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The Role of Calcium in the Cell Cycle: Facts and Hypotheses
Abstract
The regulation of cell cycle progression is a complex process which involves kinase cascades, protease action, production of second messengers and other operations. Increasing evidence now compellingly suggests that changes in the intracellular Ca2+ concentration may also have a crucial role. Ca2+ transients occur at the awakening from quiescence, at the G/S transition, during S-phase, and at the exit from mitosis. They may lead to the activation of Ca2+ binding proteins like S-100, but the key decoder of the Ca2+ signals in the cycle is calmodulin. Activation of calmodulin leads to the stimulation of protein kinases, i.e., CaM-kinase II, and of the CaM-dependent protein phosphatase calcineurin. Ample evidence now indicates the G/S transition, the progression from G2 to M, and the metaphase/anaphase transition as specific points of intervention of CaM-kinase II. Another attractive possibility for the role of Ca2+ in the cycle is through the activation of the Ca(2+)-dependent protease calpain: other proteases (e.g., the proteasome) have been suggested to be responsible for the degradation of some of cyclins, which is essential to the progression of the cycle. One of the cyclins, however, (D1) is instead degraded by calpain, which has been shown to promote both mitosis and meiosis when injected into somatic cells or oocytes.
... In the G1/S transition, the tracellular Ca2+ concentration increases. Under environmental conditions of high calciu concentration, calmodulin can bind to eEF2K and activate it so that the cells enter th phase ( Figure 3) [51]. Another function of cellular calcium is to upregulate cAMP lev [52]. ...
... In the G1/S transition, the intracellular Ca2+ concentration increases. Under environmental conditions of high calcium concentration, calmodulin can bind to eEF2K and activate it so that the cells enter the S phase ( Figure 3) [51]. Another function of cellular calcium is to upregulate cAMP levels [52]. ...
... Structures of the eEF2K activators ritonavir, lopinavir, and resveratrol(49)(50)(51). ...
Eukaryotic elongation factor 2 kinase (eEF2K or Ca2+/calmodulin-dependent protein kinase, CAMKIII) is a new member of an atypical α-kinase family different from conventional protein kinases that is now considered as a potential target for the treatment of cancer. This protein regulates the phosphorylation of eukaryotic elongation factor 2 (eEF2) to restrain activity and inhibit the elongation stage of protein synthesis. Mounting evidence shows that eEF2K regulates the cell cycle, autophagy, apoptosis, angiogenesis, invasion, and metastasis in several types of cancers. The expression of eEF2K promotes survival of cancer cells, and the level of this protein is increased in many cancer cells to adapt them to the microenvironment conditions including hypoxia, nutrient depletion, and acidosis. The physiological function of eEF2K and its role in the development and progression of cancer are here reviewed in detail. In addition, a summary of progress for in vitro eEF2K inhibitors from anti-cancer drug discovery research in recent years, along with their structure–activity relationships (SARs) and synthetic routes or natural sources, is also described. Special attention is given to those inhibitors that have been already validated in vivo, with the overall aim to provide reference context for the further development of new first-in-class anti-cancer drugs that target eEF2K.
... Increasingly, Ca^^ is becoming recognised as a key regulator o f several checkpoints within the cell cycle (for review, see Santella, 1998). [Ca^^Jj transients have been observed in late Gi prior to the initiation o f S phase, in G2 before entry into M phase, during mitosis at the transition from metaphase to anaphase and during cytokinesis. ...
... halts its progression. Similarly, changes in the level o f IP3, which releases Ca^^ from intracellular stores, has been shown to correlate with the resumption o f the cell cycle in fertilized sea urchin eggs (for comprehensive reviews, see Santella, 1998 andSantella et al., 1998). Ca^^ activates Ca^^-binding proteins such as the key Ca^^ signal decoder calmodulin, which stimulates the kinase CaMkinase II. ...
... halts its progression. Similarly, changes in the level o f IP3, which releases Ca^^ from intracellular stores, has been shown to correlate with the resumption o f the cell cycle in fertilized sea urchin eggs (for comprehensive reviews, see Santella, 1998 andSantella et al., 1998). Ca^^ activates Ca^^-binding proteins such as the key Ca^^ signal decoder calmodulin, which stimulates the kinase CaMkinase II. ...
This thesis investigates the roles of neurotransmitters and their receptors, changes in intracellular calcium ([Ca2+]i), and the retinal pigment epithelium (RPE) in the regulation of cell proliferation and migration in the developing chick retina. Confocal imaging of retinae is used to show that before synapses are formed, cells in the ventricular zone (VZ) display intermittent spontaneous [Ca2+]i transients that depend upon the endogenous release of neurotransmitters. Purinergic and muscarinic receptor- evoked transients occur in a mixed population of interphase and mitotic cells. Those produced by GABAergic and glutamatergic receptors are mostly restricted to the interphase population. Muscarinic receptor activation is shown to slow down, and purinergic activation to speed up, mitosis. These actions may result from the [Ca2+]i transients these agonists evoke. GABA and glutamate receptor activation are without effect on mitosis. The nuclei of retinal progenitor cells (PCs) migrate back-and-forth across the retina in a process called interkinetic nuclear migration (INM). To study the possible influence of neurotransmitter receptor activation on INM, a 'gene gun' technique was used to label cells in the VZ; the speed of movement of some cells is influenced by neurotransmitters. [Ca2+]i transients occur in cells during INM, which may be important in its regulation. Gap junctional communication between the RPE and the neural retina was investigated. Ca2+ -imaging experiments show that gap junctions support the spread of spontaneous Ca2+ signals between neighbouring cells. Whole-cell patch clamp recording was used to fill VZ cells with a combination of gap junction-permeable and impermeable dyes. These injections show that gap junctions couple PCs into clusters that largely exclude differentiated neurons. Coupling was also observed between cells in the RPE and the neural retina. These pathways may be important in the regulation of proliferation. The RPE is shown to express both purinergic and muscarinic receptors and to have a profound influence on the rate of cell proliferation in the neural retina. The RPE may speed mitosis in the retina through the release of ATP and other factors.
... Transient changes in intracellular calcium ([Ca 2+ ] i ) occur at the exit from quiescence in the early G 1 phase, at the G 1 /S transition, during the S phase, and at the exit from the M phase. In the early G 1 phase and at the G 1 /S transition, the cells are most sensitive to depletion of extracellular Ca 2+ [8,[12][13][14] . ...
... Diverse studies have shown that activation of the Ca 2+ -binding protein calmodulin (CaM) regulates the activity of phosphodiesterases, adenylyl cyclases, protein kinases (Ca 2+ /CaM-dependent kinase type II or CaM-kinase II), and phosphatases (calcineurin). Interestingly, the G 1 /S transition, the progression from G 2 to M, and the metaphase/anaphase transition are specific points of intervention of CaM-kinase II [13,16] . Activated CaM-kinase II promotes the phosphorylation of proteins such as Ca 2+ / cAMP response element-binding protein (CREB, a nuclear Ca 2+ -responsive transcription factor) and Ras (a cytosolic Ca 2+ -responsive transcription factor), which are required for initiating and maintaining the cell cycle [17] . ...
... Activated CaM-kinase II promotes the phosphorylation of proteins such as Ca 2+ / cAMP response element-binding protein (CREB, a nuclear Ca 2+ -responsive transcription factor) and Ras (a cytosolic Ca 2+ -responsive transcription factor), which are required for initiating and maintaining the cell cycle [17] . Additionally, CaM-kinase II and calcineurin-regulating nuclear factors such as cyclin-dependent kinases and cyclins are involved in the DNA division machinery [13,16] . ...
Extensive research is currently underway, seeking better diagnostic methods and treatments and a better understanding of the molecular mechanisms involved in cancer, from the role of specific genetic mutations to the intricate biochemical and molecular pathways involved. Because of their role in regulating relevant physiological events such as cell proliferation, migration, and invasion, ion channels have recently been recognized as important elements in cancer initiation and progression. Moreover, it has been reported that pharmacological intervention in ion channel activity might provide protection against diverse types of cancer, and that ion channels could be used as targets to counteract tumor growth, prevent metastasis, and overcome the therapy resistance of tumor cells. In this context, Ca2+ channels have been found to play a role in tumorigenesis and tumor progression. Specifically, L-type Ca2+ channel inhibition may affect cell proliferation, differentiation, and apoptosis. This review aims to provide insights into the potential role of these channels in cancer cell lines, emphasizing their participation in cell proliferation, migration, and autophagy induction, as well as their potential as rational targets for new cancer therapeutics.
... Therefore, we will go to illustrate the cell cycle regulatory pathway by calcium ions as shown in (Fig. 1). First, the calcium sensor calmodulin (CaM) is essential for driving cell cycle [8,9]; however, it is scarcely mentioned in literature of the cell cycle events checkpoints. Then, the impact of those calcium ions on DNA replication, mitosis, and cytokinesis will be mentioned. ...
... The Cdk1 cyclin-driven breakdown of the nuclear membrane and all that goes with it are triggered by the strictly localized release of calcium from thousands of endoplasmic reticular vesicles crowding around the nucleus [8,38]. Preventing the calcium surge prevents prophase, while the calcium-mobilizing caffeine or microinjected Ins(1,4,5)P3 triggers premature prophase [38,39]. ...
... The calcium barrage may trigger nuclear envelope breakdown at least in part by directly stimulating the protease calpain [8,20]. In fact, inhibiting calpain blocks the G2/M transition at least in some cells [41]. ...
The story of the cell commonder, calcium, reaches into all corners of the cell and controls cell proliferation, differentiation, function, and even death. The calcium-driven eukaryotic revolution is one of the great turning points in the life history, happened about two billion years later when it was converted from a dangerous killer that had to be kept out of cell into the cell master which drives the cell. This review article will take the readers to a tour of tissues chosen to best show the calcium’s many faces (proliferator, differentiator, and killer). The reader will first see calcium and its many helpers, such as the calcium-binding signaler protein calmodulin, directing the key events of the cell cycle. Then the tour will move onto the colon to show calcium driving the proliferation of progenitor cells, then the differentiation and ultimately the programmed death of their progeny. Moreover, the reader will learn of the striking disabling and bypassing of calcium-dependent control mechanisms during carcinogenesis. Finally, recommendations should be taken from the underlying mechanisms through which calcium masters the presistance, progression, and even apoptosis of colorectal cancer cells. Thus, this could be of great interest for designing of chemoprevention protocols.
... Ca 2+ signal has a critical role in cell cycle progression during the different phases (G1, S and G2 / M) (Santella 1998); where progression in the cell cycle is mainly regulated by calcium/calmodulin pathways (Kahl and Means 2003;Choi and Husain 2006). It has been demonstrated that during each phase of cell cycle, the levels of free intracellular calcium were changed (Pande, Kumar et al. 1996). ...
... Le signal calcique joue un rôle critique dans la progression du cycle cellulaire au cours des différentes phases (G1, S et G2 / M) (Santella 1998), la progression du cycle cellulaire est principalement régulée par les voies calcium / calmoduline (Kahl and Means 2003;Choi and Husain 2006). De plus, il a été démontré que la SOCE était nécessaire pour la transition G1 / S via la SOCE médiée par Stim1-Orai1 ). ...
Alteration in glycosylation pattern is one of the hallmarks of breast cancer. The levels and the abnormal expressions of glycan were found in breast cancer patients. Glycosylation defect can affect different glycosylated proteins which are implicated in cancerogenesis. Changes in intracellular Ca2+ levels can regulate different cellular processes. SOC channels are implicated in breast cancer proliferation, migration and survival. CO-OCS is a new glycosylation inhibitor with more selectivity toward theα- glucosidases exhibited anti-cancer activities in breast cancer cells without affecting the normal mammary cells. The objective of my thesis is investigating the related molecular mechanisms by which CO-OCS induced its anti-tumour effects.CO-OCS impaired breast cancer migration through decrease β1-integrin expression and the activation of FAK and ERK1/2 signalling pathways. CO-OCS also induced anti-migratory effect via Stim1 protein expression down-regulation leading to inhibition of SOCE. Additionally, CO-OCS affected the expression of both Orai1 and Stim1 proteins leading to anti-proliferative effects and cell cycle arrest in G1 and G2/M phase respectively. Moreover, CO-OCS affected the expression of Stim1 at the protein level without affecting its transcript level. GRP78 implicated in CO-OCS apoptotic death. The expression of Stim1 regulated the apoptosis induced by CO-OCS via modulating GRP78 expression. Orai1 down-regulation promoted CO-OCS necrotic effect. CO-OCS induced ER- calcium depletion due to increase in ER calcium leak via the Translocon; Anisomycin (Translocon inhibitor) decreased the apoptosis induced by CO-OCS. In conclusion, these results show that in breast cancer, by targeting Stim1, Orai1 and GRP78, CO-OCS reduced cell proliferation and induced apoptosis and necrosis cell death. Stim1 favours CO-OCS apoptotic effect while Orai1 protected from necrosis induced by CO-OCS. The inhibition of Translocon decreased CO-OCS apoptotic cell death via restoring the ER calcium homeostasis
... They are not correlated between cells, with exception of occasionally synchronous pairs of cells, and are mostly probably generated in proliferating radial glial cells (Weissman et al., 2004). These uncorrelated Ca 2+ transients in the proliferative VZ may at least partially resemble the Ca 2+ signals that have been observed during mitosis in several cell types (Berridge, 1995;Santella, 1998). In contrast, Ca 2+ transients in the CP are synchronized between neurons, are suppressed after inhibition of neuronal activity with the Na + channel blocker tetrodotoxin (TTX), and rely on voltage-gated Ca 2+ channels (Corlew et al., 2004). ...
... While these previous observations only indicate an influence of interstitial neurotransmitter on neurogenesis, without identifying the source of these neurotransmitters and the mechanisms of their action, more recent results indicate that neuronal activity is directly involved in the regulation of neurogenesis. Ca 2+ transients occur during mitosis in several cell types and probably directly influence proliferation (Berridge, 1995;Santella, 1998), suggesting that neuronal activity and/or non-synaptic GABA/glutamate release mediate their effect on neurogenesis by interference with these Ca 2+ transients. In the VZ of mice, the frequency and spatial properties of Ca 2+ waves are directly correlated to the amount of neurogenesis (Weissman et al., 2004), suggesting that such activity transients may modulate the rate of neurogenesis. ...
Animal and human studies revealed that patterned neuronal activity is an inherent feature of developing nervous systems. This review summarizes our current knowledge about the mechanisms generating early electrical activity patterns and their impact on structural and functional development of the cerebral cortex. All neocortical areas display distinct spontaneous and sensory-driven neuronal activity patterns already at early phases of development. At embryonic stages, intermittent spontaneous activity is synchronized within small neuronal networks, becoming more complex with further development. This transition is accompanied by a gradual shift from electrical to chemical synaptic transmission, with a particular role of non-synaptic tonic currents before the onset of phasic synaptic activity. In this review article we first describe functional impacts of classical neurotransmitters (GABA, glutamate) and modulatory systems (e.g., acetylcholine, ACh) on early neuronal activities in the neocortex with special emphasis on electrical synapses, nonsynaptic and synaptic currents. Early neuronal activity influences probably all developmental processes and is crucial for the proper formation of neuronal circuits. In the second part of our review, we illustrate how specific activity patterns might interfere with distinct neurodevelopmental processes like proliferation, migration, axonal and dendritic sprouting, synapse formation and neurotransmitter specification. Finally, we present evidence that transient alterations in neuronal activity during restricted perinatal periods can lead to persistent changes in functional connectivity and therefore might underlie the manifestation of neurological and neuropsychiatric diseases.
... Additional modulation of the IP 3 -R (through kinases like Ca 2+ /calmodulin kinase (Ca 2+ -CaMK), PKC, cGMP-dependent protein kinase and cAMP-dependent protein kinase) and the fine-tuned function of cytosolic buffers allow the generation of frequency modulated Ca 2+ shifts of different amplitude, duration, intracellular location, and recovery time that specifically regulate the metabolic activation and the differential gene transcription after egg activation [124,129]. The generated Ca 2+ wave is intricately related to different processes such as: (i) β-NAD kinasemediated control of the redox state; (ii) Ca 2+ -mediated cortical granules exocytosis; (iii) pronuclear migration; (iv) activation of protein and DNA synthesis; (v) amino acid transport; and (vi) mitosis and further cleavage cycles that lead to the progression of larval development [76,[130][131][132][133][134][135][136]. The Ca 2+ -dependent activation of NHE accounts for the alkalinisation of the cytoplasm [80,120,121]. ...
... The pH i and Ca i 2+ increases are also detected before and during the cellular division and are essential for the resumption of the cell cycle mediated by an increase in the synthesis of cyclins and the phosphorylation of other key cell cycle control proteins [159]. The Ca 2+ and CaMKII are required for the assembly of the mitotic spindle, for the control of centrosome duplication/separation and for the activation of cell cycle kinases-cyclins complexes involved in the progression of meiosis to the final cytokinesis [129,131,160]. Slightly acidic pH i conditions have been seen to retard or inhibit cleavage [39]. ...
Ocean Acidification (OA) represents a major field of research and increased efforts are being made to elucidate its repercussions on biota. Species survival is ensured by successful reproduction, which may be threatened under detrimental environmental conditions, such as OA acting in synergy with other climate change related stressors. Achieving successful gametogenesis, fertilization, and the development of larvae into healthy juveniles and adults is crucial for the perpetuation of species and, thus, ecosystems’ functionality. The considerable vulnerability of the abovementioned developmental stages to the adverse conditions that future OA may impose has been shown in many species, including sea urchins which are commonly used due to the feasibility of their maintenance in captivity and the great amount of gametes that a mature adult is able to produce. In the present review, the latest knowledge about the impact of OA on various stages of the life cycle of sea urchins is summarized with remarks on the possible impact of other stressors. The cellular physiology of the gametes before, at fertilization and, at early development, is extensively described with a focus on the complex enzymatic machinery and the intracellular pH (pHi) and Ca2+ homeostasis for their vulnerability when facing adverse conditions such as acidification, temperature variations, or hypoxia.
... Further, the regulation of mineral metabolism postnatally relies upon the hormonal regulation of the expression of regulatory molecules such as calcium-transporting ATPases, calciumbinding proteins, sodium dependent phosphate transporters, and the transient receptor potential vanilloid (TRPV) family members [6][7][8][9][10][11][12]. Additionally, many studies have provided evidence for non-classical functions of phosphate, calcium, and vitamin D in multiple tissue types, with important roles in the regulation of many physiological processes including cellular metabolism, proliferation, and protein synthesis [3,7,[13][14][15][16][17]. Recent evidence suggests that many of the postnatal regulatory mechanisms for phosphate, calcium, and vitamin D signaling, metabolism, and transport are present at the ovine maternal-conceptus interface [3,[18][19][20][21]. ...
Background:
Recent evidence suggests important roles for progesterone (P4) and interferon tau in the regulation of calcium, phosphate, and vitamin D signaling in the uteri of pregnant sheep. However, the effects of P4 and estradiol (E2), with respect to the expression of their receptors PGR and ESR1, respectively, in uterine epithelia on mineral signaling during the estrous cycle has not been investigated. Estrous cycles of mature Suffolk ewes were synchronized, prostaglandin F2α was administered, and ewes were observed for estrus (designated as Day 0) in the presence of vasectomized rams. On Days 1, 9, or 14 of the estrous cycle, hysterectomies were performed.
Results:
25-hydroxyvitamin D was more abundant in plasma from ewes on Day 14 than Day 1 (P < 0.05). Expression of fibroblast growth factor receptor 2 (FGFR2), a disintegrin and metalloprotease 17 (ADAM17), and parathyroid hormone-related protein (PTHrP) mRNAs was greater in endometria on Day 9 compared to Days 1 and 14 (P < 0.01). Similarly, expression of transient receptor potential cation channel subfamily V member 6 (TRPV6) mRNA was greater in endometria on Day 9 than Day 1 (P < 0.05). ATPase plasma membrane Ca2+ transporting 4 (ATP2B4) and S100 calcium binding protein G (S100G) mRNA expression was greater in endometria on Day 14 than on Days 1 and 9 (P < 0.01). In contrast, endometrial expression of vitamin D receptor (VDR) mRNA was lower on Days 9 and 14 than Day 1 (P < 0.01). Expression of klotho (KL) (P < 0.05) and cytochrome P450 family 24 subfamily A member 1 (CYP24) (P < 0.01) mRNAs was lower on Day 14 than Days 1 and 9. ADAM17, FGF23, CYP2R1, CYP27B1, KL, and VDR proteins immunolocalized to the uterine myometrium, blood vessels, and uterine luminal (LE), superficial glandular (sGE), and glandular (GE) epithelia. S100A9 protein was weakly expressed in the uterine myometrium, LE, sGE, and GE. Immunoreactivity of CYP2R1 and KL proteins in uterine LE and sGE was less on Day 1 than on Days 9 and 14. In contrast, S100G protein was expressed exclusively by GE, and immunoreactive S100G protein was less on Day 9. S100A12 protein localized to stromal cells of the uterine stratum spongiosum and blood vessels, but not by uterine epithelial cells.
Conclusion:
Collectively, these results implicate E2, P4, and PGR in the regulation of phosphate, calcium, and vitamin D signaling in cyclic ewes.
... Therefore, the molecular assortment of the distinct STIM/Orai and InsP 3 R isoform, as well as cell-to-cell variability in their expression, subcellular distribution, or posttranslational regulation, could contribute to pattern a heterogenous array of Ca 2+ signatures in WI-38 adult lung fibroblast (Ishida et al., 2014;Guzmán-Silva et al., 2015;Bartok et al., 2019;Wilson et al., 2020). Conversely, cell cycle asynchrony is an unlikely explanation of the cell-to-cell heterogeneity of histamine-evoked Ca 2+ waves because our experiments were performed in fibroblasts devoid of serum for 48 h, which causes cell cycle arrest in G0 phase (Santella, 1998). Similarly, previous studies in fibroblasts and other cell types have reported that this variability in the intracellular Ca 2+ dynamics is not due to cell cycle asynchrony (Ambler et al., 1988;Byron and Villereal, 1989;Dragoni et al., 2011;Guzmán-Silva et al., 2015;Okumura et al., 2022). ...
Histamine is an inflammatory mediator that can be released from mast cells to induce airway remodeling and cause persistent airflow limitation in asthma. In addition to stimulating airway smooth muscle cell constriction and hyperplasia, histamine promotes pulmonary remodeling by inducing fibroblast proliferation, contraction, and migration. It has long been known that histamine receptor 1 (H1R) mediates the effects of histamine on human pulmonary fibroblasts through an increase in intracellular Ca ²⁺ concentration ([Ca ²⁺ ] i ), but the underlying signaling mechanisms are still unknown. Herein, we exploited single-cell Ca ²⁺ imaging to assess the signal transduction pathways whereby histamine generates intracellular Ca ²⁺ signals in the human fetal lung fibroblast cell line, WI-38. WI-38 fibroblasts were loaded with the Ca ²⁺ -sensitive fluorophore, FURA-2/AM, and challenged with histamine in the absence and presence of specific pharmacological inhibitors to dissect the Ca ²⁺ release/entry pathways responsible for the onset of the Ca ²⁺ response. Histamine elicited complex intracellular Ca ²⁺ signatures in WI-38 fibroblasts throughout a concentration range spanning between 1 µM and 1 mM. In accord, the Ca ²⁺ response to histamine adopted four main temporal patterns, which were, respectively, termed peak, peak-oscillations, peak-plateau-oscillations, and peak-plateau. Histamine-evoked intracellular Ca ²⁺ signals were abolished by pyrilamine, which selectively blocks H1R, and significantly reduced by ranitidine, which selectively inhibits H2R. Conversely, the pharmacological blockade of H3R and H4R did not affect the complex increase in [Ca ²⁺ ] i evoked by histamine in WI-38 fibroblasts. In agreement with these findings, histamine-induced intracellular Ca ²⁺ signals were initiated by intracellular Ca ²⁺ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate (InsP 3 ) receptors (InsP 3 R) and sustained by store-operated Ca ²⁺ channels (SOCs). Conversely, L-type voltage-operated Ca ²⁺ channels did not support histamine-induced extracellular Ca ²⁺ entry. A preliminary transcriptomic analysis confirmed that WI-38 human lung fibroblasts express all the three InsP 3 R isoforms as well as STIM2 and Orai3, which represent the molecular components of SOCs. The pharmacological blockade of InsP 3 and SOC, therefore, could represent an alternative strategy to prevent the pernicious effects of histamine on lung fibroblasts in asthmatic patients.
... Phosphorous and calcium, two of the most abundant minerals in the body, have critical roles in the development and post-natal function of both the kidney and skeleton. Additionally, these mineral ions have important roles in the regulation of many physiological processes including cellular metabolism, proliferation, and protein synthesis [1][2][3][4][5][6][7]. ...
Minerals are required for the establishment and maintenance of pregnancy and regulation of fetal growth in mammals. Lentiviral-mediated RNA interference (RNAi) of chorionic somatomammotropin hormone (CSH) results in both an intrauterine growth restriction (IUGR) and a non-IUGR phenotype in sheep. This study determined the effects of CSH RNAi on the concentration and uptake of calcium, phosphate, and vitamin D, and the expression of candidate mRNAs known to mediate mineral signaling in caruncles (maternal component of placentome) and cotyledons (fetal component of placentome) on gestational day 132. CSH RNAi Non-IUGR pregnancies had a lower umbilical vein-umbilical artery calcium gradient (p < 0.05) and less cotyledonary calcium (p < 0.05) and phosphate (p < 0.05) compared to Control RNAi pregnancies. CSH RNAi IUGR pregnancies had less umbilical calcium uptake (p < 0.05), lower uterine arterial and venous concentrations of 25(OH)D (p < 0.05), and trends for lower umbilical 25(OH)D uptake (p = 0.059) compared to Control RNAi pregnancies. Furthermore, CSH RNAi IUGR pregnancies had decreased umbilical uptake of calcium (p < 0.05), less uterine venous 25(OH)D (vitamin D metabolite; p = 0.055), lower caruncular expression of SLC20A2 (sodium-dependent phosphate transporter; p < 0.05) mRNA, and lower cotyledonary expression of KL (klotho; p < 0.01), FGFR1 (fibroblast growth factor receptor 1; p < 0.05), FGFR2 (p < 0.05), and TRPV6 (transient receptor potential vanilloid member 6; p < 0.05) mRNAs compared to CSH RNAi Non-IUGR pregnancies. This study has provided novel insights into the regulatory role of CSH for calcium, phosphate, and vitamin D utilization in late gestation.
... Most of this work, but not all (Swierenga et al., 1976), employed cultured cell lines, presumably due to: 1) Ease of use and availability; 2) Technical barriers to culturing and manipulating primary cells and 3) Cultured cell lines being transformed, dividing rapidly, therefore well-suited to the study of mitosis. There are many excellent reviews that cover the work on mammalian cells, in detail, including those of Whitaker (Whitaker and Larman, 2001;Whitaker, 2006b), Hepler (Hepler, 1994), Silver (Silver, 1990;Silver, 1996) and Santella (Santella, 1998) therefore it will only be summarised here. Manipulation of calcium levels was shown to impact on mitosis (Izant, 1983) and calcium signals were observed and correlated, as for other model systems, with mitotic processes such as NEB and metaphase → anaphase transition (Poenie et al., 1986;Ratan et al., 1986). ...
The transformation of a single fertilised egg into an adult human consisting of tens of trillions of highly diverse cell types is a marvel of biology. The expansion is largely achieved by cell duplication through the process of mitosis. Mitosis is essential for normal growth, development, and tissue repair and is one of the most tightly regulated biological processes studied. This regulation is designed to ensure accurate segregation of chromosomes into each new daughter cell since errors in this process can lead to genetic imbalances, aneuploidy, that can lead to diseases including cancer. Understanding how mitosis operates and the molecular mechanisms that ensure its fidelity are therefore not only of significant intellectual value but provide unique insights into disease pathology. The purpose of this review is to revisit historical evidence that mitosis can be influenced by the ubiquitous second messenger calcium and to discuss this in the context of new findings revealing exciting new information about its role in cell division.
... Calcium and phosphorus are two of the most abundant minerals in the body, with approximately 99 and 85%, respectively, stored in the form of hydroxyapatite crystals in bone [1,2]. These minerals have essential roles in the skeletal and renal systems, and in the regulation of many processes including protein synthesis, cellular metabolism, and cellular proliferation [2][3][4][5][6][7][8]. ...
This study aimed to determine whether the acceleration of conceptus development induced by administration of exogenous progesterone (P4) during the pre-implantation period of pregnancy alters calcium, phosphate, and vitamin D signaling at the maternal-conceptus interface. Suffolk ewes (n = 48) were mated to fertile rams and received daily intramuscular injections of either corn oil vehicle (CO) or 25 mg progesterone in CO (P4) for the first 8 days of pregnancy and hysterectomized on either Day 9 (CO n = 5; P4 n = 6), 12 (CO n = 9; P4 n = 4) or 125 (CO n = 14; P4 n = 10) of gestation. Expression of S100A12 (P < 0.05) and FGFR2 (P < 0.01) mRNAs was lower in endometria from P4-treated ewes on Day 12. Expression of ADAM10 (P < 0.05) mRNA was greater in endometria from P4-treated ewes on Day 125. Expression of ADAM10 (P < 0.01), FGFR2 (P < 0.05), SLC20A1 (P < 0.05), TRPV5 (P < 0.05), and TRPV6 (P < 0.01) mRNAs was greater, but KL mRNA expression was lower (P < 0.05) in placentomes from P4-treated ewes at Day 125. There was lower endometrial and greater placentomal expression of mRNAs involved in mineral metabolism and transport in twin compared to singleton pregnancies. Further, expression of mRNAs involved in mineral metabolism and transport was greater in P4-treated twin placentomes. KL, FGF23, VDR, S100A9, S100A12, S100G, and CYP27B1 proteins were immunolocalized in endometria and placentomes. Exogenous P4 in early pregnancy altered expression of regulators of calcium, phosphate, and vitamin D on Day 125 of pregnancy indicating a novel effect of P4 on mineral transport at the maternal-conceptus interface.
... As a result, Ca 2+ signals must be tightly regulated during the cell cycle. Indeed, previous studies have characterized Ca 2+ transients during the cell cycle [8,9]. For example, Pande CaN dephosphorylates the NFAT transcription factor, which in turn activates p21, cyclin D1, CDK4, c-myc, and cyclin A. CaN also stabilizes cyclin D1 by dephosphorylation. ...
Calcineurin, a calcium-dependent serine/threonine phosphatase, integrates the alterations in intracellular calcium levels into downstream signaling pathways by regulating the phosphorylation states of several targets. Intracellular Ca2+ is essential for normal cellular physiology and cell cycle progression at certain critical stages of the cell cycle. Recently, it was reported that calcineurin is activated in a variety of cancers. Given that abnormalities in calcineurin signaling can lead to malignant growth and cancer, the calcineurin signaling pathway could be a potential target for cancer treatment. For example, NFAT, a typical substrate of calcineurin, activates the genes that promote cell proliferation. Furthermore, cyclin D1 and estrogen receptors are dephosphorylated and stabilized by calcineurin, leading to cell proliferation. In this review, we focus on the cell proliferative functions and regulatory mechanisms of calcineurin and summarize the various substrates of calcineurin. We also describe recent advances regarding dysregulation of the calcineurin activity in cancer cells. We hope that this review will provide new insights into the potential role of calcineurin in cancer development.
... In all eukaryotic cells, Ca 2+ is required in both the extracellular environment and intracellular stores for cell growth and division [38,39]. The transient increase in the concentration of free Ca 2+ in the cytosol and its spread to the nucleus lead to cell activation, which is involved in the binding of a broad range of stimuli including mitogenic factors and other transcription factors, and further initiates many signal transduction processes [40,41]. The transport systems that control the concentration of Ca 2+ in the nucleus are of great importance for the cell. ...
Alexandrium pacificum is a typical dinoflagellate that can cause harmful algal blooms, resulting in negative impacts on ecology and human health. The calcium (Ca2+) signal transduction pathway plays an important role in cell proliferation. Calmodulin (CaM) and CaM-related proteins are the main cellular Ca2+ sensors, and can act as an intermediate in the Ca2+ signal transduction pathway. In this study, the proteins that interacted with CaM of A. pacificum were screened by two-dimensional electrophoresis analysis and far western blots under different growth conditions including lag phase and high phosphorus and manganese induced log phase (HPM). The interactive proteins were then identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Four proteins were identified, including Ca2+/CaM-dependent protein kinase, serine/threonine kinase, annexin, and inositol-3-phosphate synthase, which all showed high expression levels under HPM. The gene expression levels encoding these four proteins were also up-regulated under HPM, as revealed by quantitative polymerase chain reaction, suggesting that the identified proteins participate in the Ca2+ transport channel and cell cycle regulation to promote cell division. A network of proteins interacting with CaM and their target proteins involved in the regulation of cell proliferation was raised, which provided new insights into the mechanisms behind the explosive growth of A. pacificum.
... Calcium is one of the most abundant minerals in the body, with approximately 99% of this calcium stored in bone in the form of hydroxyapatite (Ca 10 [PO 4 ] 6 [OH] 2 ) [1]. Calcium has an essential role in the skeletal and renal systems, and in the regulation of many processes including cellular proliferation, protein synthesis, and cellular metabolism [2][3][4][5][6][7]. Calcium transport, absorption, and homeostasis are regulated by several mechanisms. ...
Mineralization of the fetal mammalian skeleton requires a hypercalcemic gradient across the placenta from mother to fetus. However, the mechanisms responsible for maintaining the placental transport of calcium remain poorly understood. This study aimed to identify calcium and vitamin D regulatory pathway components in ovine endometria and placentae across gestation. Suffolk ewes were bred with fertile rams upon detection of estrus (Day 0). On Days 9, 12, 17, 30, 70, 90, 110, and 125 of pregnancy (n = 3–14/Day), ewes were euthanized and hysterectomized. Calcium abundance was influenced by gestational day in uterine flushings and allantoic fluid (P < 0.05). The expression of S100G, S100A9, S100A12, ATP2B3, ATP2B4, TRPV5, TRPV6, CYP11A1, CYP2R1, CYP24, and VDR mRNAs known to be involved in calcium binding, calcium transport, and vitamin D metabolism were quantified by qPCR. Mediators of calcium and vitamin D signaling were expressed by Day 17 conceptus tissue, and endometria and placentae across gestation. Gestational day influenced the expression of S100G, S100A9, S100A12, TRPV6, VDR, and CYP24 mRNAs in endometria and placentae (P < 0.05). Gestational day influenced endometrial expression of ATP2B3, and placental expression of TRPV5, ATP2B4, and CYP11A1 (P < 0.05). VDR protein localized to the endoderm and trophectoderm (Day 17 conceptus) and was expressed in endometria and placentae throughout gestation. The observed spatiotemporal profile suggests a potential role of calcium and vitamin D in the establishment of pregnancy and regulation of fetal and placental growth, providing a platform for further mechanistic investigation.
... In starfish, 1-MA exposure could trigger a transient Ca 2+ increase in the cytoplasm of immature oocytes and from the cell extract enriched with plasma membrane [74]. Nonetheless, the failure to detect 1-MA-induced Ca 2+ changes in several species of starfish has led to the conclusion that GVBD and the continuation of maturation (meiotic) cycle were not Ca 2+ -dependent [75,76]. However, results from our laboratory have shown that, in addition to a cytoplasmic Ca 2+ transient, 1-MA induces a nuclear Ca 2+ increase which was essential for the continuation of meiosis. ...
Much of the scientific knowledge on oocyte maturation, fertilization, and embryonic development has come from the experiments using gametes of marine organisms that reproduce by external fertilization. In particular, echinoderm eggs have enabled the study of structural and biochemical changes related to meiotic maturation and fertilization owing to the abundant availability of large and transparent oocytes and eggs. Thus, in vitro studies of oocyte maturation and sperm-induced egg activation in starfish are carried out under experimental conditions that resemble those occurring in nature. During the maturation process, immature oocytes of starfish are released from the prophase of the first meiotic division, and acquire the competence to be fertilized through a highly programmed sequence of morphological and physiological changes at the oocyte surface. In addition, the changes in the cortical and nuclear regions are essential for normal and monospermic fertilization. This review summarizes the current state of research on the cortical actin cytoskeleton in mediating structural and physiological changes during oocyte maturation and sperm and egg activation in starfish and sea urchin. The common denominator in these studies with echinoderms is that exquisite rearrangements of the egg cortical actin filaments play pivotal roles in gamete interactions, Ca2+ signaling, exocytosis of cortical granules, and control of monospermic fertilization. In this review, we also compare findings from studies using invertebrate eggs with what is known about the contributions made by the actin cytoskeleton in mammalian eggs. Since the cortical actin cytoskeleton affects microvillar morphology, movement, and positioning of organelles and vesicles, and the topography of the egg surface, these changes have impacts on the fertilization process, as has been suggested by recent morphological studies on starfish oocytes and eggs using scanning electron microscopy. Drawing the parallelism between vitelline layer of echinoderm eggs and the zona pellucida of mammalian eggs, we also discuss the importance of the egg surface in mediating monospermic fertilization.
Graphical abstract:
... The Ca 2+ dependence of KCa3.1 activation directly links this channel to an important second messenger and various Ca 2+ effector proteins regulating proliferation [63,64]. Ca 2+ oscillations occur during G1 phase, G1/S and G2/M transitions as well as between metaphase and anaphase [64][65][66][67]. Cell cycle progression depends on regulated Ca 2+ entry. ...
Several tumor entities have been reported to overexpress KCa3.1 potassium channels due to epigenetic, transcriptional, or post-translational modifications. By modulating membrane potential, cell volume, or Ca2+ signaling, KCa3.1 has been proposed to exert pivotal oncogenic functions in tumorigenesis, malignant progression, metastasis, and therapy resistance. Moreover, KCa3.1 is expressed by tumor-promoting stroma cells such as fibroblasts and the tumor vasculature suggesting a role of KCa3.1 in the adaptation of the tumor microenvironment. Combined, this features KCa3.1 as a candidate target for innovative anti-cancer therapy. However, immune cells also express KCa3.1 thereby contributing to T cell activation. Thus, any strategy targeting KCa3.1 in anti-cancer therapy may also modulate anti-tumor immune activity and/or immunosuppression. The present review article highlights the potential of KCa3.1 as an anti-tumor target providing an overview of the current knowledge on its function in tumor pathogenesis with emphasis on vasculo- and angiogenesis as well as anti-cancer immune responses.
... Furthermore, K + channel activity exerts an enormous function on signal pathways, among cell proliferation, differentiation and fusion [26,27]. It is necessary to promote calcium entry that increased K + channel activity and enhanced potassium efflux maintenance membrane hyperpolarization [28]. Besides, additional pathways for potassium channels such as to control the cell volume, are thought to involve in cell proliferation for which the membrane hyperpolarization is an essential requirement [29,30]. ...
Background:
Transcranial ultrasonic stimulation is a novel noninvasive tool for neuromodulation, and has high spatial resolution and deep penetration. Although it can increase excitation of neurons, its effects on neuron are poorly understood. This study was to evaluate effect of ultrasonic stimulation (US) on neurons in vitro. In this paper, the effect of US on the excitability and voltage-dependent [Formula: see text] currents of CA1 pyramidal neurons in the rat hippocampus was studied using patch clamp.
Results:
Our results suggest that US increased the spontaneous firing rate and inhibited transient outward potassium current ([Formula: see text]) and delayed rectifier potassium current ([Formula: see text]. Furthermore, US altered the activation of [Formula: see text] channels, inactivation and recovery properties of [Formula: see text] channels. After US, the [Formula: see text] activation curves significantly moved to the negative voltage direction and increased its slope factor. Moreover, the data showed that US moved the inactivation curve of [Formula: see text] to the negative voltage and increased the slope factor. Besides, US delayed the recovery of [Formula: see text] channel.
Conclusions:
Our data indicate that US can increase excitation of neurons by inhibiting potassium currents. Different US decreased the voltage sensitivity of [Formula: see text] activation differentially. Moreover, the more time is needed for US to make the [Formula: see text] channels open again after inactivating. US may play a physiological role by inhibiting voltage-dependent potassium currents in neuromodulation. Our research can provide a theoretical basis for the future clinical application of ultrasound in neuromodulation.
... These trials showed that patients who received a CDK4/6 inhibitor in addition to letrozole or fulvestrant had progression-free survival durations that were at least twice those of patients who received letrozole or fulvestrant alone (130)(131)(132). The FDA has now approved all three CDK4/6 inhibitors for the treatment of postmenopausal women with HR-positive, HER2-negative metastatic breast cancer (133)(134)(135)(136). Despite these promising clinical advances, a major limitation in the use of CDK4/6 inhibitors is the lack of reliable biomarkers to identify patients with intrinsic and/or acquired resistance to these agents. ...
Cyclin E, a regulatory subunit of cyclin-dependent kinase 2 (CDK2), is central to the initiation of DNA replication at the G1/S checkpoint. Tight temporal control of cyclin E is essential to the coordination of cell-cycle processes and the maintenance of genome integrity. Overexpression of cyclin E in human tumors was first observed in the 1990s and led to the identification of oncogenic roles for deregulated cyclin E in experimental models. A decade later, low-molecular-weight cyclin E (LMW-E) isoforms were observed in aggressive tumor subtypes. Compared with full-length cyclin E, LMW-E hyperactivates CDK2 through increased complex stability and resistance to the endogenous inhibitors p21CIP1 and p27KIP1. LMW-E is predominantly generated by neutrophil elastase–mediated proteolytic cleavage, which eliminates the N-terminal cyclin E nuclear localization signal and promotes cyclin E's accumulation in the cytoplasm. Compared with full-length cyclin E, the aberrant localization and unique stereochemistry of LMW-E dramatically alters the substrate specificity and selectivity of CDK2, increasing tumorigenicity in experimental models. Cytoplasmic LMW-E, which can be assessed by IHC, is prognostic of poor survival and predicts resistance to standard therapies in patients with cancer. These patients may benefit from therapeutic modalities targeting the altered biochemistry of LMW-E or its associated vulnerabilities. Cancer Res; 1–11. ©2018 AACR.
... Introduction Ca 2+ and Ca 2+ -binding proteins (CaBPs) such as Calmodulin (CaM), Centrins and Annexins have been implicated in cell cycle regulation and progression in many eukaryotes [1,2]. The in-vivo and in-vitro cell culture studies have revealed spikes in the levels of Ca 2+ at the G1 and G1/S boundaries [3,4]. The nuclear localized CaM plays the role of a major signal-transducing factor during the cell cycle [2,5]. ...
Author summary
E. histolytica, the etiological agent of amoebiasis, is a protozoan parasite responsible for around 100,000 deaths per year in developing nations. Though the organism has been identified more than 100 years back, there is not much understanding about the biology of this organism. Calcium signaling plays an important role in the biology of this organism. Here we show structure-functional relationship of one of the Ca²⁺-binding proteins (abbreviated as EhCaBP6) and suggest its involvement in cell division in this parasite. EhCaBP6, a nucleo-cytosolic Ca²⁺-binding protein, is a microtubule end binding protein and overexpression of its gene induces an increase in number of microtubular assemblies in E. histolytica. Cell division cycle in E. histolytica occurs along the microtubular structures without disruption of nuclear envelope. Occurrence of multinucleated cells in culture suggests duplication and reduplication of nuclear DNA without cytokinesis. Although Kinesin like protein (Klp1), Formin1 and EhCaBP6 were shown to be part of the microtubular assembly, their role in regulation of the cell cycle is not yet documented. Further, E. histolytica does not have a typical CaM like protein. However, the 3D structure of EhCaBP6 with two Ca²⁺-binding sites is similar to CaM, in spite of their low sequence similarity. Here, we demonstrate that EhCaBP6 regulates cell cycle specifically by facilitating DNA synthesis, transition from G1 to S phase and cytokinesis. The structural and functional similarity between EhCaBP6 and CaM suggests EhCaBP6 to be a functional homologue of nuclear CaM with important roles in regulation of cell cycle.
... However, the above data was recorded in non-synchronized cells and, therefore, we cannot rule out that oscillations that could be recorded at some specific point during cell cycle. For instances, Ca 2+ transients have been observed in late G 1 prior to the initiation, in G 2 before entry into the M phase, during mitosis between metaphase and anaphase, and during cytokinesis [62] and just before nuclear envelope breakdown (NEBD) in sea urchin eggs [63]. However, it still remains unclear whether these Ca 2+ transients are dependent on external calcium, hence on calcium influx. ...
... Increased intracellular calcium is implicated in the regulation of cell cycle specifically via the activity of calcium binding calmodulinkinase II, which influences S and G2/M progression. Interestingly, our data show that CD147 depleted cells are arrested in S/G2 phase [33]. Taken together, the inhibition of CD147 interactions leads to alterations in calcium homeostasis and global changes to the metabolome and proteome that synergistically affect vital cellular processes such cell cycle arrest and consequent cell growth. ...
Increased expression of CD147 in pancreatic cancer has been proposed to play a critical role in cancer progression via CD147 chaperone function for lactate monocarboxylate transporters (MCTs). Here, we show for the first time that CD147 interacts with membrane transporters beyond MCTs and exhibits a protective role for several of its interacting partners. CD147 prevents its interacting partner's proteasome-dependent degradation and incorrect plasma membrane localization through the CD147 transmembrane (TM) region. The interactions with transmembrane small molecule and ion transporters identified here indicate a central role of CD147 in pancreatic cancer metabolic reprogramming, particularly with respect to amino acid anabolism and calcium signaling. Importantly, CD147 genetic ablation prevents pancreatic cancer cell proliferation and tumor growth in vitro and in vivo in conjunction with metabolic rewiring towards amino acid anabolism, thus paving the way for future combined pharmacological treatments.
... Furthermore, we identified that activation of IP 3 R1 by TFP can be critical for the cell viability of GBL cells (Fig. 4B). TFPinduced Ca 2þ increase is also associated with CaM, which is involved in modulation of Ca 2þ signaling through interaction with Ca 2þ (28,29). We found that CaM2 is the major type among three types of CaM and is overexpressed in GBL cell lines compared with NSCs (Fig. 4C). ...
Calcium (Ca2+) signaling is an important signaling process, implicated in cancer cell proliferation and motility of the deadly glioblastomas that aggressively invade neighboring brain tissue. We have previously demonstrated that caffeine blocks glioblastoma invasion and extends survival by inhibiting Ca2+ release channel inositol 1,4,5-trisphosphate receptor (IP3R) subtype 3. Trifluoperazine (TFP) is an FDA-approved antipsychotic drug for schizophrenia. Interestingly, TFP has been recently reported to show a strong anticancer effect on lung cancer, hepatocellular carcinoma, and T-cell lymphoma. However, the possible anticancer effect of TFP on glioblastoma has not been tested. Here, we report that TFP potently suppresses proliferation, motility, and invasion of glioblas-toma cells in vitro, and tumor growth in in vivo xenograft mouse model. Unlike caffeine, TFP triggers massive and irreversible release of Ca2+ from intracellular stores by IP3R subtype 1 and 2 by directly interacting at the TFP-binding site of a Ca2+-binding protein, calmodulin subtype 2 (CaM2). TFP binding to CaM2 causes a dissociation of CaM2 from IP3R and subsequent opening of IP3R. Compared with the control neural stem cells, various glioblastoma cell lines showed enhanced expression of CaM2 and thus enhanced sensitivity to TFP. On the basis of these findings, we propose TFP as a potential therapeutic drug for glioblastoma by aberrantly and irreversibly increasing Ca2+ in glioblastoma cells. (C) 2016 AACR.
... Ca 2? functions in the transmission of internal and external information in cells as a second messenger, in biosynthesis and the release of neurotransmitters, and in metabolism and [31,32]. When the dynamic balance of Ca 2? is destroyed in the endoplasmic reticulum, Caspase12 can be directly activated. ...
The objective of our study was to assess the radioprotective effect of flavonoids extracted from Rosa roxburghii Tratt (FRT) and investigate the role of Bcl-2(Ca2+)/Caspase-3/PARP-1 pathway in radiation-induced apoptosis. Cells and mice were exposed to 60Co γ-rays at a dose of 6 Gy. The radiation treatment induced significant effects on tissue pathological changes, apoptosis, Ca2+, ROS, DNA damage, and expression levels of Bcl-2, Caspase-3 (C-Caspase-3), and PARP-1. The results showed that FRT acted as an antioxidant, reduced DNA damage, corrected the pathological changes of the tissue induced by radiation, promoted the formation of spleen nodules, resisted sperm aberration, and protected the thymus. FRT significantly reduced cell apoptosis compared with the irradiation group. The expression of Ca2+ and C-Caspase-3 was decreased after FRT treatment compared with the radiation-treated group. At the same time, expression of prototype PARP-1 and Bcl-2 increased, leading to a decrease in the percentage of apoptosis cells in FRT treatment groups. We conclude that FRT acts as a radioprotector. Apoptosis signals were activated via the Bcl-2(Ca2+)/Caspase-3/PARP-1 pathway in irradiated cells and FRT inhibited this pathway of apoptosis by down-regulation of C-Caspase-3 and Ca2+ and up-regulation of prototype PARP-1 and Bcl-2.
... Кроме того, кальций-зависимая фосфолипаза А 2 может влиять на синтез икосаэндров, которые играют важную роль в воспалительных реакциях и дисфункции клеток хозяина [29]. Плейотропные эффекты фосфолипазы А 2 также включают стимуляцию гладкой мускулатуры и эндотелиальных клеток [27,56]. Однако, несмотря на значительные успехи, достигнутые в понимании патогенеза парвовирус В19-обусловленного миокардита, этот процесс требует дальнейшего изучения с целью разработки новых подходов к диагностике и методов терапевтического воздействия. ...
The problem of inflammatory cardiomyopathy is discussed. The etiology, pathogenesis, diagnosis and treatment of inflammatory cardiomyopathy are considered with focus on the role of parvovirus B19.
... Calcium signaling regulates many fundamental steps of the cell cycle, including the transitions between the various phases of the cell cycle, the transcription of immediate early genes, and the regulating events that control quiescence and cell division [123][124][125]. In the brain, the niche of proliferating cells comprise primarily three cell types: type B cells (most stem-like), type C cells (transient amplifying cells), and type A cells (migrating neuroblasts) (Fig. 2). ...
... However, the above data was recorded in non-synchronized cells and, therefore, we cannot rule out that oscillations that could be recorded at some specific point during cell cycle. For instances, Ca 2+ transients have been observed in late G 1 prior to the initiation, in G 2 before entry into the M phase, during mitosis between metaphase and anaphase, and during cytokinesis (Santella 1998) and just before nuclear envelope breakdown (NEBD) in sea urchin eggs (Steinhardt and Alderton 1988). However, it still remains unclear whether these Ca 2+ transients are dependent on external calcium, hence on calcium influx. ...
Increases in both the basal cytosolic calcium concentration and cytosolic calcium transients play a major role in cell cycle progression, cell proliferation, and cell division. Calcium influx and calcium release from the endoplasmic reticulum are the major routes involved in the variations in cytosolic calcium concentration, and past studies have clearly shown that calcium influx controls cell growth and proliferation in several cell types. Furthermore, various studies in the past 30 years have demonstrated that cell-specific calcium channel expression levels are determinant in these physiological processes. Cell proliferation is directly linked to cell cycle progression, and it rapidly became evident that calcium channel expression interferes in this physiological process. It is also clear that the relationship between calcium influx and cell proliferation can be uncoupled in transformed and cancer cells, resulting in an external calcium-independent proliferation. Other divalent cations such as iron and zinc involved in cell proliferation permeating some calcium channels may interfere in this cellular process. Finally, we make the assumption that protein expression could be more important rather than channel function to trigger cell proliferation and that additional channel functions may be discovered soon.
... However, oocytes removed from the follicle with or without their surrounding cumulus cells resume meiosis spontaneously in the absence of gonadotropic stimulation (Edwards, 1965). However, Ca 2+ has been found to be involved in germinal vesicle breakdown (GVBD) of both spontaneous oocyte maturation and in vivo gonadotropin induced maturation (Batta and Knudsen, 1980;Bae, 1981;De Felici and Siracusa, 1982;Bae and Channing 1985;Santella, 1998). ...
The immunocytochemical method was used to identify the existence of voltage‐dependent Ca2+‐channels in mouse follicular oocytes. Three types of voltage‐dependent Ca2+‐channels were shown to exist in the follicular oocytes for the first time, the P/Q‐type Ca2+‐channel, the N‐type Ca2+‐channel, and the L‐type Ca2+‐channel. Among proven Ca2+ ‐channels, distributions of the P/Q‐type Ca2+‐channel and L‐type Ca2+ ‐channel showed localized staining (clustered pattern) on the oolemma. The distribution of the P/Q‐type Ca2+ ‐channel showed all localized staining, and the range of localized staining was from 1 to 8 in staining intensity. As the staining intensity increased from 1 to 8, the number of localized staining decreased. The L‐type Ca2+‐channel are homogeneously stained (29.4%‐54.2%), while some of them (around 28.7%‐44.1%) showed localized staining on the oolemma. However, the rest of them showed no staining at all (17.1%‐ 26.5%). On the contrary, the N‐type Ca2+‐channel showed mostly homogeneous staining, while non‐staining oocytes were around 33.8%. The rest showed localized staining (10%). However, staining intensity was much weaker than those of the P/Q‐type and L‐type Ca2+ ‐channel. In fact, the N‐type Ca ‐channel has been known to exist only in neurons (from ectoderm origin), but it is unknown how the N‐type Ca2+ ‐channel exists in the follicular oocytes (from mesoderm origin). Further studies are needed to examine the expression of Ca2+‐channels during the developmental stages of the oocytes
... [18,19]). During the cell cycle, Ca 2+ transients have been characterized during G1 and mitosis [20]. Ca 2+ is required early in G1, as cells re-enter the cell cycle, to promote the activation of AP1 (FOS and JUN) transcription factors, c-AMP-responsive element binding (CREB) protein, and the nuclear factor of activated T-cell (NFAT). ...
The study of calcium channels in molecular mechanisms of cancer transformation is still a novel area of research. Several studies, mostly conducted on cancer cell lines, however support the idea that a diversity of plasma membrane channels participates in the remodeling of Ca2 + homeostasis, which regulates various cancer hallmarks such as uncontrolled multiplication and increase in migration and invasion abilities. However few is still understood concerning the intracellular signaling cascades mobilized by calcium influx participating to cancer cell behavior. This review intends to gather some of these pathways dependent on plasma membrane calcium channels and described in prostate, breast and lung cancer cell lines. In these cancer cell types, the calcium channels involved in calcium signaling pathways promoting cancer behaviors are mostly non-voltage activated calcium channels and belong to the TRP superfamily (TRPC, TPRPV and TRPM families) and the Orai family. TRP and Orai channels are part of many signaling cascades ongoing in some cases such as the activation of transmembrane receptors by extracellular ligand from the tumor environment. TRPV can sense changes in the physical and chemical environment of cancer cells and TRPM7 are stretch activated and sensitive to cholesterol. Changes in activation and or expression of plasma-membrane calcium channels affect calcium-dependent signaling processes relevant to tumorigenesis. The studies cited in this review suggest that an increase in plasma membrane calcium channel expression and/or activity sustain an elevated calcium entry (constitutive or under the control of extracellular signals) promoting higher cell proliferation and migration in most cases. A variety of non-voltage-operated calcium channels display change expression and/or activity in a same cancer type and cooperate to the same process relevant to cancer cell behavior, or can be involved in a different sequence of events during the tumorigenesis. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.
... ELF MF could affect the concentration of intracellular Ca 2 , and an increase in the level of this ion is linked with the changes in the release of neurotransmitters (Augustine 2001). Namely, the change in the Ca 2 ions concentration affects the metabolic processes (Santella 1998) and consequently the activity of neurosecretory neurons, resulting in an increased synthesis and release of the secretory vesicles (Karabakhtsian et al. 1994). Ca 2 ions are Mouritsen and Ritz 2005). ...
Abstract PURPOSE: External magnetic fields (MFs) interact with organisms at all levels, including the nervous system. Bioelectrical activity of antennal lobe neurons of adult Morimus funereus was analyzed under the influence of extremely low frequency MF (ELF MF, 50 Hz, 2 mT) of different characteristics (exposure duration and waveform).
Neuronal activity (background/neuronal population and those nearest to the recording electrode) in adult longhorn beetles was registered through several phases of exposure to the sine wave and square wave MFs for 5, 10 and 15 min.
The sine wave MF, regardless of the exposure duration, did not change the reversibility factor of antennal lobe neuronal activity in adult M. funereus. In contrast, reversibility factors of the nearest neurons were significantly changed after the exposure to square wave MF for 10 and 15 min.
M. funereus individuals are sensitive to both sine wave and square wave ELF MFs (50 Hz, 2 mT) of different duration, whereby their reactions depend on the characteristics of the applied MF and specificity of each individual.
Despite advances in its treatment, heart failure remains a major cause of morbidity and mortality, evidencing an urgent need for novel mechanism-based targets and strategies. Myocardial hypertrophy, caused by a wide variety of chronic stress stimuli, represents an independent risk factor for the development of heart failure, and its prevention constitutes a clinical objective. Recent studies performed in preclinical animal models support the contribution of the Ca2+-dependent cysteine proteases calpains in regulating the hypertrophic process and highlight the feasibility of their long-term inhibition as a pharmacological strategy. In this review, we discuss the existing evidence implicating calpains in the development of cardiac hypertrophy, as well as the latest advances in unraveling the underlying mechanisms. Finally, we provide an updated overview of calpain inhibitors that have been explored in preclinical models of cardiac hypertrophy and the progress made in developing new compounds that may serve for testing the efficacy of calpain inhibition in the treatment of pathological cardiac hypertrophy.
Jasmonic acid (JA) is a phytohormone that plays a central role in plant defense against necrotrophic pathogens. JA signaling stimulates the increase of cytosolic calcium ion (Ca2+) and implicates the activity of mitogen-activated protein kinases (MPKs). We previously characterized that Ca2+/calmodulin (CaM) activates MPKs by inhibiting a CaM-regulated dual-specificity protein phosphatase1 (DsPTP1) at the biochemical level. In this study, we reported that Ca2+/CaM-mediated DsPTP1 negatively regulates the resistance to necrotrophic pathogens through the inhibition of JA-responsive MPK6. To elucidate the physiological function of inhibiting DsPTP1 activity by Ca2+/CaM, we constructed transgenic plants overexpressing DsPTP1 wild type (DsPTP1WT OX) and CaM deregulated mutant (DsPTP1K166E OX). Interestingly, the MPK6 activity was significantly reduced in DsPTP1K166E OX plants in response to JA compared to DsPTP1WT OX plants. Moreover, transcript levels of JA-responsive gene PDF1.2 and VSP1 were also highly decreased in DsPTP1K166E OX plants compared to DsPTP1WT OX plants. Furthermore, DsPTP1K166E OX plants showed more susceptibility to necrotrophic pathogens than DsPTP1WT OX plants. Conclusively, these results suggest that Ca2+/CaM activates the JA-responsive MPKs by inhibiting DsPTP1 for the resistance to the necrotrophic pathogen.
Normal calcium and bone homeostasis in the adult is virtually fully explained by the interactions of several key regulatory hormones, including parathyroid hormone, 1,25 dihydroxy vitamin D3, fibroblast growth factor-23, calcitonin, and sex steroids (estradiol and testosterone). In utero, bone and mineral metabolism is regulated differently from the adult. During development, it is the placenta and not the fetal kidneys, intestines, or skeleton that is the primary source of minerals for the fetus. The placenta is able to meet the almost inexhaustible needs of the fetus for minerals by actively driving the transport of calcium and phosphorus from the maternal circulation to the growing fetus. These fundamentally important minerals are maintained in the fetal circulation at higher concentrations than those in maternal blood. Maintenance of these inordinately higher fetal levels is necessary for the developing skeleton to accrue sufficient minerals by term. Importantly, in livestock species, prenatal mineralization of the skeleton is crucial for the high levels of offspring activity soon after birth. Calcium is required for mineralization, as well as a plethora of other physiological functions. Placental calcium and phosphate transport are regulated by several mechanisms that are discussed in this review. It is clear that phosphate and calcium metabolism is intimately interrelated and, therefore, placental transport of these minerals cannot be considered in isolation.
Calcineurin is a eukaryotic Ca ²⁺ - and calmodulin-dependent serine/threonine protein phosphatase. It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, which contains an active site dinuclear metal center, and a tightly associated, myristoylated, Ca ²⁺ -binding subunit, calcineurin B. The primary sequence of both subunits and heterodimeric quaternary structure is highly conserved from yeast to mammals. As a serine/threonine protein phosphatase, calcineurin participates in a number of cellular processes and Ca ²⁺ -dependent signal transduction pathways. Calcineurin is potently inhibited by immunosuppressant drugs, cyclosporin A and FK506, in the presence of their respective cytoplasmic immunophilin proteins, cyclophilin and FK506-binding protein. Many studies have used these immunosuppressant drugs and/or modern genetic techniques to disrupt calcineurin in model organisms such as yeast, filamentous fungi, plants, vertebrates, and mammals to explore its biological function. Recent advances regarding calcineurin structure include the determination of its three-dimensional structure. In addition, biochemical and spectroscopic studies are beginning to unravel aspects of the mechanism of phosphate ester hydrolysis including the importance of the dinuclear metal ion cofactor and metal ion redox chemistry, studies which may lead to new calcineurin inhibitors. This review provides a comprehensive examination of the biological roles of calcineurin and reviews aspects related to its structure and catalytic mechanism.
The beautiful mitotic waves that characterize nuclear divisions in the early Drosophila embryo have been the subject of intense research to identify the elements that control mitosis. Calcium waves in phase with mitotic waves suggest that calcium signals control this synchronized pattern of nuclear divisions. However, protein targets that would translate these signals into mitotic control have not been described. Here we investigate the role of the calcium-dependent protease Calpain A in mitosis. We show that impaired Calpain A function results in loss of mitotic synchrony and ultimately halted embryonic development. The presence of defective microtubules and chromosomal architecture at the mitotic spindle during metaphase and anaphase and perturbed levels of Cyclin B indicate that Calpain A is required for the metaphase-to-anaphase transition. Our results suggest that Calpain A functions as part of a timing module in mitosis, at the interface between calcium signals and mitotic cycles of the Drosophila embryo.
Free calcium ions (Ca2+) are known in cell physiology to play an ubiquitous role as messengers. Very small and brief fluctuations of their intracellular concentration take part in the transmission of a specific message through the cell. In fact, it is the pulsatility of these fluctuations, designated as “calcium oscillations”, which imparts their messenger activity to the calcium ions. Nowadays, more and more teams working on different cellular models are interested in the possible existence of calcium signaling located inside the nucleus (called “germinal vesicle” or GV in the special case of the oocyte), as well as the existence and functionality of a nuclear phosphoinositide cycle.
Since the early studies on sea urchin egg activation [1] and on starfish oocytes [2], to the more recent discoveries of the Ca2+-mobilizing activities of cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) in sea urchin egg homogenates [3–6], echinoderm gametes have remained a widely investigated system in the area of egg activation. In addition to fertilization, and at variance with sea urchin, starfish oocytes have also provided an exceptional model to investigate the re-initiation of the meiotic cycle (e.g., maturation) due to their synchrony, transparency and ease of handling. Maturation, which is induced by the hormone 1-methyladenine, takes these oocytes from the germinal vesicle stage (4n chromosomes, first prophase stage of meiosis) where they remain arrested to the spawning period at which they can be fertilized. During meiosis, reinitiated oocytes undergo a number of structural and biochemical changes, which prepare them for successful fertilization. Thus, oocytes have been a useful tool in investigations of the intracellular mechanisms regulating the prophase/metaphase transition. They are also a unique source of highly purified cell cycle control elements e.g., purified M-phase promoting factor [7, 8].
Although the blockage of a cell cycle by specific inhibitors of Ca2+/calmodulin-dependent protein kinase II (CaMK-II) is well known, the activity profile of CaMK-II during the cell cycle in the absence of any direct effecters of the enzyme is unclear. The activity of native CaMK-II in NM 3T3 cells was examined by the use of cell cycle-specific arresting and synchronizing methods. The total catalytic activity of CaMK-II in arrested cells was decreased about 30% in the M phase, whereas the Ca2+-independent autonomous activity increased about 1.5-fold in the M phase and decreased about 50% at the G1/S transition. The in vivo phosphorylation level of CaMK-II was lowest at G1/S and highest in M. The CaMK-II protein level was unchanged during the cell cycle, When the cells were synchronized, the autonomous activity was increased only in M. These results indicate that the physiologically relevant portion of CaMK-II is activated only in M, and that the net activation of CaMK-II is required in mitosis.
Dormancy is the main obstacle for the commercial production of temperate fruits in warm-winter regions and the protected fruit production now developing rapidly in China. Hydrogen cyanamide and high temperature are effective in the breakage of dormancy in nectarine and other deciduous trees, however, the details of mechanism are still unknown. Enhancements in the levels of H2O2 and elimination of high cytosolic Ca2+ are important processes in the dormancy release mechanism. Here, we reported that both hydrogen cyanamide and high temperature induced the increase of H2O2 in dormant nectarine floral buds. Exogenous application of H2O2 caused a shift of Ca2+ influx towards an efflux in dormant nectarine floral primordia. The observation indicated the possible involvement of H2O2 induced cytosolic Ca2+ elimination in dormancy release mechanism and provided new visions on H2O2/Ca2+ cooperation. Further experiments showed that the H2O2 induced Ca2+ flux shift could be inhibited by the pretreatment of LaCl3 (inhibitor of Ca2+-permeable channels) but not erythrosin B (inhibitor of Ca2+-ATPase), suggesting the key role of Ca2+-permeable channels in the process. In addition, H2O2 application also influenced the transportation of H+ and Mg2+, which was associated with cell cycle regulations and could also be involved in dormancy release mechanism. No significant transporting changes of Na+ and K+ were detected following the application of H2O2.
Introduction In addition to intrinsic control mechanisms (see Chapter 5 and Cepko et al., 1996), the production of neurons by progenitor cells and the determination of their fate are regulated via an array of diffusible factors, two families of which are considered in this chapter: neurotransmitters and neurotrophins. Neurotrophins are now known to play an essential role in both the formation and the maintenance of the nervous system throughout development and adult life. There is growing evidence that besides their role as molecules mediating communication between nerve cells in the mature nervous system, a variety of both slow and fast neurotransmitters also play important roles during neuronal development. This chapter reviews recent evidence that demonstrates that a number of non-synaptic neurotransmitter release mechanisms, together with many neurotransmitters and their receptors, are present in the developing retina prior to the onset of synapse formation and that these early neurotransmitters act to modulate a range of events in neural development. Their precise mechanisms of action are still being elucidated but, as described here, the ability to modulate [Ca2+]i is one feature common to these early neurotransmitter systems, and is thought to underlie a number of their developmental actions. It is becoming clear that both neurotransmitters and neurotrophins play important regulatory roles in the early stages of retinal development, including the modulation of proliferation, differentiation, cell survival and circuit formation.
In order to explore regulatory function of H2O2 in bud dormancy release, main effects of three dormancy-breaking treatments (high temperature, hydrogen cyanamide and TDZ) on H2O2 metabolism were determined, and impacts of H2O2 on Ca2+ transport were tested using non-invasive micro-test technique. The results showed that both high temperature and hydrogen cyanamide induced H2O2 accumulation and CAT inhibition were efficient in breaking dormancy during deep dormancy period. However, TDZ showed little impacts on H2O2 metabolism and was much less effective in breaking dormancy. Dormant floral primordium was absorbing state to exogenous Ca2+ due to active calcium channels. The Ca2+ transport could be changed by exogenous H2O2. H2O2 of low concentration reduced the absorption rate of Ca2+, and at high concentration, it changed the Ca2+ transport direction from absorption to release. The results indicated that H2O2 signals were related with Ca2+ signals in dormant buds. Ca2+ signal regulated by H2O2 accumulation might be important in the dormancy-breaking signal transduction process induced by high temperature and hydrogen cyanamide.
Many inorganic elements are recognized as being essential for the growth of all living organisms. Transfer of nutrients and waste material from cells and tissues in the biological systems are accomplished through a functional vasculature network. Maintenance of the vascular system is vital to the wellbeing of organisms, and its alterations contribute to pathogenesis of many diseases. This article is the first part of a review on the functional role of inorganic elements including nitrogen, iron, selenium, phosphorus, gold, and calcium in angiogenesis. The methods of exposure, structure, mechanisms, and potential activity of these elements are briefly summarized. An electronic search was performed on the role of these elements in angiogenesis from January 2005 to April 2014. The recent aspects of the relationship between different elements and their role in angiogenesis, and production of pro- and anti-angiogenic factors were assessed. Several studies emphasized the role of these elements on the different phases of angiogenesis process in vivo. These elements can either enhance or inhibit angiogenesis events. Nitrogen in combination with bisphosphonates has antiangiogenic effects, while nitric oxide promotes the production of angiogenic growth factors. Iron deficiency can stimulate angiogenesis, but its excess suppresses angiogenesis events. Gold nanoparticles and selenium agents have therapeutic effects due to their anti-angiogenic characteristics, while phosphorus and calcium ions are regarded as pro-angiogenic elements. Understanding how these elements impact angiogenesis may provide new strategies for treatment of many diseases with neovascular component.
Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Three new plakortides, 7,8-dihydroplakortide E (1), 2, and 10, along with known natural products 3, 4, spongosoritin A (5), 6-8, and plakortide P (9), were isolated from Brazilian specimens of Plakortis angulospiculatus. Compounds 2, 3, 5, and 7-9 displayed cytotoxic activities with IC50 values ranging from 0.2 to 10 μM. Compounds that contained a dihydrofuran ring were generally less active and displayed time dependence in their activity. The activities of compounds 2 and 7-9, carboxylic acids bearing a common six-membered endoperoxide, were higher overall than for compounds 3 and 5. The modes underlying the cytotoxic actions of plakortides 2, 3, 5, 7, and 9 were further investigated using HCT-116 cells. While dihydrofurans 3 and 5 induce a G0/G1 arrest, six-membered peroxides 2, 7, and 9 delivered a G2/M arrest and an accumulation of mitotic figures, indicating a distinctly different antimitotic response. Confocal analysis indicated that microtubules were not altered after treatment with 2, 7, or 9, therein suggesting that the mitotic arrest may be unrelated to cytoskeletal targets. Overall, we find that two related classes of natural products obtained from the same extract offer cytostatic activity, yet they do so through discrete pathways.
CaMKs link transient increases in intracellular Ca2 + with biological processes. In myeloid leukemia cells, CaMKII, activated by the bcr-abl oncogene, promotes cell proliferation. Inhibition of CaMKII activity restricts cell proliferation, and correlates with growth arrest and differentiation. The mechanism by which the inhibition of CaMKII results in growth arrest and differentiation in myeloid leukemia cells is still unknown. We report that inhibition of CaMKII activity results in an upregulation of CaMKIV mRNA and protein in leukemia cell lines. Conversely, expression of CaMKIV inhibits autophosphorylation and activation of CaMKII, and elicits G0/G1cell cycle arrest, reducing of cell proliferation. Furthermore, U937 cells expressing CaMKIV show elevated levels of Cdk inhibitors p27kip1 and p16ink4a and reduced levels of cyclins A, B1 and D1.These findings were also confirmed in the K562 leukemic cell line.The relationship between CaMKII and CaMKIV was also observed in primary acute myeloid leukemia (AML) cells, that correlated with their immunophenotype profile. Indeed, immature MO/M1 AML showed increased CaMKIV expression and decreased pCaMKII, whereas highly differentiated M4/M5 AML showed decreased CaMKIV expression and increased pCaMKII.Our data reveal a novel cross-talk between CaMKII and CaMKIV and suggest that CaMKII suppresses the expression of CaMKIV to enable leukemia cell proliferation.
We investigate comparatively the damaging effects of EMP, S-HPM and X-HPM on rat Sertoli cells.Primary Sertoli cells were exposed to EMP, S-HPM and X-HPM respectively. Light microscope and electromicroscope were used to observe the pathological and ultrastructural changes. Flow cytometry was used to examine apoptosis of Sertoli cells. MTT test was performed to assess the general metabolic activity of Sertoli cells.Three bands of Electromagnetic radiation could lead to histological and ultrastructural changes， could induce apoptosis and decrease metabolic activity of Sertoli cells. The rank of injury effects are as follows: EMP> X-HPM>S-HPM. INTRODUCTION Electromagnetic radiation has threatened human health [1-3]. And frequency of electromagnetic radiation in environment is composite. Testis is one of the most hypersensitive target organs for electromagnetic radiation [4]. Sertoli cells play important roles on spermatogenesis. However, the damaging effects of electromagnetic radiation on Sertoli cells are poorly understood. In the current study, we investigate comparatively the damaging effects of EMP (electromagnetic pulse), S-HPM (S-band high power microwave) and X-HPM (X-band high power microwave) on rat Sertoli cells. Our data expand previous understanding and provide the theoretic and experimental evidence for proventing the damaging effects of electromagnetic radiations.
A 50-kDa protein, which binds to the growth-regulated gene (2A9) product, calcyclin in a calcium-dependent manner, was purified from bovine lung. Partial amino acid sequencing of the protein revealed it to be the bovine equivalent of rabbit lung CAP-50 (calcyclin-associated protein, 50 kDa), which is a member of the annexin family and binds to calcyclin in a calcium-dependent manner. Specific polyclonal antibodies to bovine lung CAP-50 were prepared. Comparative studies between CAP-50 and synexin (annexin VII) on the immunoreactivity against anti-CAP-50 antibodies and the ability of binding to calcyclin revealed that CAP-50 was a distinct molecule from synexin. Using specific polyclonal antibodies to bovine lung CAP-50, tissue distribution and subcellular distribution of CAP-50 were investigated. In most rat tissues, except those in the central nervous systems and kidney, CAP-50 is expressed at a high or moderate level. Both studies by subcellular fractionation and by indirect immunofluorescence staining of the rat embryonic fibroblast cell line, 3Y1, revealed that CAP-50 mainly localized in nuclei. Moreover, between the cells at interphase and at mitotic phase, different distributions of CAP-50 were observed. That is, in the cells at interphase, CAP-50 seemed to localize throughout the nucleoplasm. On the other hand, in the cells during mitosis, CAP-50 was concentrated at the loop-like structure around the mitotic apparatus. CAP-50 was found in isolated 3Y1 nuclei lacking outer nuclear membranes, and approximately 50% of CAP-50 was extracted from the nuclei by chelating calcium. Thus, CAP-50, a unique annexin, localizes in nuclei.
A chicken calmodulin (CaM) gene has been expressed in mouse C127 cells using a bovine papilloma virus (BPV)‐based vector (BPV‐CM). The vector‐borne genes produce a mature mRNA of the expected size that is present on cytoplasmic polyribosomes. In clonal cell lines transformed by BPV‐CM, expression of the CaM gene produced CaM levels 2‐ to 4‐fold above those observed in cells transformed by BPV alone. Increased intracellular CaM caused a reduction of cell cycle length that is solely due to a reduction in the length of the G1 phase. A comparison of six cell lines revealed a linear relationship between the intracellular CaM concentration and the rate of G1 progression. These data provide the first evidence that specific elevation of CaM levels directly affects the rate of cell proliferation.
In order to examine the consequences of a transient increase or decrease in intracellular calmodulin (CaM) levels, two bovine‐papilloma‐virus (BPV)‐based expression vectors capable of inducibly synthesizing CaM sense (BPV‐MCM) or anti‐sense (BPV‐CaMAS) RNA have been constructed and used to stably transform mouse C127 cells. Upon addition of Zn2+, cells containing the BPV‐MCM vector have transiently increased CaM mRNA and protein levels. Cells carrying the BPV‐CaMAS vector transiently produce CaM anti‐sense RNA resulting in a significant decrease in intracellular CaM concentration. Increased CaM caused a transient acceleration of proliferation, while the anti‐sense RNA induced decrease in CaM caused a transient cell cycle arrest. Flow cytometric analysis showed that progression through G1 and mitosis was affected by changes in CaM levels. These data indicate that CaM levels may limit the rate of cell‐cycle progression under normal conditions of growth.
During early development, intracellular Ca 2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca 2+ were examined during meiotic maturation and re-initiation at parthenogenetic activation and during first mitosis in a single species using the same methodolo-gies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca 2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca 2+ removal , but were blocked by intracellular Ca 2+ chela-tors. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca 2+ and showed no associated Ca ~+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca 2+ transient and was dependent on Ca 2 § mobilization. We conclude that GVBD is Ca 2+ independent, but that the dependence of NEBD on Ca 2+ suggests regulation by more than one pathway. As cells develop from Ca2+-independent ger-minal vesicle oocytes to internal Ca 2 § pro-nuclear eggs, internal Ca 2 § pools increase by approximately fourfold.
A more complete understanding of calcium's role in cell division requires knowledge of the timing, magnitude, and duration of changes in cytoplasmic-free calcium, [Ca2+]i, associated with specific mitotic events. To define the temporal relationship of changes in [Ca2+]i to cellular and chromosomal movements, we have measured [Ca2+]i every 6-7 s in single-dividing Pt K2 cells using fura-2 and microspectrophotometry, coupling each calcium measurement with a bright-field observation. In the 12 min before discernable chromosome some separation, 90% of metaphase cells show at least one transient of increased [Ca2+]i, 72% show their last transient within 5 min, and a peak of activity is seen at 3 min before chromosome separation. The mean [Ca2+]i of the metaphase transients is 148 +/- 31 nM (61 transients in 35 cells) with an average duration of 21 +/- 14 s. The timing of these increases makes it unlikely that these transient increases in [Ca2+]i are acting directly to trigger the start of anaphase. However, it is possible that a transient rise in calcium during late metaphase is part of a more complex progression to anaphase. In addition to these transient changes, a gradual increase in [Ca2+]i was observed starting in late anaphase. Within the 2 min surrounding cytokinesis onset, 82% of cells show a transient increase in [Ca2+]i to 171 +/- 48 nM (53 transients in 32 cells). The close temporal correlation of these changes with cleavage is consistent with a more direct role for calcium in this event, possibly by activating the contractile system. To assess the specificity of these changes to the mitotic cycle, we examined calcium changes in interphase cells. Two-thirds of interphase cells show no transient increases in calcium with a mean [Ca2+]i of 100 +/- 18 nM (n = 12). However, one-third demonstrate dramatic and repeated transient increases in [Ca2+]i. The mean peak [Ca2+]i of these transients is 389 +/- 70 nM with an average duration of 77 s. The necessity of any of these transient changes in calcium for the completion of mitotic or interphase activities remains under investigation.
Calcium-dependent regulator protein is a low molecular weight (17,000), thermostable, calcium binding protein which is structurally homologous to skeletal muscle troponin C. This protein is present in all nonmuscle cells and has been shown to decorate stress fibers in interphase cells by indirect immunofluorescence. Using this procedure we have investigated the distribution of the protein during mitosis of eukaryotic cells. As the cells enter prophase, the distinct cytoplasmic localization disappears commensurate with the dissolution of the cytoskeleton. The regulator protein seems to be randomly distributed throughout the prophase cell, including the region around the condensed chromosomes. However, at prometaphase, it is localized in association with the half-spindles of the mitotic apparatus. Through metaphase and most of anaphase, the protein remains localized between the chromosomes and the poles of the spindle. During late anaphase the protein is also found in the interzone region but rapidly condenses into two small regions, one on each side of the midbody that separates the daughter cells. The regulator protein is not localized in the cleavage furrow during telophase, whereas actin is demonstrable in this region. Indeed, placement of the protein during mitosis is distinct from both that of actin and that of tubulin. The localization of calcium-dependent regulator protein during mitosis suggests that it may mediate the calcium effects on the mitotic apparatus and thus play a role in chromosome movement.
Fertilization or ionophore activation of Lytechinus pictus eggs can be monitored after injection with the Ca-sensitive photoprotein aequorin to estimate calcium release during activation. We estimate the peak calcium transient to reach concentrations of 2.5-4.5 & free calcium 45-60 set after activation and to last 2-3 min, assuming equal Ca2+ release throughout the cytoplasm. Calcium is released from an intracellular store, since similar responses are obtained during fertilization at a wide range of external calcium concentrations or in zero- calcium seawater in ionophore activations. In another effort to estimate free calcium at fertilization, we isolated egg cortices, added back calcium quantitatively, and fixed for obser- vation with a scanning electron microscope. In this way, we determined that the threshold for discharge of the cortical granules is between 9 and 16 @f CaZ+. Therefore, the threshold for the in vitro cortical reaction is about five times the amount of free calcium, assuming equal distribution in the egg. This result suggests that transient calcium release is confined to the inner subsurface of the egg.
We have microinjected a mAb specifically directed to phosphatidylinositol 4,5-bisphosphate (PIP2) into one blastomere of two-cell stage Xenopus laevis embryos. This antibody binds to endogenous PIP2 and reduces its rate of hydrolysis by phospholipase C. Antibody-injected blastomeres undergo partial or complete arrest of the cell cycle whereas the uninjected sister blastomeres divided normally. Since PIP2 hydrolysis normally produces diacylglycerol (DG) and inositol 1,4,5-triphosphate (Ins[1,4,5]P3), we attempted to measure changes in the levels of DG following stimulation of PIP2 hydrolysis in antibody-injected oocytes. The total amount of DG in antibody-injected oocytes was significantly reduced compared to that of water-injected ones following stimulation by either acetylcholine or progesterone indicating that the antibody does indeed suppress PIP2 hydrolysis. We also found that the PIP2 antibodies greatly reduced the amount of intracellular Ca2+ released in the egg cortex during egg activation. As an indirect test for Ins(1,4,5)P3 involvement in the cell cycle we injected heparin which competes with Ins(1,4,5)P3 for binding to its receptor, and thus inhibits Ins(1,4,5)P3-induced Ca2+ release. Microinjection of heparin into one blastomere of the two-cell stage embryo caused partial or complete arrest of the cell cycle depending upon the concentration of heparin injected. We further investigated the effect of reducing any [Ca2+]i gradients by microinjecting dibromo-BAPTA into the blastomere. Dibromo-BAPTA injection completely blocked mitotic cell division when a final concentration of 1.5 mM was used. These results suggest that PIP2 turnover as well as second messenger activity influence cell cycle duration during embryonic cell division in frogs.
A transient increase in intracellular free calcium is believed to be the signal responsible for the stimulation of the egg metabolism at fertilization and the resumption of the cell cycle. We have studied how the polyphosphoinositides (PPI) turn over at fertilization in sea urchin eggs, in order to determine the relationship between the metabolism of these lipids and the calcium signal. We compare the patterns of PPI turnover that occur during the first minute following fertilization in eggs in which PPI are labelled to steady state with [3H]inositol or [3H]arachidonate with that in which PPI are labelled for a shorter period with [3H]inositol. When eggs are labelled to apparent isotopic equilibrium with either [3H]inositol or [3H]arachidonate, no early increase in [3H]PtdInsP2 occurs while PtdIns decreases slightly. On the contrary, when not labelled to isotopic equilibrium, all [3H]PPI increase during the first 15 seconds following fertilization. We find that, within seconds, fertilization triggers a 600-fold increase in the turnover of PPI, producing an amount of InsP3 apparently sufficient to trigger calcium release. We suggest that phosphoinositidase C and PtdInsP kinase, responsible respectively for the hydrolysis and synthesis of PtdInsP2, are both stimulated to a comparable degree in the first 30 seconds following fertilization and that net changes in the amount of PtdInsP2 at fertilization are very sensitive to the relative levels of activation of the two enzymes. Activating the eggs with the calcium ionophore A23187 showed that both these enzymes are sensitive to calcium, suggesting that calcium-dependent InsP3 production might play a role in the initiation and/or the propagation of the fertilization calcium wave.(ABSTRACT TRUNCATED AT 250 WORDS)
Calcium/calmodulin dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase. We have created a calcium/calmodulin independent form of this enzyme by truncation. Expression of this enzyme fragment in a rabbit reticulocyte lysate yields a constitutive enzyme with specific activity similar to the activated native enzyme. We have established mammalian cell lines that transiently express this constitutive enzyme using the glucocorticoid-inducible mouse mammary tumor virus long terminal repeat. The transient increase in kinase activity results in a complete cessation of cell cycle progression. This block develops as a consequence of a specific arrest of the cell cycle in G2. During the block, increases in histone H1 kinase activity present in p13 beads or anti-cdc2 immunoprecipitates are seen in parallel with the accumulation of cells at G2, arguing that the arrest is not due to a failure to activate cdc2 as a histone H1 kinase. These results suggest that other changes in serine/threonine protein phosphorylation besides those involved in activation of cdc2 as a histone H1 kinase may be necessary for proper G2-M transition.
We report here that the negative cell cycle regulator protein p53 is an in vivo and in vitro substrate for protein kinase C, a cellular receptor for the tumor-promoter phorbol esters. We also demonstrate that p53 interacts in a calcium-dependent manner with S100b, a member of the S100 protein family involved in cell cycle progression and cell differentiation, and that such an interaction inhibits in vitro p53 phosphorylation by protein kinase C. The interaction between p53 and S100b was utilized for the purification of cellular and recombinant murine p53 by affinity chromatography with S100b-Sepharose. Furthermore, and of particular interest, we have shown that purified p53 undergoes temperature-dependent oligomerization and that the interaction between S100b and p53 not only induces total inhibition of p53 oligomerization but also promotes disassembly of the p53 oligomers. We suggest that these effects result from the binding of S100b to the multifunctional basic C-terminal domain of p53 and propose that p53 may be a cellular target for the S100 protein family members involved in the control of the cell cycle at the G0-G1/S boundary.
During early development, intracellular Ca2+ mobilization is not only essential for fertilization, but has also been implicated during other meiotic and mitotic events, such as germinal vesicle breakdown (GVBD) and nuclear envelope breakdown (NEBD). In this study, the roles of intracellular and extracellular Ca2+ were examined during meiotic maturation and reinitiation at parthenogenetic activation and during first mitosis in a single species using the same methodologies. Cumulus-free metaphase II mouse oocytes immediately resumed anaphase upon the induction of a large, transient Ca2+ elevation. This resumption of meiosis and associated events, such as cortical granule discharge, were not sensitive to extracellular Ca2+ removal, but were blocked by intracellular Ca2+ chelators. In contrast, meiosis I was dependent on external Ca2+; in its absence, the formation and function of the first meiotic spindle was delayed, the first polar body did not form and an interphase-like state was induced. GVBD was not dependent on external Ca2+ and showed no associated Ca2+ changes. NEBD at first mitosis in fertilized eggs, on the other hand, was frequently, but not always associated with a brief Ca2+ transient and was dependent on Ca2+ mobilization. We conclude that GVBD is Ca2+ independent, but that the dependence of NEBD on Ca2+ suggests regulation by more than one pathway. As cells develop from Ca(2+)-independent germinal vesicle oocytes to internal Ca(2+)-dependent pronuclear eggs, internal Ca2+ pools increase by approximately fourfold.
A 50-kDa protein, which binds to the growth-regulated gene (2A9) product, calcyclin in a calcium-dependent manner, was purified from bovine lung. Partial amino acid sequencing of the protein revealed it to be the bovine equivalent of rabbit lung CAP-50 (calcyclin-associated protein, 50 kDa), which is a member of the annexin family and binds to calcyclin in a calcium-dependent manner. Specific polyclonal antibodies to bovine lung CAP-50 were prepared. Comparative studies between CAP-50 and synexin (annexin VII) on the immunoreactivity against anti-CAP-50 antibodies and the ability of binding to calcyclin revealed that CAP-50 was a distinct molecule from synexin. Using specific polyclonal antibodies to bovine lung CAP-50, tissue distribution and subcellular distribution of CAP-50 were investigated. In most rat tissues, except those in the central nervous systems and kidney, CAP-50 is expressed at a high or moderate level. Both studies by subcellular fractionation and by indirect immunofluorescence staining of the rat embryonic fibroblast cell line, 3Y1, revealed that CAP-50 mainly localized in nuclei. Moreover, between the cells at interphase and at mitotic phase, different distributions of CAP-50 were observed. That is, in the cells at interphase, CAP-50 seemed to localize throughout the nucleoplasm. On the other hand, in the cells during mitosis, CAP-50 was concentrated at the loop-like structure around the mitotic apparatus. CAP-50 was found in isolated 3Y1 nuclei lacking outer nuclear membranes, and approximately 50% of CAP-50 was extracted from the nuclei by chelating calcium. Thus, CAP-50, a unique annexin, localizes in nuclei.
Although rare, a recessive temperature-sensitive calmodulin mutant has been isolated in Saccharomyces cerevisiae. The mutant carries two mutations in CMD1, isoleucine 100 is changed to asparagine and glutamic acid 104 is changed to valine. Neither mutation alone conferred temperature sensitivity. A single mutation that allowed production of an intact but defective protein was not identified. At the nonpermissive temperature, the temperature-sensitive mutant displayed multiple defects. Bud formation and growth was delayed, but this defect was not responsible for the temperature-sensitive lethality. Cells synchronized in G1 progressed through the cell cycle and retained viability until the movement of the nucleus to the neck between the mother cell and the large bud. After nuclear movement, less than 5% of the cells survived the first mitosis and could form colonies when returned to permissive conditions. The duplicated DNA was dispersed along the spindle, extending from mother to daughter cell. Cells synchronized in G2/M lost viability immediately upon the shift to the nonpermissive temperature. At a semipermissive temperature, the mutant showed approximately a 10-fold increase in the rate of chromosome loss compared to a wild-type strain. The mitotic phenotype is very similar to yeast mutants that are defective in chromosome disjunction. The mutant also showed defects in cytokinesis.
Primer recognition proteins (PRP) stimulate the activity of DNA polymerase alpha on DNA substrates with long single-stranded template containing few primers. Purified PRP from HeLa cells and human placenta are composed of two subunits of 36,000 (PRP 1) and 41,000 (PRP 2) daltons. By amino acid sequence homology, we have identified PRP 2 as the glycolytic enzyme 3-phosphoglycerate kinase. Here we present data that establishes PRP 1 to be the protein-tyrosine kinase substrate, calpactin I heavy chain. Amino acid sequence analysis of six tryptic peptides of PRP 1 followed by homology search in a protein sequence data base revealed 100% identity of all six peptides with the deduced amino acid sequence of human calpactin I heavy chain. The activities of PRP and calpactin I coelute on gel filtration columns, and a high correlation of PRP and calpactin I activities was seen at different stages of purification. A rabbit polyclonal anti-chicken calpactin I antibody was shown to cross-react with PRP 1 polypeptide at various stages of PRP purification, and the homogeneous preparation of PRP exhibits 3-phosphoglycerate kinase (PRP 2) and calpactin I (PRP 1) activities. PRP activity is neutralized by a mouse monoclonal anti-calpactin II antibody although having no effect on the polymerase alpha activity itself. Calpactin II has a 50% amino acid sequence homology with calpactin I. However, PRP 1 is not calpactin II as shown by lack of cross-reaction to a monoclonal anti-calpactin II antibody on Western blots. Calpactin I and 3-phosphoglycerate kinase, purified independently, cannot be efficiently reconstituted into the PRP complex, indicating that their association in the PRP complex involves specific protein-protein interactions that remain to be elucidated. The biochemical and immunological data presented here revealing the identity of PRP 1 as calpactin I provide evidence for one physiological role of calpactin I in the cell.
Exit from M phase, which requires cyclin degradation, is prevented from occurring in unfertilized eggs of vertebrates arrested at second meiotic metaphase due to a cytostatic factor recently identified as p39mos, the product of the proto-oncogene c-mos. Calpain can destroy both p39mos and cyclin in vitro in extracts prepared from metaphase-arrested Xenopus eggs, but only when free Ca2+ concentration is raised to the millimolar range. When free Ca2+ concentration is raised for only 30 s to the micromolar range, as occurs in physiological conditions after fertilization, cyclin degradation is induced, byt p39mos is not degraded. Cyclin proteolysis at micromolar free Ca2+, is not inhibited by calpastatin, and therefore does not involve calpain. A cyclin mutant modified in the destruction box is found to be resistant at micromolar, but not millimolar free Ca2+, suggesting that the ubiquitin pathway mediates cyclin degradation at micromolar Ca2+ concentration whereas calpain is involved at the millimolar level. A synthetic peptide which binds Ca(2+)-calmodulin with high affinity suppresses cyclin degradation at micromolar but not millimolar free Ca2+, and this only when it is present in the extract during the first 30 s after raising free Ca2+ concentration. The inhibition of the cyclin degradation pathway by the Ca(2+)-calmodulin binding peptide can be overcome by adding calmodulin. These results strongly suggest that a Ca(2+)-calmodulin process is required as an early event following fertilization to release the cyclin degradation pathway from inhibition in metaphase-arrested eggs. In contrast, p39mos degradation is not required.
In Xenopus embryos, previous results failed to detect changes in the activity of free calcium ions (Ca2+i) during cell division using Ca2(+)-selective microelectrodes, while experiments with aequorin yielded uncertain results complicated by the variation during cell division of the aequorin concentration to cell volume ratio. We now report, using Ca2(+)-selective microelectrodes, that cell division in Xenopus embryos is accompanied by periodic oscillations of the Ca2+i level, which occur with a periodicity of 30 min, equal to that of the cell cycle. These Ca2+i oscillations were detected in 24 out of 35 experiments, and had a mean amplitude of 70 nM, around a basal Ca2+i level of 0.40 microM. Ca2+i oscillations did not take place in the absence of cell division, either in artificially activated eggs or in cleavage-blocked embryos. Therefore, Ca2+i oscillations do not represent, unlike intracellular pH oscillations (Grandin, N., and M. Charbonneau. J. Cell Biol. 111:523-532. 1990), a component of the basic cell cycle ("cytoplasmic clock" or "master oscillator"), but appear to be more likely related to some events of mitosis.
Global Ca2+ transients have been observed to precede nuclear envelope breakdown and the onset of anaphase in Swiss 3T3 fibroblasts in 8% (vol/vol) FBS. The occurrence of these Ca2+ transients was dependent on intracellular stores. These Ca2+ transients could be (a) abolished by serum removal without halting mitosis, and (b) eliminated by increasing intracellular Ca2+ buffering capacity through loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffer, via the tetra(acetoxymethyl) ester, without hindering the transition into anaphase. Microinjection of sufficient concentrations of BAPTA buffer could block nuclear envelope breakdown. Pulses of Ca2+ generated by flash photolysis of intracellularly trapped nitr-5, a "caged" Ca2+, could precipitate precocious nuclear envelope breakdown in prophase cells. In metaphase cells, photochemically generated Ca2+ pulses could cause changes in the appearance of the chromosomes, but the length of time required for cells to make the transition from metaphase to anaphase remained essentially unchanged regardless of whether a Ca2+ pulse was photoreleased during metaphase. The results from these photorelease experiments were not dependent on the presence of serum in the medium. Discharging intracellular Ca2+ stores with ionomycin in the presence of 1.8 mM extracellular Ca2+ doubled the time for cells to pass from late metaphase into anaphase, whereas severe Ca2+ deprivation by treatment with ionomycin in EGTA-containing medium halted mitosis. Our results collectively indicate that Ca2+ is actively involved in nuclear envelope breakdown, but Ca2+ signals are likely unnecessary for the metaphase-anaphase transition in Swiss 3T3 fibroblasts. Additional studies of intracellular Ca2+ concentrations in mitotic REF52 and PtK1 cells revealed that Ca2+ transients are not observed at all mitotic stages in all cells. The absence of observable global Ca2+ transients, where calcium buffers can block and pulses of Ca2+ can advance mitotic stages, may imply that the relevant Ca2+ movements are too local to be detected.
Changes in Ca2+ levels in echinoderm and medaka eggs were measured in relation to their cleavage cycles. Fertilized echinoderm eggs were treated with a calcium-ionophore (A23187), and Ca2+ efflux into the ambient solution was measured by the luminescence of a photoprotein (aequorin). The Ca2+ efflux changed cyclically with the same period as that of the cleavage cycle. The Ca2+ efflux decreased at the time of furrowing of every cleavage. These cyclic changes in Ca2+ efflux may reflect corresponding changes in the Ca2+ concentration in eggs. Luminescence of aequorin injected into medaka eggs indicated that the intracellular Ca2+ concentration changed cyclically with the same period as the cleavage cycle. The intracellular Ca2+ concentration was lowest at the time of furrowing. These results suggest that the constriction of the cleavage furrow is regulated by Ca2+ in an inhibitory manner. ATP concentration in echinoderm eggs was measured using thermoluminescence of the luciferin-luciferase system. The ATP concentration remained almost constant following fertilization and throughout the cleavage cycles.
Yeast calmodulin (CaM) is required for the progression of nuclear division (Ohya, Y. and Y. Anraku. 1989. Curr. Genet. 15:113-120), although the precise mechanism and physiological role of CaM in this process are unclear. In this paper we have characterized the phenotype caused by a temperature-sensitive lethal mutation (cmdl-101) in the yeast CaM. The cmdl-101 mutation expresses a carboxyl-terminal half of the yeast CaM (Met72-Cys147) under the control of an inducible GAL1 promoter. Incubation of the cmdl-101 cells at a nonpermissive temperature causes a severe defect in chromosome segregation. The rate of chromosome loss in the cmdl-101 mutant is higher than wild-type cell even at permissive temperature. The primary visible defect observed by immunofluorescence and electron microscopic analyses is that the organization of spindle microtubules is abnormal in the cmdl-101 cells grown at nonpermissive temperature. Majority of budded cells arrested at the high temperature contain only one spindle pole body (SPB), which forms monopolar spindle, whereas the budded cells of the same strain incubated at permissive temperature all contain two SPBs. Using the freeze-substituted fixation method, we found that the integrity of the nuclear morphology of the cmdl-101 mutant cell is significantly disturbed. The nucleus in wild-type cells is round with smooth contours of nuclear envelope. However, the nuclear envelope in the mutant cells appears to be very flexible and forms irregular projections and invaginations that are never seen in wild-type cells. The deformation of the nuclear becomes much more severe as the incubation at nonpermissive temperature continues. The single SPB frequently localizes on the projections or the invaginations of the nuclear envelope. These observations suggest that CaM is required for the functions of SPB and spindle, and the integrity of nucleus.
Global Ca2+ transients have been observed to precede nuclear envelope breakdown and the onset of anaphase in Swiss 3T3 fibroblasts in 8% (vol/vol) FBS. The occurrence of these Ca2+ transients was dependent on intracellular stores. These Ca2+ transients could be (a) abolished by serum removal without halting mitosis, and (b) eliminated by increasing intracellular Ca2+ buffering capacity through loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffer, via the tetra(acetoxymethyl) ester, without hindering the transition into anaphase. Microinjection of sufficient concentrations of BAPTA buffer could block nuclear envelope breakdown. Pulses of Ca2+ generated by flash photolysis of intracellularly trapped nitr-5, a "caged" Ca2+, could precipitate precocious nuclear envelope breakdown in prophase cells. In metaphase cells, photochemically generated Ca2+ pulses could cause changes in the appearance of the chromosomes, but the length of time required for cells to make the transition from metaphase to anaphase remained essentially unchanged regardless of whether a Ca2+ pulse was photoreleased during metaphase. The results from these photorelease experiments were not dependent on the presence of serum in the medium. Discharging intracellular Ca2+ stores with ionomycin in the presence of 1.8 mM extracellular Ca2+ doubled the time for cells to pass from late metaphase into anaphase, whereas severe Ca2+ deprivation by treatment with ionomycin in EGTA-containing medium halted mitosis. Our results collectively indicate that Ca2+ is actively involved in nuclear envelope breakdown, but Ca2+ signals are likely unnecessary for the metaphase-anaphase transition in Swiss 3T3 fibroblasts. Additional studies of intracellular Ca2+ concentrations in mitotic REF52 and PtK1 cells revealed that Ca2+ transients are not observed at all mitotic stages in all cells. The absence of observable global Ca2+ transients, where calcium buffers can block and pulses of Ca2+ can advance mitotic stages, may imply that the relevant Ca2+ movements are too local to be detected.
Animal cells are cleaved by the formation and contraction of an extremely thin actomyosin band. In most cases this contractile band seems to form synchronously around the whole equator of the cleaving cell; however in giant cells it first forms near the mitotic apparatus and then slowly grows outwards over the cell. We studied the relationship of calcium to such contractile band growth using aequorin injected medaka fish eggs: we see two successive waves of faint luminescence moving along each of the first three cleavage furrows at approximately 0.5 micron/s. The first, narrower waves accompany furrow extension, while the second, broader ones, accompany the subsequent apposition or slow zipping together of the separating cells. If the first waves travel within the assembling contractile band, they would indicate local increases of free calcium to concentrations of about five to eight micromolar. This is the first report to visualize high free calcium within cleavage furrows. Moreover, this is also the first report to visualize slow (0.3-1.0 micron/s) as opposed to fast (10-100 microns/s) calcium waves. We suggest that these first waves are needed for furrow growth; that in part they further furrow growth by speeding actomyosin filament shortening, while such shortening in turn acts to mechanically release calcium and thus propagates these waves as well as furrow growth. We also suggest that the second waves act to induce the exocytosis which provides new furrow membrane.
The role of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in nuclear envelope breakdown (NEB) was investigated in sea urchin eggs. The eggs contain a 56-kD polypeptide which appears to be a homologue of neuronal CaM kinase. For example, it undergoes Ca2+/calmodulin-dependent autophosphorylation that converts it to a Ca2(+)-independent species, a hallmark of multifunctional CaM kinase. It is homologous to the alpha subunit of rat brain CaM kinase. Autophosphorylation and substrate phosphorylation by the sea urchin egg kinase are inhibited in vitro by CaMK(273-302), a synthetic peptide corresponding to the autoinhibitory domain of the neuronal CaM kinase. This peptide inhibited NEB when microinjected into sea urchin eggs. Only one mAb to the neuronal enzyme immunoprecipitated the 56-kD polypeptide. Only this antibody blocked or significantly delayed NEB when microinjected into sea urchin eggs. These results suggest that sea urchin eggs contain multifunctional CaM kinase, and that this enzyme is involved in the control of NEB during mitotic division.
The nucleus (germinal vesicle) of starfish oocytes can be injected in vivo to introduce into it calcium indicators and various effectors or inhibitors of calcium signalling pathways. This is advantageous to the study of the debated problem of nuclear calcium homeostasis, which is related to that of the function of calcium in the nucleus. The work described here has shown that, at variance with other cell types, the nuclear envelope of starfish oocytes is relatively impermeable to calcium and to calcium sensitive dyes. It has also shown that a rise in free nuclear calcium is required for the reinitiation of meiosis induced by 1-methyladenine. Bom inositol 1, 4, 5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPr) receptors are present and functional in the membrane enveloping the nucleus. The chief processor of the calcium signal, caknodulin, interacts in the nucleus with the heterogeneous ribonucleoprotein particles and could thus play an important role in the processing of pre-mRNA.
Starfish oocyte maturation is triggered by a natural hormone, 1-methyladenine (1-MeAde), produced in the follicle cells, or artificially by dithiothreitol (DTT). These substances act on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which induces germinal vesicle breakdown (GVBD) and subsequent processes of meiotic maturation. Further, MPF is amplified in immature oocytes that have received the injection of MPF. In this paper the effect of leupeptin and antipain, protease inhibitors of microbial origin, on starfish oocyte maturation was investigated. The protease inhibitors were found to inhibit 1-MeAde-induced maturation when they were applied externally or injected into oocytes. DTT-induced maturation was also inhibited by injection of leupeptin. However, leupeptin did not inhibit the maturation-inducing action of MPF or MPF amplification. These results show that the protease inhibitors suppress the production of MPF by 1-MeAde or DTT, suggesting that some endogenous protease(s) acts in the production of MPF.
In the blue mussel Mytilus edulis, shed oocytes are arrested at metaphase I of meiosis until fertilization. In this study, the mechanisms involved for maintaining the oocytes in metaphase were investigated. Analysis of 35-methionine-labelled proteins separated by 1D SDS-PAGE reveals that two protein bands of apparent MW of 50 and 54 kDa periodically appear and disappear during meiotic and mitotic cycles. Moreover, the 50 kDa protein band, clearly detected in unfertilized oocytes, is seen to disappear within 15 min after fertilization. Unfertilized or fertilized oocytes treated with emetine have a protein synthesis rate reduced to less than half the normal rate observed in the untreated cells. Addition of this protein synthesis inhibitor at various times after fertilization inhibits the normal progression through the cell cycles. However, emetine added to unfertilized oocytes induces the completion of first meiotic maturation, polar body extrusion, and the decondensation of chromosomes which form one or two large pronuclei. This process is accompanied by DNA synthesis, and is preceded by the early disappearance of the 50 kDa protein band, seen to cycle after fertilization. These results indicate that metaphase arrest, in mussel oocytes, requires the continuous synthesis of short-lived proteins, the destruction of which is sufficient to induce meiosis resumption followed by DNA synthesis.
Ca²⁺ and Ca²⁺-binding proteins are involved in running the cell cycle. Ca²⁺ spikes and signals from integrin-activated focal adhesion complexes and Ca²⁺ receptors on the cell surface along with cyclic AMP begin the cycle of cyclin-dependent protein kinases (PKs). These transiently expressed PKs stimulate the coordinate expression of DNA-replicating enzymes, activate replication enzymes, inactivate replication suppressors (e.g., retinoblastoma susceptibility protein), activate the replicator complexes at the end of the G1 build-up, and when replication is complete they and a Ca²⁺ spike trigger mitotic prophase. Another Ca²⁺ surge at the end of metaphase triggers the destruction of the prophase-stimulating PKs and starts anaphase. Ca²⁺ finally stimulates cytoplasmic division (cytokinesis).
Calcium-containing solutions were microinjected into dividing PtK1 cells to assess the effect of calcium ion concentration on the morphology and physiology of the mitotic spindle. Solutions containing 50 M or more CaCl2 are immediately and irreversibly toxic to PtK1 cells. Those containing 5–10 M CaCl2 cause reversible reduction in spindle birefringence followed by normal anaphase and cytokinesis. Microinjection of 5 M or less CaCl2 into anaphase PtK1 cells has no detectable effect on the rate or extent of chromosome movement. Metaphase cells tend to enter anaphase 4–5 min after injection with 1–10 M CaCl2, compared with an average of 16 min after injection with calcium-free buffer. Reducing the intracellular calcium concentration by injection of EGTA-CaCl2 buffers increases the lag between injection and anaphase to 20 min or more. Microinjection of calcium solutions does not promote precocious chromatid separation in nocodazole-arrested metaphase cells, indicating that the increase in calcium concentration does not induce centromere separation directly. An increase in the concentration of free calcium ions during metaphase appears to stimulate the onset of anaphase. Such an increase, regulated by the cell itself, may contribute to the initiation of chromosome separation in mammalian cells.
The concentrations of Ca2+, Na+ and H+ in echinoderm oocytes and eggs were measured during maturation and activation using ion-selective microelectrodes. In both oocytes and eggs, from three species of starfish and two species of sea urchin, the resting level of cytosolic Ca2+ was about 10-7
M. We did not detect any change in Ca2+ concentration either during hormone-induced oocyte maturation (starfish) or during egg activation (starfish and sea urchin) induced by spermatozoa or chemical agents. During 1-methyl-adenine induced maturation of starfish oocytes the intracellular level of Na+ increased from 12–35 mM to 40–90 mM, while the pH changed from 6.6–6.8 to 7.0–7.2 Aged oocytes, with intact germinal vesicles, also had elevated levels of Na+ and pH.
A protease involved in oocyte maturation of a starfish, Asterina pectinifera, was explored. Trypsin-like and chymotrypsin-like activities of the 650-kDa protease in oocyte extract were revealed to increase more than twice under the influence of 1-methyladenine before germinal vesicle breakdown (GVBD) during maturation. The inhibitory potencies of leupeptin and its five analogs against the chymotrypsin-like activity, but not the trypsin-like activity, of this protease was well in accord with those against GVBD (Takagi Sawada et al. (1989). Dev. Biol.133, 609–612). These results indicate that the chymotrypsin-like activity of the 650-kDa protease (most probably 20 S proteasome) plays a key role in starfish oocyte maturation.
Eggs from the sea urchin, Lytechinus pictus, were injected with either EGTA or EDTA, and were subsequently fertilized. EGTA prevented cortical vesicle discharge and formation of the fertilization membrane. EDTA had either no effect, or sometimes retarded the elevation of the fertilization membrane, or reduced the percentage of eggs with elevated membranes. Theoretical considerations lead to estimates of the probable effects of EGTA and EDTA on the internally released calcium which triggers the cortical reaction. Whether or not cytoplasmic calcium buffers are considered, it is concluded: (1) that normally several times the threshold calcium concentration for the cortical reaction is released into a subsurface space; (2) that if a rapidly-equilibrating high-affinity buffer is present, it is locally saturated by the calcium released internally; (3) the injected EDTA reduces the subsurface free calcium concentration normally reached to approximately threshold for the cortical reaction, while injected EGTA reduces the calcium concentration to below this threshold; and (4) a rise in the internal ionic calcium concentration is a necessary step in the activation of the cortical reaction at fertilization.
The calmodulin content of synchronized Chinese hamster ovary (CHO-K1) cells was determined at each phase of the cell cycle. The calmodulin content was minimum in the G1 phase, increased after the cells entered S phase and reached the maximum level at the late G2 or early M phase. When 30 μM of W-7 (calmodulin antagonist) was added at the S phase, the cell cycle was blocked at the late G2 or early M phase. The addition of W-7 also prevented the morphological changes caused by cholera toxin. These results suggest that calmodulin plays an important role in the phases through S to M, possibly in the initiation of DNA synthesis and in the mitosis.
Inhibition of subsite-substituted leupeptin analogs, potent trypsin inhibitors, on 1-methyladenine-induced germinal vesicle breakdown was investigated in a starfish, Asterina pectinifera. Of benzyloxycarbonyl(Z)-Leu-P2-argininals, the analog with Ser at P2 residue was the strongest inhibitor, and those with Pro, Leu > Thr > Gly were followed in this order. In Z-P3-Ser-argininals, ranking of the inhibitory ability was as follows: Phe > Leu ⪢ Pro > Ala at P3 residue. Among 11 analogs synthesized, Z-Phe-Ser-argininal showed the strongest inhibition. The inhibitory potency of the analog was 100-fold stronger than that of leupeptin (acetyl-Leu-Leu-argininal). Thus, trypsin-like enzyme possessing a narrow subsite specificity participates in oocyte maturation in the starfish.
The biochemical pathways underlying the prophase/metaphase transition have been studied extensively in isolated starfish oocytes. These cells are released from their meiotic prophase arrest by 1-methyladenine. This hormone interacts with plasma membrane receptors coupled to heterotrimeric G-proteins. Early events following 1-methyladenine/receptor interaction include a decrease of cAMP concentration and possibly involve tyrosine kinases, phospholipases A2 and C, proteases and phosphatase 2A. Later events include the activation of an M phase-Promoting Factor (MPF) through dephosphorylation/phosphorylation of its p34cdc2 and cyclin B subunits, MPF translocation to the nucleus and activation of the MAP-kinase p44mpk. Starfish oocytes provide an exceptional model to investigate a hormone-regulated cell cycle phase as well as a unique source for the purification of cell cycle control elements.
THE oocytes of most vertebrates and invertebrates develop to a particular stage of meiosis and then stop1. Various external stimuli, such as hormones, sperm or specific proteases2 cause them to resume maturation, but it is not known by what mechanism these act. Indirect evidence suggests the involvement of Ca2+ in triggering this process in various invertebrate oocytes3-6, and in a few species Ca2+ is known to act directly on the oocyte. Such oocytes include those not normally associated with follicular cells, in particular the annelids Pomatoceros7, Hydroides5 and Chaetopterus8 or the lamellibranchs Barnaea4,6 and Spisula9. The reinitiation of meiosis under the influence of the divalent cation ionophore A23187 has been demonstrated with oocytes of Spisula9, Nereis10 and Chaetopterus11 or follicle-free Xenopus oocytes12-13 in the presence of external Ca2+. The assumption that Ca2+ can trigger meiosis in starfish3,14 has been questioned because it seems that follicle cells release a meiosis-inducing substance when isolated in normal or Ca2+-enriched artificial seawater (ASW)15, and because the ionophore also activates germinal vesicle-bearing oocytes without inducing nuclear maturation16. Here we present evidence that Ca2+ can act directly on starfish oocytes and that a transient release of intracellular Ca2+ is a major step in the action of 1-methyladenine (1-MeAde), the natural hormone which triggers meiosis17.
The involvement of calmodulin in the proliferation of Chinese hamster embryo fibroblast cells has been studied with a specific monoclonal antibody to calmodulin. We observed that calmodulin levels increase 2-fold in the late G1 period in these cells, and this coincides with the increase in DNA polymerase alpha activity as the cells progress synchronously from a quiescent state in the G1 to the S phase. However, there is a concurrent 10-fold enhancement of thymidine kinase activity, which is tightly coupled to the entry of cells into the S phase. Incubation of permeabilized S-phase cells with calmodulin-specific murine monoclonal antibody resulted in a dose-dependent inhibition of DNA replication. This inhibitory effect of anti-calmodulin antibodies on DNA replication is completely reversed by the addition of exogenously purified calmodulin. These observations provide evidence for the involvement of calmodulin in DNA replication and, therefore, in cell proliferation during the S phase.
Little is known about the relative intracellular localizations of the calcium-dependent proteases, calpains, and their naturally occurring inhibitor, calpastatin. In the present study, the intracellular localization of mu-calpain, m-calpain, and calpastatin was studied at the light microscopic level in proliferating A431 cells. Highly specific antibodies against the three antigens revealed distinct staining patterns in interphase and mitotic cells. Most notably, calpastatin in interphase cells was localized near the nucleus in tube-like, or large granular structures, while the calpains were more uniformly distributed through the cytoplasm in either a fibrillar form (mu-calpain) or a diffuse or fine granular form (m-calpain). The distribution patterns of the two calpain isozymes were distinctly different during mitosis. m-Calpain was concentrated at the mitotic spindle poles and midbody, while mu-calpain appeared to accumulate at the cell membrane and the spindles. Four other human cell lines as well as normal human monocytes were examined to determine if the calpains-calpastatin segregation patterns are common to other cells or are unique to the A431 line. With the exception of abundant nuclear mu-calpain in the C-33A cervical carcinoma, the segregation of the proteins was similar to that of A431. These studies indicate that calpains may be localized at regions which are relatively poor in calpastatin content. Proteins at these sites may be susceptible to calpain-catalyzed cleavage.
Although the mechanism of calcium regulation is not understood, there is evidence that calcium plays a role in mitosis. Experiments conducted show that: (1) the spindle apparatus contains a highly developed membrane system that has many characteristics of sarcoplasmic reticulum of muscle; (2) this membrane system contains calcium; and (3) there are ionic fluxes occurring during mitosis which can be seen by a variety of fluorescence probes. Whether the process of mitosis can be modulated by experimentally modulating calcium is discussed.
The retinoblastoma-susceptibility gene product (RB) undergoes cell cycle-dependent phosphorylation and dephosphorylation. We characterized RB phosphorylation after mitogenic stimulation of primary human T lymphocytes, initially arrested in the G0 state. RB is phosphorylated in at least three steps when T cells are driven into the cell cycle. The first event occurs during mid G1 phase, the second during S phase, and the third in G2/M. Tryptic phosphopeptide mapping indicates that the different phosphorylation events occur, at least in part, on different residues in RB. Given the known relationship of the RB phosphorylation state to function, it is possible that RB regulates growth at multiple points in the cell cycle.
In human vascular endothelial cells, both growth factor-induced DNA synthesis and retinoblastoma gene product (RB) phosphorylation are absolutely dependent on extracellular Ca2+, and are potently inhibited by an active calmodulin antagonist, W-7, but not an inactive analogue, W-12. A reduction in the extracellular Ca2+ or an addition of W-7 as late as 8 h after growth factor stimulation still inhibits both RB phosphorylation and DNA synthesis to the full extent. However, once RB phosphorylation occurs 12-16 h after addition of the growth factors, it is not reversed by subsequent Ca2+ reduction or W-7. These results suggest the existence of a Ca2+/calmodulin-dependent process relatively late in the mitogenic signalling cascade, at a step proximal to RB phosphorylation reaction itself.
Recently, it has been shown that the product of the c-mos proto-oncogene is a component of cytostatic factor, an activity present in unfertilized eggs from vertebrates that arrests the cell cycle in metaphase of the second meiotic division (metaphase II) possibly by stabilizing maturation-promoting factor (MPF). We have studied the behavior of the c-mos product in metaphase II mouse oocytes and soon after activation. The amount of c-mos in the oocyte was still very high after second polar body extrusion, when cyclin B has been degraded and MPF activity had decreased dramatically. Degradation of c-mos takes place later, during the G1 phase of the first cell cycle and a residual amount of c-mos is detectable during the first zygotic interphase. Our data show that the degradation of c-mos is not involved in the release from the metaphase arrest.
Microtubule-associated protein 2 (MAP2) isolated from porcine brains stimulated the activity of DNA polymerase alpha immunopurified from calf thymus or human lymphoma cells, in a dose-dependent manner. This stimulation was pronounced when activated DNA or poly(dA).(dT)10 was used as the template-primer. DNA polymerase alpha bound to a MAP2-immobilized column, whereas preincubation of the enzyme with unbound MAP2 prevented binding to the column. These events suggested that a physical binding occurred between the polymerase and MAP2. Kinetic analyses revealed that MAP2 decreased the Km value of the polymerase for deoxyribonucleotides, irrespective of the species of template-primer. A concomitant increase in Vmax was observed; however, the extent of the increase depended on the species of template-primer. MAP2 also decreased the Km value of the polymerase for template-primers when activated DNA of poly(dA).(dT)10 was used as the template-primer. Product analyses showed that MAP2 did not significantly alter the processivity of the polymerase and the increment of Vmax is considered to be due to an increase in the frequency of initiation of DNA synthesis. The stimulation by MAP2 occurred specifically in the activity of DNA polymerase alpha, but not DNA polymerases beta, gamma, and I from Escherichia coli. Other MAPs, tau and 190-kDa MAP, could substitute for MAP2. Thus, the specific stimulation of DNA polymerase alpha by MAPs supports the notion of a possible involvement of MAPs or MAP-like proteins in DNA replication, in vivo.
The involvement of calcium ions in the mechanism of meiotic resumption has been studied in mouse oocytes made resistant to the lethal effects of calcium‐free medium (CFM) by zona pellucida removal (De Felici et al., '89). We show here that such oocytes undergo meiotic resumption in CFM (as evaluated by germinal vesicle breakdown, GVBD) at a rate comparable to that shown by oocytes cultured in medium containing 1.7 m M Ca ²⁺ . The addition to CFM of 50 u M Quin2/AM (a membrane permeable, high affinity Ca ²⁺ chelator) totally prevents GVBD, while purported antagonists of Ca ²⁺ release from intracellular stores, such as 150 u M 8‐(N, N‐diethylamino)octyl‐3‐4‐5 trimethoxybenzoate (TMB‐8) or 300 u M chlortetracycline, only cause a slight meiotic delay. On the other hand, if the oocytes are pre‐incubated for 30 min in CFM supplemented with 100 u M TBM‐8 plus 0.2 m M dibutyryl‐cyclic AMP (dbcAMP, a reversible inhibitor of GVBD), and then cultured in the same medium, without dbcAMP, a sustained inhibition of meiotic maturation is obtained.
Our observations suggest that an increase in intracellular free Ca ²⁺ is essential for meiotic resumption by mouse oocytes; in the experimental absence of external Ca ²⁺ , release of the cation from internal stores is sufficient to allow meiotic resumption.
We describe the characterization of a novel cDNA, mbh1 (myc basic motif homolog-1), which was found during a search for candidate factors which might interact with the c-Myc oncoprotein. Embedded within the amino acid sequence encoded by mbh1 is a region distantly related to the basic/helix-loop-helix (B/HLH) DNA-binding motif and a potential nuclear localization signal. Mbh1 encodes a polypeptide structurally similar to the actin-severing proteins gelsolin and severin. Translation of mbh1 RNA in rabbit reticulocyte extracts produces an approximately 45 kd protein capable of binding actin-coupled agarose beads in vitro in a Ca2(+)-dependent manner. Antiserum raised to a trpE/mbh1 bacterial fusion protein recognizes an approximately 45 kb protein in murine 3T3 fibroblasts, suggesting that the cDNA encodes the complete Mbh1 protein. Examination of Mbh1 localization in 3T3 fibroblasts by indirect immunofluorescence reveals a larger cell population showing diffuse staining, and a smaller population exhibiting a distinct nuclear stain. Western analysis corroborates this intracellular localization and indicates that total cellular levels and localization of Mbh1 are not affected by the cell growth state. The data suggest that Mbh1 may play a role in regulating cytoplasmic and/or nuclear architecture through potential interactions with actin.
Elongation factor 2 (eEF-2) is a 100-kD protein that catalyzes the ribosomal translocation reaction, resulting in the movement of ribosomes along mRNA. eEF-2 is the target for a very specific Ca2+/calmodulin-dependent eEF-2 kinase. Phosphorylation of eEF-2 makes it inactive in translation, which suggests that protein synthesis can be regulated by Ca2+ through eEF-2 phosphorylation. Recent data demonstrate that eEF-2 phosphorylation can be involved in cell-cycle regulation and other processes where changes of intracellular Ca2+ concentration induce a new physiological state of a cell. The main role of eEF-2 phosphorylation in these processes is temporary inhibition of overall translation in response to transient elevation of the Ca2+ concentrations in the cytoplasm. Temporary inhibition of translation may trigger the transition of a cell from one physiologic state into another because of the disappearance of short-lived repressors and thus the activation of expression of new genes.
Calmodulin is a small Ca(2+)-binding protein proposed to act as the intracellular Ca2+ receptor that translates Ca2+ signals into cellular responses. We have constructed mutant yeast calmodulins in which the Ca(2+)-binding loops have been altered by site-directed mutagenesis. Each of the mutant proteins has a dramatically reduced affinity for Ca2+; one does not bind detectable levels of 45Ca2+ either during gel filtration or when bound to a solid support. Furthermore, none of the mutant proteins change conformation even in the presence of high Ca2+ concentrations. Surprisingly, yeast strains relying on any of the mutant calmodulins not only survive but grow well. In contrast, yeast strains deleted for the calmodulin gene are not viable. Thus, calmodulin is required for growth, but it can perform its essential function without the apparent ability to bind Ca2+.
The cell division cycle of the early sea urchin embryo is basic. Nonetheless, it has control points in common with the yeast and mammalian cell cycles, at START, mitosis ENTRY and mitosis EXIT.
Progression through each control point in sea urchins is triggered by transient increases in intracellular free calcium. The Cai transients control cell cycle progression by translational and post-translational regulation of the cell cycle control proteins pp34 and cyclin. The START Cai transient leads to phosphorylation of pp34 and cyclin synthesis. The mitosis ENTRY Cai transient triggers cyclin phosphorylation. The motosis EXIT transient causes destruction of phosphorylated cyclin.
We compare cell cycle regulation by calcium in sea urchin embryos to cell cycle regulation in other eggs and oocytes and in mammalian cells.