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Down-Regulation of Tricarboxylic Acid (TCA) Cycle Genes Blocks Progression Through the First Mitotic Division in Caenorhabditis Elegans Embryos

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Down-Regulation of Tricarboxylic Acid (TCA) Cycle Genes Blocks Progression Through the First Mitotic Division in Caenorhabditis Elegans Embryos Down-regulation of tricarboxylic acid (TCA) cycle genes blocks progression through the first mitotic division in Caenorhabditis elegans embryos Mohammad M. Rahman, Simona Rosu, Daphna Joseph-Strauss, and Orna Cohen-Fix1 Laboratory of Cell and Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 Edited* by Angelika Amon, Massachusetts Institute of Technology, Cambridge, MA, and approved January 9, 2014 (received for review June 19, 2013) The cell cycle is a highly regulated process that enables the accurate We have previously conducted a visual screen in C. elegans transmission of chromosomes to daughter cells. Here we uncover a embryos for genes that when down-regulated by RNAi lead to an previously unknown link between the tricarboxylic acid (TCA) cycle abnormal nuclear morphology (9). Most genes whose inactiva- and cell cycle progression in the Caenorhabditis elegans early em- tion affected early embryonic development did so without ar- bryo. We found that down-regulation of TCA cycle components, in- resting cell cycle progression. It was therefore striking when we cluding citrate synthase, malate dehydrogenase, and aconitase, came across a set of genes, coding for enzymes of the tricarboxylic resulted in a one-cell stage arrest before entry into mitosis: pronu- acid (TCA) cycle, that when down-regulated, led to a one-cell clear meeting occurred normally, but nuclear envelope breakdown, stage arrest with paired nuclei. The TCA cycle, also known as the centrosome separation, and chromosome condensation did not take Krebs cycle, uses the oxidation of acetate (in the form of acetyl place. Mitotic entry is controlled by the cyclin B–cyclin-dependent CoA) derived from carbohydrates, proteins, or lipids, to generate kinase 1 (Cdk1) complex, and the inhibitory phosphorylation of intermediates (i.e., NADH and FADH2) that are used by the Cdk1 must be removed in order for the complex to be active. We electron transport chain for ATP production. Intermediates of found that following down-regulation of the TCA cycle, cyclin B the TCA cycle are also important for various anabolic pathways, levels were normal but CDK-1 remained inhibitory-phosphorylated such as fatty acid synthesis, and the synthesis of nucleotides. In in one-cell stage-arrested embryos, indicative of a G2-like arrest. this study, we examine the relationship between TCA cycle down- Moreover, this was not due to an indirect effect caused by check- regulation and cell cycle progression in the one-cell C. elegans point activation by DNA damage or replication defects. These obser- embryo. Our data suggest that down-regulation of the TCA cycle leads to a G2-like arrest at the one-cell stage embryo by pre- vations suggest that CDK-1 activation in the C. elegans one-cell venting the activation of cyclin B–Cdk1. embryo is sensitive to the metabolic state of the cell, and that down-regulation of the TCA cycle prevents the removal of CDK-1 Results inhibitory phosphorylation. The TCA cycle was previously shown to Down-Regulation of the C. elegans Citrate Synthase Ortholog Leads be necessary for the development of the early embryo in mammals, to a One-Cell Stage Embryonic Arrest Before Nuclear Envelope but the molecular processes affected were not known. Our study Breakdown. demonstrates a link between the TCA cycle and a specific cell cycle Recently we conducted an RNAi screen in C. elegans transition in the one-cell stage embryo. for genes that affect nuclear morphology by targeting genes that were reported to cause embryonic lethality when mutated or down-regulated by RNAi (9). In the course of these studies we he developmental program of any organism must be precisely uncovered an unusual phenotype caused by the down-regulation Texecuted. In Caenorhabditis elegans embryos, immediately after of ORF T20G5.2 that codes for CTS-1, an ortholog of the eu- fertilization, two pronuclei form at opposite poles of the embryo: karyotic citrate synthase (Fig. S1). When cts-1 was down-regulated one containing the maternal chromosomes and the other con- taining the paternal ones (1, 2). These pronuclei then move toward Significance each other, and at the same time centrosomes separate and begin to assemble a spindle. After pronuclear meeting, the cell enters its Cell division is driven by the cell cycle machinery, which first mitosis, resulting in nuclear envelope breakdown, chromatin responds in an unknown fashion to the metabolic and nutrient condensation, and the subsequent alignment of chromosomes on state of the cell. We uncovered a previously unknown link the metaphase plate, followed by chromosome segregation (2). – between the cell cycle machinery and the tricarboxylic acid Entry into mitosis depends on the mitotic cyclin B cyclin-dependent (TCA) cycle (also known as the Krebs cycle), which forms kinase 1 (Cdk1) complex. The activity of this complex is regu- intermediates required for ATP production and other anabolic lated by both cyclin B levels and regulatory phosphorylation of pathways. We show that in Caenorhabditis elegans embryos, Cdk1. In particular, Cdk1 activity is inhibited by Wee1 phos- down-regulation of the TCA cycle inhibits entry into the first phorylation, which is removed at the onset of mitosis by the mitotic division by preventing the removal of inhibitory Cdc25 phosphatase (3, 4). Cdk1 activation is also subjected to phosphorylation on cyclin-dependent kinase 1. Our data sug- various checkpoints that inhibit mitotic progression in the pres- gest that in the one-cell stage embryo, the cell cycle machinery ence of intracellular damage (5). However, in organisms that is sensitive to the metabolic state of the cell, a phenomenon undergo rapid embryonic divisions, including C. elegans, check- that may also exist in mammalian embryos. points are inoperative during the first few cell cycles (6). Although it is clear that cell cycle progression requires energy, Author contributions: M.M.R., S.R., D.J.-S., and O.C.-F. designed research; M.M.R., S.R., and the link, if any, between metabolic pathways and progression D.J.-S. performed research; M.M.R., S.R., and D.J.-S. contributed new reagents/analytic through mitosis is poorly understood. Genes and proteins in- tools; M.M.R., S.R., D.J.-S., and O.C.-F. analyzed data; and M.M.R., S.R., D.J.-S., and O.C.-F. volved in various aspects of metabolism (e.g., nucleotide bio- wrote the paper. synthesis and lipid metabolism) are regulated by the cell cycle The authors declare no conflict of interest. machinery, and cells will not commit to a new cell cycle if *This Direct Submission article had a prearranged editor. nutrients are scarce (7). However, to what extent the metabolic 1To whom correspondence should be addressed. E-mail: [email protected]. state of the cell is sensed by the cell cycle machinery once cells This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. have passed into S phase is not clear (8). 1073/pnas.1311635111/-/DCSupplemental. 2602–2607 | PNAS | February 18, 2014 | vol. 111 | no. 7 www.pnas.org/cgi/doi/10.1073/pnas.1311635111 Downloaded by guest on September 30, 2021 by RNAi, embryos accumulated at the one-cell stage with paired stage-arrested embryos were indistinguishable from the germ nuclei (Fig. 1A). line of control animals (Fig. S4A). Meiosis appeared to have During normal development, embryos at the one-cell stage taken place normally, as all six chromosome pairs connected by with paired nuclei are relatively rare: they are present transiently chiasmata were visible at diakinesis in oocytes from animals following maternal and paternal pronuclear meeting and before treated with either control RNAi or cts-1 RNAi (Fig. S4B). nuclear envelope breakdown (1). Adult C. elegans animals have Moreover, 100% of the one-cell stage-arrested embryos (n = 60) one or no such embryos on each side of the uterus (that is, the contained two polar bodies, indicating that both meiosis I and II uterine half that is between a spermatheca and the vulva); most divisions took place. Finally, polarity cues, as judged by the embryos in the uterus contain four or more cells, as was also distribution of the asymmetrically localized P granules (10) and observed in our control RNAi-treated animals (Fig. 1 A and B the polo-like kinase PLK-1 (11), appeared normal in one-cell and Fig. S2). In contrast, a 40 h treatment with RNAi against cts-1 stage-arrested embryos (Fig. S5). The one difference we noticed resulted in a dramatic increase in one-cell stage embryos with between control and cts-1 RNAi-treated animals at the point paired nuclei (Fig. 1 A and B and Fig. S2). The percentage of when the one-cell stage-arrested embryos were assayed (i.e., 40 h animals exhibiting this phenotype (i.e., at least two or more one- of RNAi treatment) was a reduced rate of egg laying in the cts-1 cell stage embryos with paired nuclei per uterine half; hence- RNAi-treated worms: 39% fewer eggs laid per animal per 40 h in forth, “animals with arrested embryos”) varied from experiment the cts-1 RNAi-treated worms (n = 38) compared with control to experiment, ranging from 50% to 90%. The reason for this worms (n = 16). The reduced rate of egg laying could be in- partial penetrance is not known. The RNAi treatment resulted in dicative of starvation or muscle/neuronal dysfunction (12), both a ∼60% reduction in CTS-1 levels (Fig. S3). Although CTS-1 levels plausible consequences of TCA cycle down-regulation. None- in embryos from cts-1 RNAi-treated animals that did not accu- theless, the cell division events leading to the formation of one- mulate one-cell embryos was slightly higher than that of arrested cell stage embryos appeared to be overall normal.
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