Down-regulation of tricarboxylic acid (TCA) cycle 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 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 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 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 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 BIOLOGY 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 DEVELOPMENTAL 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.

www.pnas.org/cgi/doi/10.1073/pnas.1311635111 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 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. embryos (Fig. S3), the difference was very small. This difference To examine mitotic cell cycle events after fertilization, we may be sufficient to bypass the one-cell stage arrest, but it is also followed chromosomes using histone H2B fused to mCherry possible that embryos that can progress past the one-cell stage (H2B::mCR) and nuclear envelope dynamics using nuclear pore despite low CTS-1 levels have somehow adapted to low CTS-1 protein NPP-1::GFP. In embryos from animals treated with levels. In animals with arrested embryos, the average fraction of control RNAi, chromosome condensation happened as the ma- embryos at the one-cell stage with paired nuclei was over 50% of ternal and paternal pronuclei met, followed immediately by nu- the total number of embryos per each uterine half (Fig. 1B and Fig. clear envelope breakdown and metaphase plate formation (less S2). This was not accompanied by an accumulation of two- and than 7 min after pronuclear meeting; Fig. 1C). In contrast, in four-cell stage embryos (Fig. 1B and Fig. S2), arguing that the embryos from cts-1(RNAi) animals, maternal and paternal down-regulation of cts-1 did not cause a general slowing down of pronuclear meeting occurred normally, but nuclear envelope embryonic development. Embryos from all cts-1 RNAi-treated breakdown did not happen, even after a prolonged time (greater animals that did not arrest at the one-cell stage continued to de- than 30 min, n = 8; for example, Fig. 1D). In these animals, velop but arrested before hatching, with the exception of a small chromatin condensation did not take place and a metaphase fraction of embryos that hatched but arrested as larvae. Thus, plate never formed (compare Fig. 1 C with D). down-regulation of cts-1 resulted in one of two fates: an arrest at We also examined nuclear envelope dynamics using additional the one-cell stage with paired nuclei, or a developmental arrest at markers, including LMN-1 (the single C. elegans B-type lamin a multicellular embryo or early larva stage. homolog) (13), NPP-9 (another nuclear pore complex component) Cell cycle events that preceded fertilization were also exam- (14), and LEM-2 (a LEM-domain protein that resides in the ined. The germ line of cts-1 RNAi animals containing one-cell nuclear envelope and is a component of the nuclear lamina)

A control RNAi cts-1 RNAi CDcontrol RNAi cts-1 RNAi E NPP-9::GFP - 4 min - 3 min 0 min 0 min

B 100 F 4 min

control RNAi 4 min LMN-1::YFP LEM-2::GFP cts-1 RNAi

50 7 min 7 min

NPP-1::GFP H2B::mCR

0 34 min % embryos / uterine half 1-cell 2-cell 4-cell multi-cell NPP-1::GFP H2B::mCR

Fig. 1. Down-regulation of cts-1 results in an accumulation of one-cell stage embryos with paired nuclei. (A) Embryos from animals after 40 h of RNAi treatment with either control RNAi (Left)orcts-1 RNAi (Right). The animals (strain OCF3) expressed NPP-1 fused to GFP (NPP-1::GFP) and histone H2B fused to mCherry (H2B::mCR). (B) The percentages of one-, two-, four-, or multicell stage embryos per uterine half of OCF3 animals treated with control or cts-1 RNAi (n = 16 and n = 19 animals, respectively) are shown. For cts-1 RNAi-treated animals, only those that accumulated one-cell stage embryos (i.e., two or more per gonad arm) were included. Shown are the results from a typical experiment, where 50% of animals accumulated one-cell stage-arrested embryos following cts-1 RNAi (see Fig. S2 for additional experiments). Error bars indicate SD. The difference in the percentage of embryos in the one-cell stage between control and cts-1(RNAi) animals was statistically significant (P < 0.05, Student t test). (C) Time-lapse images of an embryo from a control RNAi-treated OCF3 animal. Time 0 was defined as the time point when the maternal and paternal pronuclei met. (D) Time-lapse images of an embryo from a cts-1 RNAi-treated OCF3 animal. Outlines indicate the edges of the embryos. (E) The uterus of a cts-1 RNAi-treated animal expressing NPP-9::GFP. (F) Embryos from cts-1 RNAi-treated animals expressing the indicated fusion proteins, imaged 30 min following pronuclear meeting. (Scale bar: 10 μm.)

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1311635111 Rahman et al. Downloaded by guest on September 25, 2021 (15). Following cts-1 RNAi, all three proteins remained at the A dlat-1 RNAi dlat-1+cts-1 RNAi nuclear envelopes of the paired nuclei in one-cell stage-arrested embryos for at least 30 min following pronuclear meeting (Fig. 1 E and F). Thus, down-regulation of cts-1 causes a general defect in nuclear envelope breakdown.

Down-Regulation of Additional TCA Cycle Components Leads to an Embryonic One-Cell Stage Premitotic Arrest. To determine whether the one-cell stage embryonic arrest observed following cts-1 RNAi was due to CTS-1’s function in the TCA cycle, we exam- ined whether down-regulation of other TCA cycle components resulted in a similar phenotype. Acetyl CoA is made from py- B 100 C54G4.8C54G4 8 R RNAiNAi ruvate by the pyruvate dehydrogenase (PDH) complex. A re- controlcontrol RRNAiNAi duction in PDH complex activity would likely limit the amount of cts-1cts-1 RNAi acetyl CoA available for conversion to citrate by citrate synthase, dlat-1dlat-1 RNAi thus reducing overall TCA cycle activity (16). The PDH complex cts-1cts-1 + dlat-1dlat-1 consists of three enzymes: PDH, dihydrolipoyl transacetylase, doubledouble RNRNAiAi and dihydrolipoyl dehydrogenase (17). The sequence of C. elegans 50 ORF F23B12.5, coding for DLAT-1, is highly similar to verte- brate dihydrolipoyl dehydrogenase, which is also part of the oxoglutarate dyhedrogenase complex (also in the TCA cycle) and the alpha-keto acid dehydrogenase complex. Down-regula- tion of dlat-1 by RNAi resulted in a paired-nuclei phenotype in

one-cell stage-arrested embryos, similar to the phenotype ob- % embryos / uterine half 0 served following cts-1 down-regulation (Fig. 2 A and B). More- 1-cell 2-cell2-cell 4-cell multi-cellmulti-cell over, the fraction of animals with arrested embryos and the percentage of one-cell stage-arrested embryos in these animals Fig. 2. Down-regulation of TCA cycle components results in a paired-nuclei was similar to that of cts-1(RNAi) animals (Fig. 2B). A double phenotype in a one-cell stage-arrested embryo. (A) Embryos from an OCF3 BIOLOGY RNAi treatment against dlat-1 and cts-1 resulted in a similar animal treated with RNAi against dlat-1 (Left)ordlat + cts-1 (Right). Control and DEVELOPMENTAL μ phenotype as cts-1 RNAi or dlat-1 RNAi alone (Fig. 2B and Fig. cts-1 RNAi alone are the same as shown in Fig. 1A.(Scalebar:10 m.) (B)OCF3 S6), suggesting that DLAT-1 and CTS-1 affect mitotic progression animals were treated with control RNAi or RNAi against cts-1, dlat-1, or both, and the percentages of embryos in the indicated stages in each uterine half in the one-cell stage embryo through the same metabolic pathway. were scored. Shown is a typical RNAi experiment (additional experiments are in Consistent with this, we found that down-regulation of additional Fig. S6). For each treatment, uterine halves from at least 10 animals were scored. TCA cycle components, malate dehydrogenase, aconitase, and The percentages of animals with arrested embryos in this experiment were succinate dehydrogenase, also resulted in an accumulation of 50%, 61.9%, and 40% for RNAi against cts-1, dlat-1,andcts-1 + dlat-1,re- one-cell stage embryos with paired nuclei (Fig. S7). Thus, down- spectively. Error bars indicate SD. cts-1, dlat-1,andcts-1 + dlat-1 RNAi treat- regulating the TCA cycle blocks the first embryonic cell cycle ments led to the accumulation of one-cell stage embryos that was statistically before mitotic entry. significantly higher than in the control (P < 0.05, Student t test). The difference betweencontrol and thethree RNAi treatments in the percentageof multistage Down-Regulation of the TCA Cycle Affects Centrosome Separation in embryos was also statistically significant (P < 0.05, Student t test). the C. elegans One-Cell Stage Embryos. Centrosomes are necessary for the formation of a bipolar spindle in the C. elegans embryo Down-Regulation of the TCA Cycle Prevents CDK-1 Activation in the (2, 18). After fertilization, the paternally contributed centrioles One-Cell Stage Embryo. Because centrosome separation, chro- duplicate, generating two centrosomes that separate and migrate mosome condensation and nuclear envelope breakdown are to the center of the embryo, where they nucleate microtubules mechanistically independent (20, 21), it was likely that down- that ultimately form a bipolar spindle (2). To follow spindle β regulation of the TCA cycle prevented mitotic entry by blocking dynamics in vivo we used transgenic animals expressing -tubulin a common upstream regulatory event. In metazoans, entry into fused to GFP (TBB-2::GFP) and NPP-1 fused to mCherry (NPP- mitosis is largely dependent on increased activity of Cdk1, 1::mCR). In control RNAi-treated animals, before nuclear en- which involves the accumulation of nuclear cyclin B and the velope breakdown, the two centrosomes align on either side of removal of inhibitory phosphorylation from threonine (Thr14) the plane where the two pronuclei meet (Fig. 3A). In contrast, in and tyrosine (Tyr15) residues of Cdk1 (residue numbers ac- embryos from cts-1 RNAi-treated animals, the position of the cording to the human Cdk1) by the Cdc25 phosphatase (3, 4). centrosomes was abnormal: over half of the embryos had either = Cyclin B builds up in the nucleus in G2 and begins to be de- one or two closely associated centrosomes (53.8%, n 78), graded upon entry into mitosis (22). Thus, in G2 cells, Cdk1 whereas in the remaining embryos the centrosomes were sepa- activity is held in check primarily by inhibitory phosphorylation rated but abnormally positioned (Fig. 3B). Live imaging of on Thr14/Tyr15 (4). To examine the status of Cdk1 in the one- transgenic animals expressing a centrosomal component, SPD-2, cell stage arrest induced by down-regulation of the TCA cycle, fused to GFP (SPD-2::GFP) (19) confirmed that cts-1 down- we examined the levels of C. elegans cyclin B (CYB-1) and the regulation prevented the timely separation of centrosomes in the phosphorylation state of CDK-1 in embryos following control one-cell stage embryo (n = 4; Fig. 3C, Insets). A similar trend was or cts-1 RNAi using antibodies against CYB-1 (23) and anti- observed following dlat-1 down-regulation by RNAi: of the 57 bodies against human inhibitory phosphorylated Cdk1(Thr14/ embryos arrested in the one-cell stage with paired nuclei, 50.9% Tyr15) that also recognize the C. elegans phospho-Cdk1(Thr32/ had centrosomes that failed to separate, 7% had centrosomes Tyr33) (Fig. S8). that separated but were abnormally positioned, and in 42.1% the In control RNAi-treated animals, cyclin B was visible at the centrosomes were separated and positioned correctly. Thus, one-cell stage before nuclear envelope breakdown (Fig. 4A), and down-regulation of the TCA cycle not only blocked nuclear en- it exhibited the expected pattern in later cell cycle stages (see, for velope breakdown and chromosome condensation, but also inhibi- example, its absence from a metaphase cell in the four-cell stage ted the timely separation of centrosomes, another event associated embryo). One-cell stage-arrested embryos from cts-1(RNAi) with mitotic entry. animals also had significant levels of CYB-1 (in 59 embryos of 65

Rahman et al. PNAS Early Edition | 3of6 Downloaded by guest on September 25, 2021 A TBB-2::GFP NPP-1::mCR merged and consequently inhibition of CDK-1 activation. The G2/M control RNAi transition is subjected to regulation by DNA damage response pathways, and specifically by the Chk1 (24, 25). In the presence of DNA damage, activated Chk1, which is phos- phorylated on Ser345 (residue number according to the human B protein), inhibits the Cdc25 protein phosphatase (26). This, in cts-1 RNAi turn, prevents the removal of inhibitory phosphorylation of Cdk1 that is critical for Cdk1 activation, as discussed in the In- troduction. In C. elegans, a similar CHK-1 activation pathway, through the phosphorylation of the corresponding Ser344, has been reported (27, 28). Although the DNA damage and repli- cation checkpoints are thought to be inactive in the one-cell

no separation stage embryo (28, 29), it was formally possible that down-regu- lation of the TCA cycle led to unscheduled CHK-1 activation at this stage. To test this, we examined one-cell stage-arrested embryos from cts-1 RNAi-treated animals for the presence of CHK-1 phosphorylated at Ser344 (p-CHK-1S344) by immuno- fluorescence using antibodies against human phospho-Chk1 at Ser345 (27). As a positive control for the presence of p-CHK- 1S344, we down-regulated the large subunit of ribonucleotide mis-placed

A control RNAi C 0 min 6 min 10 min 13 min 1-cell prior rotation 1-cell after rotation 4-cell control RNAi DAPI

0 min 4 min 9 min 13 min cts-1 RNAi SPD-2::GFP

Fig. 3. Down-regulation of cts-1 results in failure in centrosome separation. anti p-Cdk1 anti cyclin B Embryos from a control (A)orcts-1 (B) RNAi-treated animals (strain OCF28) expressing β-tubulin fused to GFP and NPP-1 fused to mCherry. (C, Upper)An B cts-1 RNAi embryo from a control RNAi-treated animal expressing SPD-2::GFP (strain OC534) showing two centrosomes shortly following duplication (0 min), and as they move to the center of the embryo. Images were taken at the indicated time points. (C, Lower) An embryo from the same strain treated with cts-1 (RNAi), showing duplicated centrosomes (see Insets, which are an enlargement of the boxed areas in the first and last time points). (Scale bar: 10 μm.)

analyzed; Fig. 4B), comparable to interphase nuclei in embryos from control RNAi-treated animals. Thus, the inability to enter mitosis following cts-1 down-regulation was not due to a defect in CYB-1 accumulation. However, although in one-cell stage em- bryos from the control RNAi-treated animals the levels of in- hibitory phosphorylated CDK-1 diminished as the two pronuclei rotated (Fig. 4A, Center) and were absent once mitosis had begun (Fig. S9), the levels of inhibitory phosphorylated CDK-1 in one- cell stage-arrested embryos from cts-1(RNAi) animals were un- usually high (Fig. 4B). This suggested that the one-cell stage anti p-Cdk1 anti Cyclin B DAPI arrest is due to persistent inhibitory phosphorylation of CDK-1. The high level of inhibitory phosphorylated CDK-1, along with Fig. 4. Down-regulation of cts-1 results in a G2-arrest in the one-cell stage the simultaneous presence of nuclear localized CYB-1, suggests embryo. (A) One-cell stage embryos before (Left) or after (Center) pro- that the one-cell stage-arrested embryos from cts-1 RNAi- nuclear rotation, and a four-cell stage embryo, all from control RNAi-treated animals, were coimmunostained with antibodies against cyclin B (Middle) treated animals are in a G2-like state. and phosphospecific antibodies against Cdk1 inhibitory phosphorylation at Thr14 and Tyr15 (Bottom). Note that neither cyclin B nor phospho-Cdk1 is The One-Cell Stage Embryonic Arrest Following cts-1 Down-Regulation detected in the metaphase cell in the four-cell stage embryo, as shown by Is Not Due to the Activation of the DNA Damage Checkpoint or the DAPI staining (Top). (B) Two typical examples of embryos arrested in the Spindle Assembly Checkpoint. The G2-like arrest could have been one-cell stage from cts-1 RNAi-treated animal. Embryos were coimmunos- the result of a cell cycle defect that led to checkpoint activation tained as in A. (Scale bar: 10 μm.)

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1311635111 Rahman et al. Downloaded by guest on September 25, 2021 reductase (rnr-1) by RNAi, thereby reducing de novo synthesis of divisions, either in its regulation of Cdk-1 activation or in its deoxyribonucleotides, leading to stalling of DNA replication fork dependence on TCA cycle activity. For example, in mammalian progression and delaying mitotic entry via CHK-1 activation (30, embryos the TCA cycle is active during early embryonic divisions 31). As expected, down-regulation of rnr-1 resulted in activation whereas glycolysis is activated only postimplantation (see below). of CHK-1 (Fig. S10 A and B). In contrast, p-CHK-1S344 was Down-regulation of enzymes in other metabolic pathways, in- largely absent in embryos from control RNAi-treated animals cluding glycolysis [the C. elegans homologs of (coded and never detected in the one-cell–arrested embryos from cts-1 by F14B4.2), glyceraldehyde 3-phosphoate dehydrogenase and (RNAi) animals (Fig. S10 A and B). Thus, the persistence of 6-], the electron transport chain (including Cdk1 inhibitory phosphorylation following cts-1 down-regulation the CYC-1 and UCR-1 subunits of complex III and the CCO-1 is not due to the activation of the DNA damage or replication and CCO-2 subunits of complex IV) and the PHI-37, ATP-4, and checkpoint through CHK-1 activation. ATP-2 subunits of ATP synthase, all led to embryonic lethality We also examined whether the one-cell stage arrest following but did not lead to an increase above control of one-cell stage TCA cycle down-regulation depended on the spindle assembly embryos. This suggests that either the TCA cycle is predominant checkpoint, which prevents progression through mitosis in the during this phase of development, or that it has a unique in- absence of chromosome-microtubule attachments (32). If that termediate that affects cell cycle progression. It was previously were the case, then the inactivation of this checkpoint would shown that depolarizing mitochondria in human colorectal car- bypass the one-cell arrest. To test this possibility we inactivated cinoma HTC116 tissue culture cells (38) or reducing ATP levels the C. elegans MAD3 homolog by using a san-1 mutant (33, 34) in Drosophila cells (39) blocks the G1/S transition. We, however, and treated the worms with RNAi against cts-1. The absence of found that down-regulating the TCA cycle arrests cells in G2-like a functional spindle assembly checkpoint did not bypass the one- state. It is possible that the first cell division cycle in the C. ele- cell stage arrest following cts-1 RNAi (Fig. S10C), consistent gans embryo has unique features that allow it to progress past with a cell cycle arrest before mitotic entry. G1/S and on to G2 in the presence of low ATP levels. Alterna- tively, the arrest in the one-cell stage embryo following TCA Discussion cycle down-regulation could be due to changes in the levels of Metabolism affects cell cycle progression, but the extent to which a metabolite other than ATP; either the accumulation of a toxic this happens during embryogenesis, and the pathways that link intermediate or the absence of a critical one. Alterations in the metabolism and the cell cycle circuitry, are largely unknown. Here TCA cycle may also affect intracellular pH or the redox state of the cytoplasm, both of which can have a profound effect on many we show that in C. elegans, down-regulation of TCA cycle com- BIOLOGY ponents results in a one-cell stage embryonic arrest with paired different processes. nuclei. The arrested embryos fail to break down the nuclear en- The Cdc25 phosphatase, which removes the Wee1-dependent DEVELOPMENTAL velope, chromosomes remain decondensed, and centrosomes are inhibitory phosphates from Cdk1, is an obvious candidate for unseparated or abnormally separated. Taken together, these phe- being affected by the TCA cycle (40, 41). In C. elegans, CDC- notypes are consistent with a defect in mitotic entry. Indeed, the 25.1, which is homologous to the mammalian Cdc25A, is ma- arrested embryos had high levels of both cyclin B and inhibitory- ternally contributed and is proposed to promote mitotic pro- phosphorylated CDK-1, indicative of a G2-like stage arrest. Thus, gression in the early embryo (42, 43). If our model is correct, our data suggest that the cell cycle machinery of the one-cell stage then down-regulation of CDC-25 should phenocopy cts-1 RNAi embryo is sensitive to the metabolic state of the cell, and that when whereas inactivation of WEE-1 should bypass the one-cell stage the TCA cycle is down-regulated, the one-cell stage embryo fails to arrest. Unfortunately, in C. elegans, the activities of both CDC-25 enter the first mitotic division following fertilization. and WEE-1 are necessary for gametogenesis (44, 45), precluding One of the earliest defects caused by TCA cycle down-regu- us from testing these predictions at this time. lation was a defect in centrosome separation. Previously, Hachet To what extent can our findings in C. elegans embryos extend et al. proposed that centrosomes affect timely entry into mitosis to mammalian systems? Studies conducted mostly in the 1960s in the C. elegans early embryo (35). However, it is unlikely that and 1970s have shown that two-cell stage mammalian embryos centrosomes are the main target affected by down-regulation of use pyruvate and lactate through the TCA cycle, whereas gly- colysis is activated at a later embryonic, postimplantation, stage the TCA cycle because defects in centrosome maturation, caused – by down-regulation of SPD-2 or SPD-5 and which blocks cen- (46 48). Moreover, inhibitors of the TCA cycle block mamma- trosome separation and spindle assembly, do not prevent nuclear lian embryonic development at the one-cell stage, whereas in- envelope breakdown or entry into mitosis (19, 35, 36). hibition of glycolysis affects the morula to blastocyst transition At present we do not know how the TCA cycle affects the cell (49). When these studies were conducted, very little was known cycle machinery; it may cause a metabolic imbalance in the about the molecular details of the cell cycle machinery, and thus embryo that blocks Cdk1 activation, but it may also cause an the molecular link between the TCA cycle and the ability to imbalance earlier, in the germ line, which is maintained post- execute the early embryonic divisions was not known. The present fertilization. Even if the TCA cycle perturbation occurs in the study gains insight into the link between metabolism and cell cycle germ line, our data suggest that its effect on the cell cycle ma- progression in the early embryo; at least in C. elegans, down-reg- chinery occurs in the embryo rather than earlier. This is based on ulation of the TCA cycle blocks Cdk1 activation by preventing the observations that the germ line and oocytes of cts-1 RNAi the removal of its inhibitory phosphorylation. animals appear morphologically normal and that all of the Materials and Methods arrested embryos had two polar bodies, indicating that the completion of meiosis was not perturbed. Had Cdk1 been inac- Strains. All C. elegans strains were maintained at 20 °C using standard tivated in the germ line, meiosis would not have been completed methods except where noted otherwise (50). A list of strains and their (37). Thus, the most straightforward explanation for the one-cell genotypes is in Table S1. embryonic arrest is that down-regulation of TCA cycle activity led, either directly or indirectly, to a defect in mitotic Cdk1 ac- RNAi Experiments. RNAi constructs were isolated from the RNAi feeding library (Open Biosystems) and performed using standard feeding methods. The identity tivation in the one-cell stage embryo, and this, in turn, blocked of each RNAi clone was verified by sequencing. L4-stage larvae were transferred further progression through the cell cycle. to RNAi plates, and embryos were examined after 40 h of RNAi treatment at An interesting observation is that embryos that did not arrest 20 °C. For each treatment, at least 10 animals were scored, and each experi- in the one-cell stage following TCA cycle down-regulation con- ment was repeated multiple times. Because the two gonad arms act in- tinued to develop to multicellular embryos. This indicates that dependently of each other, we scored the phenotypes of embryos per uterine although the first mitotic division is sensitive to perturbations in half, that is, between each spermatheca and the vulva. To determine embry- the TCA cycle, subsequent divisions are not. One possibility is onic lethality, 8–10 animals were transferred to new RNAi plates following 40 h that the first mitotic division is inherently different from later treatment and removed 3–6 h thereafter. Hatching was scored 24 h later.

Rahman et al. PNAS Early Edition | 5of6 Downloaded by guest on September 25, 2021 Immunofluorescence. Animals from RNAi plates were dissected on poly-L- Apo objective, four LMM5 laser merge module with diode lasers (excitation lysine–coated slides and embryos were opened by the freeze-cracking at 405, 491, 561, and 655 nm) from Applied Research, a CSU10 spinning-disk method as described previously (9). Samples were fixed in −20 °C cold unit by Yokogawa, and a C9100-13 EM-CCD camera by Hamamatsu. Immu- methanol for 2 min and blocked in 5% (vol/vol) BSA in PBS for 30 min at 4 °C nostained embryo images were taken on a Nikon E800 microscope using before overnight incubation at 4 °C with primary antibody diluted in 1% IPLab 3.9.5 software (BioVision Technologies). The microscope is equipped BSA. The following antibodies were used: anti-cyclin B at 1:50 dilution with a 60 × 1.4 N.A. Apo objective and a C4742-95 CCD camera by Hamamatsu. (Development Studies Hybridoma Bank, University of Iowa, Iowa City, IA), Images were processed with IPLab 3.9.5 and 4.0.8, ImageJ 1.44o (http://imagej. anti–phospho-Cdk1 Thr14/Tyr15 1:200 dilution (Santa Cruz Biotechnology; nih.gov/ij), Adobe Photoshop CS Version 8.0, and Adobe Illustrator CS5 sc-28435-R). Samples were washed three times in PBS containing 0.1% Version 15.1.0. Tween20 followed by 45 min incubation in secondary antibodies, Alexa Fluor 488 and 568 (Invitrogen), each at a 1:2,500 dilution. Samples were ACKNOWLEDGMENTS. We thank Andy Golden, Amy Fabritius, and Edward then washed three times in PBS containing 0.1% Tween20 and mounted Kipreos for comments on the manuscript. We also thank Andy Golden and ’ in Vectashield with DAPI (Vector Laboratories). Kevin O Connell for advice, strains, and antibodies; and Geraldine Seydoux for the PGL-1::GFP strain. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health Office Microscopy. Confocal images of live embryos were taken on a Nikon Eclipse of Research Infrastructure Programs (P40 OD010440). M.M.R., S.R., D.J.-S. TE2000U (spinning-disk confocal) microscope using IPLab 4.0.8 software and O.C.-F. were funded by an intramural grant from National Institute of (BioVision Technologies). The microscope is equipped with a 60 × 1.4 N.A. Diabetes and Digestive and Kidney Diseases.

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