Calcium and Cell Cycle Control

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Calcium and Cell Cycle Control
  Development 108, 525-542 (1990)Printed in Great Britain © The Company of Biologists Limited 1990 Review Article 525 Calcium and cell cycle control MICHAEL WHITAKER and RAJNIKANT PATEL Department of Physiology, University College London, Cower Street, London WC1E 6BT, UK Summary The cell division cycle of the early sea urchin embryo isbasic. Nonetheless, it has control points in common withthe yeast and mammalian cell cycles, at START, mitosisENTRY and mitosis EXIT.Progression through each control point in sea urchinsis triggered by transient increases in intracellular freecalcium. The Caj transients control cell cycle pro-gression by translational and post-translational regu-lation of the cell cycle control proteins pp34 and cyclin.The START Ca s transient leads to phosphorylation ofpp34 and cyclin synthesis. The mitosis ENTRY Cajtransient triggers cyclin phosphorylation. The motosisEXIT transient causes destruction of phosphorylatedcyclin.We compare cell cycle regulation by calcium in seaurchin embryos to cell cycle regulation in other eggs andoocytes and in mammalian cells. Key words: calcium, cell cycle, sea urchin, cyclin, proteinpp34, mammal. Introduction There are two sides to cell division cycle control. One isa cell's decision from the outside about whether to enterthe cell cycle. The other, on the inside, is the decisionabout when key cell cycle events, such as mitosis,should take place. In deciding to enter the cell cycle, thecell responds to external signals such as growth factors.Decisions on the inside are taken on the basis of insideinformation like cell size or the completion of genomereplication. We shall discuss cell division cycle controlfrom this internal perspective.The idea that there are particular points during thecell cycle when a cell must make up its mind and commititself to one particular outcome or another has arisenfrom work on yeast. A large number of mutant strainsof yeast with anomalous cell cycle control has beenselected. Three cell cycle control points have beenidentified by analysis of mutant phenotypes: START, atwhich a decision between mitosis, meiosis, arrest orconjugation is taken; ENTRY into mitosis after DNAsynthesis is completed and EXIT from mitosis oncechromosome segregation has occurred. The yeast cellcycle mutants have also provided the opportunity toisolate cell cycle control proteins, using molecular gen-etics techniques to identify the mutant genes. It is nowevident that all eukaryotic cells possess close homol-ogues of the key yeast cell cycle control proteins.It's very satisfying to come across so telling anexample of what used to be called the Uniformity ofNature. It makes it so much easier to justify studyingcell cycle control in frog, starfish, clam and sea urchinoocytes, eggs and embryos. Early embryos have a veryrapid and straightforward cell cycle. Immature oocytesare arrested at meiosis ENTRY and resume the cellcycle in response to hormones. In many cases, matureoocytes arrest at mitosis EXIT until they are fertilized.Cell cycle control points can be defined easily in eggsand oocytes because these cells have evolved naturalcell cycle pauses from which they are released by anexternal signal. The natural breaks in the cell cycle ineggs and oocytes are specific adaptations of cell cyclecontrol points that exist in all dividing cells. Ourexperiments with early sea urchin embryos show thatcontrol points analogous to START, mitosis ENTRYand mitosis EXIT are found in the sea urchin. We shalldiscuss the idea that the most basic internal cell cyclecontrol mechanisms involve the generation of internal signals that govern the transition from one phase of thecell cycle to the next. The virtue of the basic cell cycle inyeast is that it is amenable to analysis by genetictechniques. The virtue of the basic cell cycle in eggs andembryos is that it can be analysed at the level of thecell's physiology, in terms of the cell's internal signallingsystems.A useful way of thinking about cytoplasmic signallingis in terms of messengers that are generated within thecell to hit targets. The targets in cell cycle signalling are,by definition, a subset of the cell cycle control proteins.The messengers in cell cycle signalling are all or a subsetof the second messengers that have been identified bystudying how cells respond to hormones. Calcium is acell messenger that seems to play a central part in cellcycle control. Much of the evidence that points to  526 M. Whitaker and R. Patel calcium as a signal that triggers cell cycle START,mitosis ENTRY and mitosis EXIT comes from work onsea urchin eggs and embryos. The cell cycle and its control points A cell must generally accomplish four things during thecell cycle: grow, replicate its DNA, duplicate its centro-somes and segregate its chromosomes. In general, thedecision to begin DNA synthesis (S phase) is made bymonitoring cell growth (Killander and Zetterberg, 1965; Brookes and Shields, 1985); passage throughmitosis ENTRY is triggered only when DNA synthesisis completed (Pardee, 1974; Pardee et al. 1978) andrequires that centrosome duplication has occurred(Rattner and Phillips, 1973; Kuriyama and Borisy, 1981). Mitosis EXIT awaits the condensation and as-sembly of chromosomes on the mitotic spindle (Sluder, 1979). The G, S and M phases of the cell cycle (Fig. 1)can be thought of as different cell states. The cell cyclecontrol points are situated just prior to state transitions.START is in Gi, just before the S-phase transition,mitosis ENTRY in G2, just prior to mitosis onset andmitosis EXIT is in M, probably at the metaphase-ana-phase transition. The pattern is most obvious in yeast(Fig. 1), where cell cycle mutants have been found thatenter S phase at the wrong cell size (Nurse, 1975; Nurseand Thuriaux, 1980) (I), or fail to enter (Nurse andBissett, 1981; Russell and Nurse, 1986; Booher andBeach, 1988) (II) or exit (Moreno et al. 1988) (III)mitosis.This pattern is also found in eggs and oocytes. Thecell cycle pauses after S phase in immature oocytes(Kanatani, 1973; Masui and Clarke, 1979; Meijer andGuerrier, 1985) while waiting for the hormonal signal toresume. There is a second hiatus after oocyte matu-ration in frog and mammalian eggs at mitosis EXIT asthe cell cycle waits for the egg to be fertilized(Whitaker, 1989). The sea urchin egg is arrested ininterphase and STARTs the cell cycle at fertilization.Eggs and oocytes differ from somatic cells in two UEIOSIS YEAsAlICLAUX FR0G\STARFISHX 00CYTEs\ YEAST/HIUAUUALIAN/FROG/EGG/ MITOSIS ENTRY UEIOSIS „-,.„ MITOSIS EXIT STARTFig. 1. Cell cycle control points in yeast and cell cyclestopping points in eggs and oocytes. important respects. They do notgrow:the early embryois the same size as the egg. Nor do they think of leavingthe cell cycle: there is nowhere yet for them to go.The similarities between cell cycle control points inyeast and in oocytes are striking. It is also evident thatthe internal control points can be governed by either internal (size control in yeast) or external signals (thehormones and sperm in eggs and oocytes). The evi-dence for similar cell cycle control points in mammaliansomatic cells is on the whole more indirect, but thegeneral scheme does not need too much Procrusteanstretching to apply to their cell cycle too. There is goodevidence for a cell cycle commitment point analogous toyeast START in late G t of the somatic cell cycle(Pardee, 1974). To find cell cycle arrest at other points israre or abnormal. The best argument for START,mitosis ENTRY and mitosis EXIT as control points inthe mammalian cell cycle is that the cell cycle controlproteins isolated from yeast and oocytes have theirexact equivalents in mammalian cells (Lee and Nurse, 1987; Hunt, 1989). The cell cycle of the early sea urchin embryo The unfertilized sea urchin egg waits in interphase forthe sperm. It is haploid and metabolically quiescent.Cell cycle progression (START) is triggered by fertiliz-ation. 30min after fertilization S phase begins and iscompleted at 60min. Mitosis onset is marked by dissol-ution of the nuclear envelope (nuclear envelope break-down, NEB) and chromatin condensation, 70min afterfertilization. Mitotic anaphase occurs at 90min and thenucleus reforms at 110 min. Only the first cell cycleshows a lengthy G! phase. In subsequent cycles, Gi isalmost elided, START follows quickly on mitosis EXITand S phase of the next cell cycle begins very soon aftermitotic telophase. S phase and M phase follow oneanother helter skelter until the mid-blastula stage (12 hafter fertilization), when the cell cycle lengthens,regains its longer Gi and G 2 phases and paternal genesare first transcribed. It is perhaps worth emphasizingthat the early embryonic cell cycles take place withouttranscription: proteins are made from maternal mess-age laid down as the oocyte matures (Davidson et al. 1982). Cell cycle calcium signals STARTing the cell cycle at fertilization The cell cycle resumes abruptly at fertilization: within 10 min metabolism and protein synthesis rates haveincreased 20-fold. The cell signal responsible for cellcycle progression is a transient increase in intracellularfree calcium concentration, [Ca] ; . [Ca]j increases rapidly at fertilization from its restingvalue of 0.1/XM to 1-5 fm and declines towards theresting value over a period of 5-10 min (Steinhardt et al. 1977; Fig. 2). The source of calcium is intracellular,since the Caj transient is not affected by removingcalcium from the extracellular medium (Chambers, 1980; Chambers and Angeloni, 1981; Schmidt et al.  Ca 2+ and cell cycle control 527 200 400 600 800Time (seconds)1000 Fig. 2. Changes in Caj and pHj at fertilization in a singlesea urchin egg. The Cai change was measured by micro-injecting the fluorescent calcium indicator dye fura 2 (50 ^M) before fertilization (Swann and Whitaker, 1986). The pH;change was measured using the fluorescent pH indicatorBCECF. The measurements were made by Ian Crossley. L. piclus. 16°C. 1982; Crossley et al. 1988). Some experiments done by Steinhardt and Epel in the 1970s (Steinhardt and Epel,1974; Steinhardt et al. 1976) indicate that the increase in [Ca]i is a necessary and sufficient signal for resumptionof the cell cycle (Whitaker and Steinhardt, 1982).Calcium chelators that prevent the Caj increase blockcell cycle progression (Zucker and Steinhardt, 1978).Causing an artificial Caj increase with the calciumionophore A23187 leads to a metabolic activation and resumption of the cell cycle identical to activation at fertilization (Steinhardt and Epel, 1974; Whitaker and Steinhardt, 1982). Using a trick or two in addition,parthenogenetic activation with A23187 leads to normal(though haploid) embryonic development (Brandriff etal. 1975). These experiments demonstrate that the Caj transient is the sole causal agent for cell cycle resump-tion at fertilization. Dual ionic signals at fertilization - the increase in pH t One major and long-lasting consequence of the tran-sient Cai increase at fertilization is a sustained increasein intracellular pH (Shen and Steinhardt, 1978; Johnson et al. 1976; Johnson and Epel, 1981 and Fig. 2). The unfertilized egg cytoplasm is unusually acidic(pH 6.7-6.8). A normal cytoplasmic pH (pH7.2-7.3) is restored at fertilization by the activation of a Na-H antiporter in the egg plasma membrane (Johnson et al. 1976;Payan et al. 1983). The pHj signal is an essentialcomponent of cell cycle onset. Artificially maintainingthe cytoplasm at an acidic pH (Grainger et al. 1979) or preventing the extrusion of H + by removing externalNa + or inhibiting the antiporter (Chambers, 1976; Johnson et al. 1976; Shen and Steinhardt, 1979) blockscell cycle progression. The intracellular pH controls therate and extent of protein synthesis (Grainger et al. 1979; Winkler et al. 1980; Dube et al. 1985). The increase in the rate of protein synthesis at fertilization is largely due to the removal of the metabolic brake PtdlnsPpHjt Fig. 3. The phosphoinositide messengers at fertilization in sea urchin eggs. The fertilizing sperm stimulates hydrolysisof the plasma membrane lipid PtdInsP2, producing thephosphoinositide messengers InsP 3 and DAG and triggeringthe fertilization wave. The positive feedback necessary forthe propagation of the calcium wave is provided by furthercalcium-stimulated production of InsP3. The DAGstimulates the pHj increase via protein kinase C and theNa-H antiporter.represented by the unusually acidic pH of the unferti-lized egg (Whitaker and Steinhardt, 1982). Cell messengers at fertilization - InsP^ and DAG Microinjection of the phosphoinositide messenger InsP 3 stimulates resumption of the cell cycle in sea urchin eggs (Whitaker and Irvine, 1984; Fig. 4). This was, incidentally, the first report of the consequences of InsP 3 injection in a living cell. InsP 3 releases calciumfrom an internal store within the egg (Clapper and Lee, 1985; Crossley et al. 1988) and causes a Caj transientthat is very similar to the Caj transient at fertilization in magnitude and duration (Swann and Whitaker, 1986;Swann et al. 1987). Blocking InsP 3 production withneomycin (a not-altogether specific inhibitor of PtdInsP 2 hydrolysis) prevents cell cycle onset (Swannand Whitaker, 1986).The other arm of the phosphoinositide signallingsystem is DAG (diacylglycerol). Synthetic DAG acti- FERTIUZEDMONOSPERMIC NEB FERTILIZEDPOLYSPERMIC NEB A23187 NEB4080 120 TIME (MINUTES)160200 Fig. 4. A Ca : transient, however elicited, leads to mitosisonset. Polyspermic eggs contain 6-10 sperm. A23187concentration was 20 ^M. Final InsP3 concentration aftermicroinjection was 10 nM. NEB was scored using DICoptics. L. pictus. 16°C.  528 M. Whitaker and R. Patel vates the Na-H antiporter, causing cytoplasmic alkalin-ization (Shen and Burgart, 1986; Lau et al. 1986), as does PMA (Swann and Whitaker, 1985; Lau et al. 1986), a tumour promoter that stimulates protein kinaseC (Nishizuka, 1984). Metabolism of polyphosphoinosit-ide (PPI) lipids increases dramatically at fertilization(Turner et al. 1984) and both InsP 3 and DAG rise and fall in concert with the Caj transient (Ciapa and Whitaker, 1986). This seems to be the result of a positive feedback mechanism in which Ca; increasesstimulate further InsP 3 production (Whitaker and Aitchison, 1985; Swann and Whitaker, 1986). The messenger pathways are illustrated in Fig. 3. There are very clear parallels between InsP 3 and DAG production at fertilization and the response of cells to hormones and growth factors (Berridge, 1987a;19876). In both circumstances, an external messenger(the growth factor or the sperm) stimulate changes in the phosphoinositide cell messengers (InsP 3 , DAG, Cai). The PPI messengers must then search out the specific protein targets that are responsible for cell cycleonset. This analogy appears to be imperfect, though, in at least one respect. Signal transduction of hormoneand growth factor signals across the plasma membraneinvolves the interaction of receptors with GTP-bindingproteins at the inner surface of the plasma membrane(Gilman, 1987; Cockcroft and Stutchfield, 1988). It has been suggested that signal transduction at fertilizationoperates through a GTP-binding protein (Turner et al. 1986; Turner et al. 1987), but it seems rather that the sperm triggers the concerted increase in InsP 3 , Caj and DAG by another route (Whitaker et al. 1989). The Ca, transient at fertilization STARTs the cell cycle Whether the Cai transient in the unfertilized egg is elicited by natural means, or artificially by InsP 3 micro-injection or calcium ionophore treatment, the conse-quence is that the cell cycle resumes and mitosis onsetoccurs 80-100min later (Fig. 4). These experimentsalso illustrate the point that the sperm's main contri-bution to cell cycle control is to trigger the calciumtransient, since eliciting the calcium transient with InsP 3 or the calcium ionophore A21387 also cause resumptionof the cell cycle. Nor do markedly polyspermic eggsshow an altered cell cycle timing. The absence or excessof the sperm's DNA does not appear to affect the timing of mitosis onset. Any cell cycle anomalies in parthenogenetically activated sea urchin eggs are due to the lack of the sperm's centriole without which a mitoticspindle cannot form (Brandriff et al. 1987). Indeed, the nuclear cycle of S phase and chromatin condensationdoes not require a functional mitotic spindle (Mazia, 1974; Mazia and Ruby, 1974; Patel et al. I989a,b). Co, at mitosis ENTRY Transient calcium signals occur throughout the  first  cellcycle in sea urchin embryos (Poenie et al. 1985 and Fig. 5). In a way, the calcium signals later in cell cycleare more interesting than the START signal becausethey are an example of antonomous internal cell signal-ling. The evidence that points to the existence of a 2- FERTILIZATIONCLEAVAGE 20406080100120140TIME (MINUTES) Fig. 5. Cell cycle calcium transients. The inset shows a typical mitosis ENTRY transient in a polyspermic egg. Caj was measured by microinjecting the fluorescent calciumindicator dye fura 2 to a final concentration of 10-20 JUM. Basal Cai just prior to the mitosis ENTRY transient was 250±30IIM (mean and S.E.M., n=6). In monospermic eggs the mitosis ENTRY transient reached 350 IIM in someexperiments, but equally often could not be detected. We estimate our detection threshold at 250 nM Ca, to be 30-50 nM. In polyspermic eggs, the mitosis ENTRYtransient, when detected, ranged from 290-1050 nM (690±120nM, mean and S.E.M., n=6). L. pictus. 16°C. calcium signal at mitosis entry is shown schematically in Fig. 6. We can measure an increase in [Ca]j in eggs 1-2min before normal mitosis ENTRY (Figs 5 and 6). In polyspermic eggs, the transient is large and easilydetected using whole cell fura2 recording techniques.Causing a premature increase in Caj by microinjecting InsP 3 or calcium leads to a rapid and precocious mitosisENTRY (Twigg et al. 1988). Mitosis ENTRY is blockedby microinjection of the calcium chelators EGTA or BAPTAand the block can be relieved by microinjectingcalcium (Twigg et al. 1988; Steinhardt and Alderton, 1988). These experiments show that the Caj transient at mitosis ENTRY is a necessary signal for mitosis onset.It is also a sufficient signal, provided the egg has passedSTART and the protein synthesis requirement is met (Twigg et al. 1988; Patel et al. 1989 and Fig. 10). The immediate target of the mitosis ENTRY Ca ( transient in sea urchin embryos is calmodulin and a typeII calmodulin-regulated kinase (CAM-II kinase).Microinjection of antibodies or inhibitory peptidesdirected against the CAM-II kinase arrests the cell cycleat mitosis ENTRY (Baitinger et al. 1989; Baitinger et al. 1990). Local increases in COJ at mitosis ENTRY The timing of mitosis ENTRY is independent of the degree of polyspermy at fertilization (Fig. 4). In con- trast, the mitosis entry Caj transient is much larger in polyspermic than in monospermic eggs; it is often not easily detected in whole cell fura2 experiments aftermonospermic fertilization (Fig. 5) and is sometimes  Ca and cell cycle control 529 100-1 80-60- m UJ *40-20- o 0.6 1.4 2.3i BAPTA (mM)o Fig. 6. The mitosis ENTRY Ca| transient governs mitosis onset. TOP LEFT: the mitosis ENTRY Ca, transient recordedwith fura 2 in a single egg showing the timing of subsequent mitosis onset (nuclear envelope breakdown: NEB). TOPRIGHT: Micro-injecting the phosphoinositide messenger InsP3 (100 nM  final  concentration) stimulates a precocious calciumtransient and premature NEB. BOTTOM LEFT: microinjecting the calcium chelator BAPTA (3 mM) blocks the calciumtransient and mitosis onset. BOTTOM RIGHT: mitosis onset is prevented at a cytoplasmic BAPTA concentration above 2ITIM. undetectable. Strictly speaking, something that cannotbe detected doesn't exist. For obvious reasons, weprefer the idea that, in many normal, monospermicembryos, the Caj transient at mitosis entry is localizedto the area of the nucleus. This would make it difficultto pick out from the noise in fura2 measurements. Itseems that the mitosis ENTRY Cai transient is ampli-fied when several sperm nuclei are present in thecytoplasm, though the underlying cell cycle timingmechanisms that lead to the generation of the calciumtransient are unaffected. Meiosis ENTRY in surf clam oocytes Surf clam (Spisula) oocytes ENTER meiosis immedi-ately after fertilization in response to an internal signaltriggered by sperm (Fig. 1). Cell cycle onset throughmeiosis ENTRY is also triggered in these eggs bymicroinjection of InsP 3 (Bloom etal. 1988), presumablybecause InsP 3 causes a Caj transient. An increase in PPIturnover has been measured at fertilization (Eckbergand Szuts, 1987) and it has been suggested that DAGproduction leads to cytoplasmic alkalinization via pro-tein kinase C and a Na-H antiporter, as in sea urchineggs (Dube, 1988). Calmodulin antagonists preventmeiosis onset (Carroll and Eckberg, 1986). There arestrong similarities, therefore, with the cell signals thatoperate at START in sea urchin eggs, though in Spisula the source of the activating calcium is extracellular(Dube, 1988). Ca t and mitosis EXIT Caj transients occur in sea urchin embryos at mitosisEXIT (Poenie et al. 1985). Figure 5 shows the timing ofthe transient. Embryos micro-injected during mitosis
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