for example," membrane elution", where cells growing on a membrane come away at division). They produce less distortion but a much lower yield than induction. Published: 09 February 2005 Theoretical Biology and Medical Modelling 2005, 2:4 doi:10.1186/1742-4682-2-4 Received: 14 January 2005 Accepted: 09 February 2005 This article is available from: http://www.tbiomed.com/content/2/1/4 © 2005 Mitchison; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Theoretical Biology and Medical Modelling 2005, 2:4 http://www.tbiomed.com/content/2/1/4 Page 2 of 5 (page number not for citation ... kinase (Cdk) D Many of the negative regulator proteins of the cell cycle were discovered in what type of cells? gametes cells in G0 cancer cells 8/10 Control of the Cell Cycle stem cells C Which negative... Although the cyclins are the main regulatory molecules that determine the forward momentum of the cell cycle, there are several other mechanisms that fine-tune the 5/10 Control of the Cell Cycle. .. “turned off.” 6/10 Control of the Cell Cycle Art Connection Rb halts the cell cycle and releases its hold in response to cell growth Rb and other proteins that negatively regulate the cell cycle