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The role of calcium in cell division
Abstract
Calcium ions (Ca2+) appear to participate in the regulation of several aspects of cell division. Evidence is accumulating that transients or local gradients in the [Ca2+] contribute to different events including nuclear envelope breakdown and reformation, cleavage furrow formation and growth, and cell plate formation. At present there is little direct evidence that Ca2+ transients trigger the onset of anaphase. However, studies with exogenously applied Ca2+ indicate that spindle fibers and the movement of chromosomes at anaphase are exquisitely sensitive to the ion at physiological levels. Although Ca2+ is involved with many processes there are many gaps in our understanding, particularly pertaining to exactly when and where the ion concentration changes are expressed, which events and macromolecules are targeted, and what the processes are that control Ca2+.
... These ideas were founded on early studies employing model plant (Saunders and Hepler, 1982;Hepler and Callaham, 1987;Hepler, 1994) and animal systems (Poenie et al., 1985;Silver, 1990;Whitaker, 2006a;B. Silver, 1989;Silver, 1986;Silver, 1996) where mitotic calcium signals were observed with chemical dyes (Poenie et al., 1985) or protein-based sensors of calcium such as aequorin (Keating et al., 1994). ...
... 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). ...
... Either calcium signals were observed but there was no discernible correlation with mitotic events or calcium signals could not be reliably detected (Tombes and Borisy, 1989). This confusion led to the view that, on balance, calcium was involved in mitosis but that the functionally relevant signals might be spatially restricted and/or temporally fleeting, rendering them difficult to reproducibly detect Hepler, 1994). Sadly, this novel and potentially highly valuable area of research faded away around 15 years ago having failed to reach a consensus about the role of calcium during mitosis in mammalian cells. ...
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.
... Cell cycle transitions other than meiotic resumption at fertihsation also appear to be regulated by Ca^^. This is suggested by the presence of endogenous ceh cycle Ca^^ transients in various types of somatic cells (Hepler, 1994;Kao et al., 1990;Poenie et al., 1986;Ratan et al., 1988;Wahl and Gruenstein, 1993) and in oocytes and early embryos of a range of species including the sea urchin (Ciapa et al., 1994;Poenie et al., 1985;Steinhardt and Alderton, 1988;Whitaker and Patel, 1990), Xenopus (Snow and Nuccitelli, 1993), the medaka fish (Fluck et al., 1991) and the mouse (Carroll and Swann, 1992;Kono etal., 1996;Stachecki and Armant, 1996b;Tombes et al., 1992). In the sea urchin, the initial large increase in intracellular Ca^^ at fertihsation is followed by further smaUer increases associated with nuclear envelope breakdown (NEBD), chromosome separation at metaphase-anaphase transition and cytokinesis (Poenie et al., 1985;Whitaker and Patel, 1990). ...
... In addition to the role of Ca^^ at entry into mitosis (NEBD) (Hepler, 1994;Kono et al., 1996;Lu and Means, 1993;Tombes et al., 1992;Wilding et al., 1996), it appears that Ca^^ is necessary at other key stages during the course of mitosis. In Xenopus oocyte extracts, onset of anaphase requires the ubiquitin-dependent proteolytic pathway to destroy both mitotic cyclins (see above) and an unknown protein responsible for preventing sister chromatid separation and maintaining metaphase arrest (Holloway et al., 1993). ...
... The regulation of cell cycle transitions by Ca^^ is suggested by the presence of endogenous cell cycle Ca^^ transients in various types of somatic cells (Hepler, 1994;Kao et aL, 1990;Foenie et aL, 1986;Ratan et aL, 1988;Wahl and Gruenstein, 1993) and in oocytes and early embryos of a range of species including the sea urchin (Ciapa et aL, 1994;Steinhardt and Alderton, 1988;Whitaker and Fatel, 1990), Xenopus (Snow and Nuccitelli, 1993), the medaka fish (Pluck et aL, 1991) and the mouse (Carroll and Swann, 1992;Kono et aL, 1996;Stachecki and Armant, 1996b;Tombes et aL, 1992). In the mouse, there have been several studies on cell cycle-associated Ca^^ release and on the sensitivity of oocytes and embryos to agonists which stimulate Ca^^ release. ...
A fertilisation-induced increase in intracellular Ca2+ is responsible for initiating all of the events of egg activation. In mammals, the Ca2+ increase takes the form of a series of Ca2+ oscillations that continue for 3-4 hours. In most cells, intracellular Ca2+ is regulated by Ca2+ channels in the endoplasmic reticulum known as inositol 1,4,5-trisphosphate receptors (InsP3Rs). The aim of the experiments presented in this thesis is to investigate the role of three known isoforms of the Insp3Rs in oocyte maturation, fertilisation and early mammalian development. Western analysis revealed that the type I isoform accounts for all of the detectable InsP3R protein in unfertilised mouse oocytes. Furthermore, type I protein levels were dramatically decreased within 4 hours of fertilisation. During development to the blastocyst the level of type I did not return to prefertilisation levels and types II and III remained below our detection limit. One mechanism that leads to the inhibition of Ca2+ transients after fertilisation may be the downregulation of InsP3Rs. I examined the mechanism of this InsP3R downregulation and found that neither egg activation nor Ca2+ transients are necessary or sufficient for its stimulation. The only stimulus, besides fertilisation, that downregulated InsP3Rs was microinjection of the potent InsP3R agonist adenophostin A, suggesting that InsP3 binding is sufficient for downregulation. InsP3R downregulation was inhibited by the cysteine protease and proteasome inhibitor ALLN but not by specific proteasome inhibitors MG 132 or lactacystin. To examine the role of InsP3-induced Ca2+ release during oocyte maturation, at fertihsation and in the preimplantation embryo, I used adenophostin A to produce immature oocytes. Mil eggs and fertilised 1-cell embryos which were depleted of InsP3Rs. I found that sperm-induced Ca2+ signaling was inhibited in InsP3R-depleted Mil eggs, indicating that Ca2+ signaling at fertilisation is mediated via the InsP3R. In contrast, I found that endogenous levels of InsP3Rs are not necessary for GVBD and meiosis I of oocyte maturation or for NEED of the first cell cycle or for subsequent cell division cycles up to the morula stage. I conclude that the type I InsP3R is essential for fertilisation but that less than 5% of the endogenous receptors are necessary for the first meiotic division and the early embryonic cell division cycles. These studies suggest that the previously reported role for Ca2+ in mitosis in mammalian cells requires re-evaluation.
... However, several studies have also reported that mitosis can occur in the absence of [Ca^^Ji changes (Tombes and Borisy, 1989;Kao et al, 1990;Whitaker and Larman, 2001). As such, the explanation for this variation is unclear, though localised [Ca^^] changes have been observed during mitosis (Keith et al, 1985;Ratan et al, 1986;Wilding et al, 1996), and it has been argued that such events may be sufficient for cell-cycle progression, and may in some cases go undetected (Whitaker and Patel, 1990;Hepler, 1994). This notion is discussed further below. ...
... Two previous studies have reported that entry into the first mitotic division in mouse can be inhibited by the membrane-permeable Ca^^ chelator BAPTA-AM (Tombes et al, 1992;Kono et al, 1996), suggesting that NEBD may be Ca^^-dependent. It is known that Ca^^ chelators are differentially potent in blocking Ca^^-dependent cell-cycle events in vivo according to their affinities for Ca^^ (Speksnijder et al, 1989;Snow and Nuccitelli, 1993;Hepler, 1994). ...
In mammals, fertilisation triggers a series of intracellular Ca2+ transients which are responsible for egg activation and completion of meiosis. These oscillations are generated by InsP3-induced release of Ca2+ from the endoplasmic reticulum (ER). Ca2+ oscillations last for 3-4 hours in mouse, ceasing at the time of pronucleus formation. The subsequent breakdown of the pronuclei (NEBD) at mitosis entry is accompanied by the resumption of Ca2+ oscillations. The experiments presented in this thesis examine the relationship between ER structure and Ca2+ release in the mouse oocyte and early embryo, and investigate the role of Ca2+ release m mitosis. Using the ER-specific marker Dil, we report that germinal vesicle breakdown is associated with a dramatic microtubule-dependent redistribution of ER to the region surrounding the metaphase-I spindle. ER remains tightly packed around the spindle, during centro-cortical migration. The formation of clusters of ER in the oocyte cortex occurs around the time of polar body formation, and coincides with increased responsiveness of InsP3-mediated Ca2+ release. The decrease in cdkl-cyclin B activity which occurs following activation is both necessary and sufficient for the subsequent disappearance of ER clusters, and corresponds with diminished Ca2+ release in response to InsP3. Cortical ER clusters do not re-appear following NEBD, rather ER accumulates around the mitotic spindle. NEBD is associated with increased responsiveness of Ca2+ release both in fertilised and parthenogenetic embryos. The role of Ca2+ in mitosis was examined. Mitotic Ca2+ transients are dispensable since InsP3-receptor-downregulation and Ca2+ -chelators prohibit mitotic Ca2+ transients without affecting the first embryonic division. Microinjection of a fluorescent marker into one pronucleus reveals that nuclear membrane permeablisation begins prior to initiation of mitotic Ca2+ signals. The subsequent cessation of mitotic oscillations precedes the formation of nuclei in the two-cell embryo. No Ca transients are detected during the second mitotic division. These data demonstrate dynamic microtubule and cell cycle dependent ER reorganisations in meiosis and mitosis, in which clustering of ER in the cortex or around the spindle is associated with increased responsiveness of InsP3-releasable Ca2+ stores. Additionally, the results presented suggest that global mitotic Ca2+ transients are triggered by NEBD, rather than being the cause.
... At this stage of development, the constant participation of signaling elements, such as calcium, is necessary to mediate the process. The presence of calcium located in the vicinity of the nuclei in mitosis is possibly involved in various processes such as polymerization of microtubules, dissipation of the NE, synthesis of the mitotic spindle, and condensation of chromatin (Weisenberg 1972;Salmon 1982;Hepler 1994), and this is probably because of proteins, such as those that makeup microtubules, that are sensitive to calcium (Zhang et al. 1992). Calcium is essential for the polymerization of tubulin into microtubules and these are of vital importance in the process of nuclear mitosis, because they generate mechanical forces to dismantle the nuclear membrane (Kite 1913;Mühlhäusser and Kutay 2007). ...
... The localization of cytosolic calcium and actin filaments during the displacement of the nuclei throughout the cytoplasm of the embryo sac in the Asparagaceae family demonstrated that cytosolic calcium is probably an essential mediator for the synthesis and activation of actin as a motor protein required during nuclear migration from the chalaza to the micropyle. The results showed that the increase of cytosolic calcium around the nuclei favors the mitotic division (Weisenberg 1972;Hepler 1994). Although the molar concentrations of Ca 2+ were not determined (mitosis and nuclear migration), the results of this study generate new knowledge, helping to improve the understanding of the mechanisms involved in the early stages of endosperm development, as is calcium. ...
Calcium is a secondary messenger that regulates and coordinates the cellular responses to environmental cues. Despite calcium being a key player during fertilization in plants, little is known about its role during the development of the endosperm. For this reason, the distribution, abundance, and dynamics of cytosolic calcium during the first stages of endosperm development of Agave tequilana and Agave salmiana were analyzed. Cytosolic calcium and actin filaments detected in the embryo sacs of Agave tequilana and A. salmiana revealed that they play an important role during the division and nuclear migration of the endosperm. After fertilization, a relatively high concentration of cytosolic calcium was located in the primary nucleus of the endosperm, as well as around migrating nuclei during the development of the endosperm. Cytosolic calcium participates actively during the first mitosis of the endosperm mother cell and interacts with the actin filaments that generate the motor forces during the migration of the nuclei through the large cytoplasm of the central cell.
... Some previous studies indicated that calcium could participate in early embryonic mitotic division [24][25][26][27]. And cleavage failure was reported in four other cases, for which artificial oocyte activation (AOA, A23187 Ca 2+ ionophore) was able to overcome the problem successfully [8]. ...
... And cleavage failure was reported in four other cases, for which artificial oocyte activation (AOA, A23187 Ca 2+ ionophore) was able to overcome the problem successfully [8]. This technique would be likely to work well on our patient, since modestly elevated Ca 2+ promoted the breakdown of spindle microtubules and by the mechanism modulated chromosome move to the spindle poles [24]. Unfortunately, due to economic factors, our patient did not try another ART cycle with AOA. ...
PurposeCleavage of the zygote during human reproduction is a key event of early embryonic development. The genetic events associated with idiopathic embryonic cleavage failure are not certain. Mutations in the tubulin beta 8 class VIII (TUBB8) gene have been reported to be associated with oocyte maturation, fertilization, and developmental arrest. Here, we aimed to assess the clinical and genetic characteristics of complete cleavage failure in fertilized eggs. Methods
We have characterized a patient with a 9-year history of primary infertility in a non-consanguineous family from China. The patient presented complete cleavage failure in all two-pronuclear (2PN) fertilized oocytes after 2 cycles of in vitro fertilization (IVF). We performed Sanger sequencing of the TUBB8 gene in the patient, and further bioinformatics analysis to identify pathogenesis of gene. ResultsA novel homozygous mutation, c.322G > A (p.Glu108Lys), was detected, and this change was absent from 179 control subjects. Glutamic acid is highly conserved at this position, and replacement by lysine was predicted to be repelled by the α-tubulin positive region, disrupting the α-β tubulin interaction. Conclusions
Our findings presented a homozygous mutation of TUBB8 associated with complete cleavage failure in fertilized eggs and provided new data for the genotype-phenotype of TUBB8-related diseases.
... However, our results (table in the above text box), in accordance with the previous ones, suggest that the minerals, resulting from a high cell metabolism, are sequestered in the form of crystals. In this way, crystals represent, an internal reserve that will be mobilized during the following vegetative season and used to support cell divisions and differention (Hepler, 1994;Fromm, 2011). This may be the physiological role of the crystals found in teak cambium which were previously identified as calcium oxalate and considered as the by-products of the species high metabolism (Rao & Dave, 1984). ...
... On the other hand, crystals presence in the diffentiating tissues, at all the growth stages, indicates their role in the cell differentiation process, i.e, during the formation of the perforations plates (Hepler, 1994). Whereas crystals storage in phloem fibres indicates their contribution in the cell (fibres) wall hardening, in order to enhance the protective role of tree bark (Webb, 1999). ...
Although they currently absorb more carbon than they release, tropical forests suffer disturbances generated by human activities and amplified by global warming. Therefore, forest plantation, based on fast-growth fuelwood and timber species such as teak, becomes an option to counteract the risk of loss of these natural biomes. For its various advantages (ecological plasticity, good behaviour in plantation, fast-growth, high natural durability and wood density), teak (Tectona grandis) is introduced under different climatic conditions along the inter-tropical belt, occupying 75% of the world tropical timber plantations. However, this species remains highly sensitive to climate, suggesting that disturbances in the rainfall rate and intensity are expected to negatively affect tree growth and, consequently, induce variations in the derived wood properties. Therefore, to be successful, in response to the high demand for high quality wood, teak based reforestation programs should be underpinned with reliable and accurate information based on field data on tree growth rate and wood density dynamics. Because growth rate (reflected in ring widths) and wood density represent the main two functional traits that portray tree sensitivity/vylnerability to environment (climate). Information on the dynamics of growth and wood density remains, therefore, crucial to control and predict planted stands productivity, biomass estimate and the wood quality in regions marked by ongoing climatic variability like in Ivory Coast. Our study addressed the following questions: (i) What are the determining factors of wood formation and its fluctuations in teak planted in Ivory Coast? (ii) How does the precipitation variability influence managed and non managed tree growth? Is there a large-scale climatic signal in the tree growth rate (ring width), in addition to local the precipitation signal? (iii) How to assess the degree of variability of wood density in teak planted in Ivory Coast? Does climate variability affect teakwood density? To address these research questions, monthly cambial sampling was carried out in an evergreen forest and tree-ring analysis was performed in trees from 2 stands: a managed plantation located in an evergreen forest close to the Atlantic coast and an unmanaged semi-deciduous forest situated in the Central West Ivory Coast. Microdensity measurements were implemented on trees also from the evergreen forest, in order to assess the variations in wood density. Cambial activity showed that a 3 months dry season induced growth interruption and resulted in the formation of an annual xylem growth ring with 2 phloem rings over the same vegetative season. Anatomical quantification of the structural changes occurring in the cambial zone was closely correlated to monthly precipitations. Growth anomalies, induced by climatic and phenological variations during the growing period, were visualized through the formation of intra-annual xylem growth zones. Tree-ring analysis revealed higher growth rate in the managed trees than in the non-managed forest. In the deciduous forest, teak growth was only sensitive to the wet season precipitation while trees reacted to both wet and annual precipitation in the semi-deciduous forest. Anomalies in the sea surface temperature of the Gulf of Guinea also influenced trees growth. Moreover, growth patterns reflected the effect of three major El Niño events in the southern forest. Overall, climatic influence was more pronounced (slightly high correlation) in the non-managed teak plantation compared to the managed one as a consequence of the lack of sylvicultural intervention in the non-managed stand. This indicates the role of sylviculture in enabling trees to cope with a water deficit resulting from climate disturbances. The results reveal clear and direct effect of the broad-scale climate on tree growth, in addition to local precipitation recordings. Microdensitometrical profiling revealed that wood density was more variable within the tree than between trees. Intra-tree density variations reflected fluctuations occurring in the intra-ring xylem anatomy. Direct and significant correlations were found between October rainfall and the total ring wood density (r= 0.37; p<0.05) and latewood density (r= 0.41; p<0.05) which also correlated with the post-wet season rainfall (r=0.47; p< 0.05). The intra-ring anatomy related density changes led to the occurrence of the alternating pattern of intra-ring denstiy variation which best supported the correlation with the bimodal monthly precipitation. Inter and intra-annual density variations were high and persistent in the mature wood portion, indicating that the cambial age was not the main driving factor of the density variability. This research work, founded on original field data and unique for this region, provides highly useful results for teak plantations management in West-Africa. This scientific data evidences the sensitivity of teak growth to climate at both local and global scales. As a result of this climate sensitivity, (1) growth slowed down during years of low precipitation and (2) in response to intra-annual precipitations disturbances, trees formed intra-ring anatomy fluctuations which, in turn, result in the intra-ring density variations, the main source of the mean tree density variability. As a perspective to future research, a weekly cambial sampling could test the hypothesis on the intra-annual xylem growth zones formation which affected tree-ring density and, consequently, mean tree density. Monitoring of thinnings and pruning operations, combined with tree-ring analysis, and the monitoring of leaf area index, evapotranspiration and soil water content, could be a key to unravel the mechanism through which the managed stand trees adapt to water deficits. Heritability study could allow to establish the degree of the genetic control on wood density namely the earlywood features which could contribute to stabilize the mean tree wood density.
... Besides, emerging evidence suggests a strong correlation between ROS and calcium homeostasis (Steinhorst and Kudla 2013). Considering the importance of Ca 2+ for plant cytokinesis (Hepler 1994;Verma 2001) and ROS participation in Ca 2+ homeostasis (Steinhorst and Kudla 2013), along with the multiple effects of ROS homeostasis disturbance in cell plate formation, it may be hypothesized that ROS modulators disrupt the local Ca 2+ gradients and consequently the development of the cell plate. Disturbed cytoplasmic Ca 2+ gradient in treated cells is further supported by the altered ER distribution. ...
... Several cytokinetic cells incubated with ROS modulators lacked ER membranes in the interzone between the daughter nuclei, whereas in other cells intense ER gatherings were detected. ER contributes to Ca 2+ homeostasis in the cell plate region (Hepler 1994), and importantly, the redox status within ER is of particular importance for its function, including the release of Ca 2+ from its membranes (Görlach et al. 2006). ...
Reactive oxygen species (ROS) are emerging players in several biological processes. The present work investigates their potential involvement in plant cytokinesis by the application of reagents disturbing ROS homeostasis in root-tip cells of Triticum turgidum. In particular, the NADPH-oxidase inhibitor diphenylene iodonium, the ROS scavenger N-acetyl-cysteine, and menadione that leads to ROS overproduction were used. The effects on cytokinetic cells were examined using light, fluorescence, and transmission electron microscopy. ROS imbalance had a great impact on the cytokinetic process including the following: (a) formation of atypical "phragmoplasts" incapable of guiding vesicles to the equatorial plane, (b) inhibition of the dictyosomal and/or endosomal vesicle production that provides the developing cell plates with membranous and matrix polysaccharidic material, (c) disturbance of the fusion processes between vesicles arriving on the cell plate plane, (d) disruption of endocytic vesicle production that mediates the removal of the excess membrane material from the developing cell plate, and (e) the persistence of large callose depositions in treated cell plates. Consequently, either elevated or low ROS levels in cytokinetic root-tip cells resulted in a total inhibition of cell plate assembly or the formation of aberrant cell plates, depending on the stage of the affected cytokinetic cells. The latter failed to expand towards cell cortex and hence to give rise to complete daughter cell wall. These data revealed for the first time the necessity of ROS homeostasis for accomplishment of plant cytokinesis, since it seems to be a prerequisite for almost every aspect of this process.
... Calcium (Ca 2+ ) is an essential secondary messenger that impacts various aspects of cellular life, including cell division, muscle contraction, and neurotransmitter release (Hepler 1994;Südhof 2012;Szent-Györgyi 1975). In plants, Ca 2+ signaling plays a vital role in growth and development as well as in response to various biotic and abiotic stresses (Kong et al. 2020;Tian et al. 2019). ...
Key message
Plants exhibit a unique pattern of cytosolic Ca²⁺ dynamics to correlate with microtubules to regulate cytokinesis, which significantly differs from those observed in animal and yeast cells.
Abstract
Calcium (Ca²⁺) transients mediated signaling is known to be essential in cytokinesis across eukaryotic cells. However, the detailed spatiotemporal dynamics of Ca²⁺ during plant cytokinesis remain largely unexplored. In this study, we employed GCaMP5, a genetically encoded Ca²⁺ sensor, to investigate cytokinetic Ca²⁺ transients during cytokinesis in Nicotiana tabacum Bright Yellow-2 (BY-2) cells. We validated the effectiveness of GCaMP5 to capture fluctuations in intracellular free Ca²⁺ in transgenic BY-2 cells. Our results reveal that Ca²⁺ dynamics during BY-2 cell cytokinesis are distinctly different from those observed in embryonic and yeast cells. It is characterized by an initial significant Ca²⁺ spike within the phragmoplast region. This spike is followed by a decrease in Ca²⁺ concentration at the onset of cytokinesis in phragmoplast, which then remains elevated in comparison to the cytosolic Ca²⁺ until the completion of cell plate formation. At the end of cytokinesis, Ca²⁺ becomes uniformly distributed in the cytosol. This pattern contrasts with the typical dual waves of Ca²⁺ spikes observed during cytokinesis in animal embryonic cells and fission yeasts. Furthermore, applications of pharmaceutical inhibitors for either Ca²⁺ or microtubules revealed a close correlation between Ca²⁺ transients and microtubule organization in the regulation of cytokinesis. Collectively, our findings highlight the unique dynamics and crucial role of Ca²⁺ transients during plant cell cytokinesis, and provides new insights into plant cell division mechanisms.
... Calcium ions play important roles in the physiology of eukaryotic cells, including control of DNA synthesis, chromosomal configuration, and signal transduction (Marcum et al., 1978;Boynton et al., 1980;Krishtal et al., 1981;Lux and Nagy, 1981;Tsien, 1983;Poenie et al., 1985;Carafoli, 1987;Steinhardt and Alderton, 1988;Hepler, 1994;Sanders et al., 2002;Verret et al., 2010). Calcium ions enter the cell passively via channels Verret et al., 2010) that allow for a diffusive influx driven by a steep concentration gradient across the plasma membrane . ...
The emergence of intracellular calcification among marine unicellular eukaryotes in the Late Triassic (237-201 Ma) had profound consequences for the carbonate buffering capacity of the ocean. Research on the appearance of calcification typically focuses on the reasons that made this process successful on a global scale. The underlying environmental and physiological conditions that led to its appearance, therefore, are still obscure. Using gene tree analysis, we show that the physiological machinery for calcification was already present in non-calcifying ancestor cells. Additionally, by modelling the energy demands for calcium transport in calcifying and non-calcifying cells, we demonstrate that intracellular calcification does not require additional energy investments. Since all eukaryotic cells export calcium across the plasma membrane, our findings indicate that the onset of intracellular calcification in Earth history only required the activation of calcium transport proteins during their passage to the plasma membrane. Our work sheds new light on the physiological and biogeochemical conditions that led to one of the most important evolutionary innovations of the Mesozoic era.
... There was a linear increase in the growth parameters with an increase in calcium levels. [23], therefore, application of calcium at higher rates might have increased the vegetative growth [24]. It also enhances the cell wall formation, cell wall integrity which in turn increases the vegetative growth. ...
Aims: To evaluate the effect of various sources, levels and methods of calcium application on the growth, yield and SPAD index of carrots grown on acid soils Study Design: Factorial randomized block design (FRBD) with three replications. Place and Duration of Study: A field experiment was conducted in the farmer’s field at Takkarbaba Nagar, Ooty from February to June, 2023. Methodology: A field experiment was conducted with carrot hybrid Zubera using lime at 50% LR and dolomite at 20,40,60 and 80 kg Ca ha-1 as basal soil application and foliar spraying of 0.5% calcium EDTA and calcium citrate twice on 30 and 45 DAP in a factorial randomized block design with three replications. The growth parameters such as plant height, petiole length, number of leaves per plant, SPAD index and tuber yield were recorded as per standard protocols at different plant growth stages viz., vegetative, tuber development and harvest stages. Results: A significant linear increase in plant height, petiole length, number of leaves per plant, SPAD index and tuber yield was recorded with the supplementation of calcium at different levels. Higher plant height (26.3 cm, 51.2 cm and 56.1 cm), petiole length (13.8 cm, 23.0 cm and 35.8 cm), number of leaves per plant (7.62, 7.82 and 9.30), SPAD index (22.0, 38.0 and 35.2) and fresh tuber yield (37.2 t ha-1) was registered with NPK+ 80 kg Ca as dolomite along with two sprays of 0.5% Ca EDTA twice which was followed by the same level of calcium as dolomite + foliar spraying of 0.5% calcium citrate twice. The minimum growth and yield response of carrot at all the growth stages was noticed with NPK control. Conclusion: Application of calcium through soil and foliar spraying showed synergistic influence on the growth and SPAD index of carrot at all stages of growth. Application of NPK+ 80 kg Ca ha-1 as dolomite performed better when compared to lime applied at 50% LR. Between Ca EDTA and Ca citrate, the performance of Ca EDTA was better in improving the growth and yield of carrot. It was concluded that the combined application of soil test based NPK+80 kg Ca ha-1 as dolomite along with two sprays of 0.5% calcium EDTA on 30 and 45 DAP would be effective in improving the growth, yield and SPAD index of carrot grown on acid lateritic soils.
... Calcium ions play important roles in the physiology of eukaryotic cells, including control of DNA synthesis, chromosomal configuration, and signal transduction (Marcum et al., 1978;Boynton et al., 1980;Krishtal et al., 1981;Lux and Nagy, 1981;Tsien, 1983;Poenie et al., 1985;Carafoli, 1987;Steinhardt and Alderton, 1988;Hepler, 1994;Sanders et al., 2002;Verret et al., 2010). Calcium ions enter the cell passively via channels (Gussone et al., 2006;Verret et al., 2010) that allow for a diffusive influx driven by a steep concentration gradient across the plasma membrane (Langer et al., 2006). ...
The rise of calcifying nannoplankton in the Late Triassic (237–201 Ma) had profound consequences for the carbonate buffering capacity of the ocean. Research on the appearance of calcification typically focuses on the reasons that made this process successful on a global scale. The underlying environmental and physiological conditions that led to its appearance, therefore, are still obscure. Here we show that the rise of intracellular calcification is intrinsically linked to cellular calcium metabolism and to the prevention of calcium accumulation in the cytoplasm. Using gene tree analysis, we present evidence that the physiological machinery for calcification was already present in non-calcifying ancestor cells. Additionally, by modelling the energy demands for calcium transport in calcifying and non-calcifying cells, we demonstrate that intracellular calcification does not require additional energy investments. Since all eukaryotic cells export calcium across the plasma membrane, our findings indicate that the onset of intracellular calcification in Earth’s history required only the activation of calcium transport proteins during their passage to the plasma membrane. Hence, intracellular calcification could have occurred at any time in the geological past because physiological preconditions were already present, but a combination of favourable environmental factors may have helped calcifying nannoplankton to spread at the beginning of the Mesozoic, a time of catastrophic climate changes and mass extinctions that reshuffled life on Earth.
... Interestingly, actin bundle formation requires an elevation of Ca 2+ levels, while detachment and cortical translocation of actin is a prerequisite for normal cellular cleavage, indicating an important role for Ca 2+ -dependent actin reorganisation [167,[170][171][172]. It was suggested that heparinor age-induced hyperpolymerization of the starfish egg cortical actin disrupted cytoskeletal dynamics at fertilisation, which in turn detrimentally influenced Ca 2+ release [167,[173][174][175] Considering that the relationship between actin and Ca 2+ could be viewed as one where actin acts as a buffer to store and release Ca 2+ [161][162][163], it is thus possible that such a phenomenon could be affecting the timing of cellular cleavage apart from other events in cell division, such as cleavage furrow formation, nuclear envelope breakdown, and reformation [176]. To this degree, particle image velocimetry (PIV) detected specific rhythmic cytoplasmic movements due to contraction of the actomyosin cytoskeleton triggered by Ca 2+ oscillations. ...
Oocyte activation, a fundamental event during mammalian fertilisation, is initiated by concerted intracellular patterns of calcium (Ca2+) release, termed Ca2+ oscillations, predominantly driven by testis-specific phospholipase C zeta (PLCζ). Ca2+ exerts a pivotal role in not just regulating oocyte activation and driving fertilisation, but also in influencing the quality of embryogenesis. In humans, a failure of Ca2+ release, or defects in related mechanisms, have been reported to result in infertility. Furthermore, mutations in the PLCζ gene and abnormalities in sperm PLCζ protein and RNA, have been strongly associated with forms of male infertility where oocyte activation is deficient. Concurrently, specific patterns and profiles of PLCζ in human sperm have been linked to parameters of semen quality, suggesting the potential for PLCζ as a powerful target for both therapeutics and diagnostics of human fertility. However, further to PLCζ and given the strong role played by Ca2+ in fertilisation, targets down- and up-stream of this process may also present a significantly similar level of promise. Herein, we systematically summarise recent advancements and controversies in the field to update expanding clinical associations between Ca2+-release, PLCζ, oocyte activation and human fertility. We discuss how such associations may potentially underlie defective embryogenesis and recurrent implantation failure following fertility treatments, alongside potential diagnostic and therapeutic avenues presented by oocyte activation for the diagnosis and treatment of human infertility.
... With the addition of exogenous Ca 2+ , the reduced concentrations of total phenol, flavonoid, and mineral elements in Cd stress plants were recovered, enhancing the crop yield of Cicer arietinum (Ahmad et al. 2016). The involvement of Ca 2+ in plant cell division may be one of the probable explanations (Hepler 1994). For example, Ca 2+ supplementation alleviated a wide range of Cd-induced mitotic anomalies and enhanced the mitotic index in Vicia faba and Wedelia trilobata plants respectively (El-Ashry and Mohamed 2012; Shi et al. 2014). ...
Despite its known phytotoxicity, cadmium (Cd) is being added in most arable soils via different agricultural activities. Additionally, information is meager on the Cd-phytotoxicity mitigation strategies employing sustainable approaches. Essential plant nutrient calcium (Ca 2+) and signaling molecule nitric oxide (NO) are known as the key regulators of plant biochemical and physiological processes under stress conditions. Though there exists a close functional synergism between Ca 2+ and NO in plant stress tolerance, the role of either Ca 2+ and/or NO has been explored in plant stress tolerance in isolated reports. Given this, in addition to briefly overviewing the availability, uptake, transport and toxicity (including impact on morpho-physiological traits, photosynthesis, oxidative stress, and genotoxicity), this paper aimed to highlight the general roles, transport, and signaling of Ca 2+ and/or NO in plants, and appraises the literature available on their significance in improvements of plant growth, photosynthesis and control of oxidative stress under Cd stress. Effort was also made to present a crosstalk on Ca 2+ and NO signaling, and the significance of its outcome in plant Cd-tolerance.
... The highest amounts of Ca are found in mitochondria. It is involved in cell division and cell elongation (Helper, 1994). It is a messenger in several developmental and environmental changes (Heintz, 1960;Sanders et al., 2002). ...
The research focused on determining the economic value of a selected collection of 20 shrubs from a wild-growing population of Mahonia aquifolium (Pursh) Nutt. from Arboretum Mlyňany and Nitra region. By morphological analysis, we determined weight of fruits 0.18-0.50 g, the height of fruits 5.57-13.22 mm, the width of fruits 0.98-11.00 mm, and the number of seeds 1.67-5.30 pcs. The content of macro-and microelements was found in the fruits and leaves. M. aquifolium samples are a very valuable source of potassium as the main mineral element contained in leaves (10.437 mg.kg-1) and fruits (9.763 mg.kg-1). Microelements such as manganese and iron prevailed in leaves (80.1 mg.kg-1 of Mn and 35.0 mg.kg-1 of Fe), fruits (29.7 mg.kg-1 of Mn and 25.0 mg.kg-1 of Fe), and heavy metals (Al, As, Cd, Ni, Pb, Hg) are present only in the small amounts with the most abundant aluminium (17.6 mg.kg-1 of Al in leaves and 3.6 mg.kg-1 of Al in fruits) content and can be used as indicator suggesting the environmental pollution status in the region. We determined the antioxidant activity by the Trolox method in methanol extracts (76.2 and 101.2 mg TE.g-1 DW), in ethanol extracts (54.3 and 47.4 mg TE.g-1 DW), in acetone extracts (63.4 and 51.9 mg TE.g-1 DW) and water extracts (35.5 and 60.3 mg TE.g-1 DW) for fruits and leaves, respectively. Extraction of whole fruit (A1-WF), mashed fruit (A1-MF), and fruitless clusters (A1-CT) in structured (activated) water obtained by Kalyxx for 5 days determined a significant reduction trend pH in mashed fruits (A1-MF). The electrolytic conductivity and total dissolved solids of the extracts decreased significantly from the third day of extraction in variants A1-MF and A1-WF. Significant stability of pH, electrolytic conductivity and total dissolved solids during the experimental period was determined for the fruitless clusters' extracts (A1-CT). The results show that Mahonia aquifolium has a multifunctional practical use even in the conditions of the Slovak Republic.
... On the other hand, calcium is an important component of the cell wall and membranes, thus helping in maintaining the normal structure and function of cells and reducing or delaying damage to the cell membrane (Hocking et al., 2016). At the same time, calcium regulates the mitosis of plant cells by controlling the spatial and temporal distribution of calcium ions or their receptors (calmodulin), which is positively correlated with the consumption of organic carbon (Hepler, 1994;Zhang and Liu, 2021). Calcium also affects the ability of plants to absorb other elements, and there are antagonistic and synergistic effects between other elements (Jackson, 1967;Arif et al., 2016). ...
As a crucial element for plants, calcium (Ca) is involved in photosynthesis and nutrient absorption, and affects the growth of plants. Poplar is an important economic forest and shelter forest species in China. However, the optimum calcium concentration for its growth is still unclear. Herein, we investigated the growth, biomass, photosynthetic pigments, photosynthetic parameters and products, chlorophyll fluorescence parameters, water use efficiency (iWUE), and antioxidant enzyme activity of “Liao Hu NO.1” poplar (P. simonii × P. euphratica) seedlings at 0, 2.5, 5, 10, and 20 mmol·L⁻¹ concentrations of Ca²⁺, and further studied the absorption, distribution, and utilization of nutrient elements (C, N, P, K, and Ca) in plants. We found that with increasing calcium gradient, plant height and diameter; root, stem, leaf, and total biomasses; net photosynthetic rate (Pn); stomatal conductance (Gs); intercellular carbon dioxide (Ci) level; transpiration rate (Tr); Fv/Fm ratio; Fv/F0 ratio; chlorophyll-a; chlorophyll-b; soluble sugar and starch content; superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) levels; and long-term water use efficiency (iWUE) of poplar seedlings first increased and then decreased. These parameters attained maximum values when the calcium concentration was 5 mmol·L⁻¹, which was significantly different from the other treatments (P < 0.05). Moreover, a suitable Ca²⁺ level promoted the absorption of C, N, P, K, and Ca by various organs of poplar seedlings. The absorption of C, N, P, and K increased first and then decreased with the increased calcium concentration, but the optimum calcium concentrations for the absorption of different elements by different organs were different, and the calcium concentration in leaves, stems, and roots increased gradually. Furthermore, the increase in exogenous calcium content led to a decreasing trend in the C/N ratio in different organs of poplar seedlings. C/P and N/P ratios showed different results in different parts, and only the N/P ratio in leaves showed a significant positive correlation with Ca²⁺ concentration. In conclusion, the results of this study indicate that 5 mmol·L⁻¹ concentration of Ca²⁺ is the optimal level, as it increased growth by enhancing photosynthesis, stress resistance, and nutrient absorption.
... Calcium is responsible for regulation of plant growth and development (Cao et al., 2017). It is very important for cell walls and membranes and is necessary to membrane permeability (Hepler, 1994;Marschner, 1995). Growth parameters also varied with cultivars. ...
In the present investigation, effect of calcium chloride (CaCl2) used as foli-ar application (0, 10, 20 mM) was studied on morphological, yield, and quality of lettuce (Romaine and Green Waves cultivars) during 2017/2018 and 2018/2019seasons. Our results indicated that, foliar spray of calcium chloride either at 10 or20 mM significantly increased vegetative growth and yield parameters i.e., leaf number per plant, fresh and dry leaf weight compared to control treatment. Also ,foliar spray of calcium chloride resulted in a significant increase in yield and quality parameters i.e., TSS, chlorophyll content and Vitamin C. ‘Romaine’ cultivar was significantly superior to ‘Green Waves’ cvs in plant height, fresh and dry weight of the leaves, TSS and chlorophyll content. However, there was no significant difference in vitamin C content between the two tested cultivars.‘ Green waves’ cv. sprayed with 20 mM CaCl2 gave the highest significant leaf number per plant. Fresh and dry leaf weight of ‘Romaine ’cv recorded the highest values when the plants were sprayed with 20 mM CaCl2 in both seasons., In the first season, ‘Romaine’ cv sprayed with 20 mM of CaCl2 produced the highest total yield, while the highest total yield in the second season was obtained from plants of both tested cvs sprayed with 20 mM CaCl2. ‘Romaine’ cultivar treated with 20 mM of calcium chloride as foliar application had the highest TSS and chlorophyll content. Fascinatingly, high association existed between total yield and all studied traits, except leaf number per plant. Overall results suggest applying calcium chloride as foliar application at 20 mM for improving yield quantity and quality in both ‘Romaine’ and ‘Green Waves’ cultivars.
Keywords: cultivars, calcium chloride treatments, yield components, yield quality
... It has been reported that, under abiotic stress, the increase of cytosolic concentrations of Ca 2+ in S phase may be a signal of DNA damage and cell cycle regulation by modulating cyclin expression. Ca 2+ , at high exogenous concentrations, has been found to delay cell cycle progression and disrupt anaphase at spindle fiber and chromosome movement levels [42][43][44]. This cation is required for various structural functions in the cell wall and membranes as it can form intermolecular linkages [45] and constitute a second messenger in many biological systems [46][47][48]. ...
In view of climate change, increasing soil salinity is expected worldwide. It is therefore important to improve prediction ability of plant salinity effects. For this purpose, brackish/saline irrigation water from two areas in central and coastal Tunisia was sampled. The water samples were classified as C3 (EC: 2.01–2.24 dS m−1) and C4 (EC: 3.46–7.00 dS m−1), indicating that the water was questionable and not suitable for irrigation, respectively. The water samples were tested for their genotoxic potential and growth effects on Vicia faba seedlings. Results showed a decrease in mitotic index (MI) and, consequently, growth parameters concomitant to the appearance of micronucleus (MCN) and chromosome aberrations when the water salinity increased. Salt ion concentration had striking influence on genome stability and growth parameters. Pearson correlation underlined the negative connection between most ions in the water inappropriate for irrigation (C4) and MI as well as growth parameters. MI was strongly influenced by Mg2+, Na+, Cl−, and to a less degree Ca2+, K+, and SO42−. Growth parameters were moderately to weakly affected by K+ and Ca2+, respectively. Re-garding MCN, a very strong positive correlation was found for MCN and K+. Despite its short-term application, the Vicia-MCN Test showed a real ability to predict toxicity induced by salt ions confirming that is has a relevant role in hazard identification and risk assessment of salinity effects.
... Nearly 50% of Mg within the body is present in soft tissues, and the other half in teeth and bones. Around 90% of body Ca is concentrated in calcified structures, and small amounts are present in blood and in inter-and intracellular fluids (Hepler 1994). Approximately half of the Ca within these fluids is present as free ions or electrolytes, and is vital for a variety of processes (Denda et al. 2003). ...
Macro-elements such as potassium (K), sodium (Na), chlorine (Cl), magnesium (Mg), and calcium (Ca) are essential in marine mammals’ nutrition. These elements are involved in physiological processes. Upon consumption, they are assimilated and accumulate in tissues. For the first time, they were detected in lung, spleen, liver, kidney, muscle, uterus, ovary, and testis of 5, and in skin of 12, stranded false killer whales (Pseudorca crassidens) in sub-Antarctic waters of the South Atlantic Ocean. Results showed that testis reached the highest potassium mean concentration, 1.62 (0.25) wt% dry weight (DW) (standard deviation in parentheses), followed by muscle, 1.11 (0.12) wt% DW, and decreasing in skin to 0.351 (0.098) wt% DW. Testis and lung exhibited among the highest sodium concentrations, with 0.96 (0.20) and 0.93 (0.18) wt% DW, respectively. Chlorine concentration was highest in testis, (1.55 wt% DW) followed by uterus (1.26 wt% DW) and kidney [1.13 (0.16) wt% DW]. Magnesium reached higher concentrations in uterus (0.134 wt% DW) and muscle [0.109 (0.054) wt% DW]. Calcium was higher in lung [0.230 (0.05) wt% DW] and kidney (0.149; 0.294 wt% DW). Hepatic levels of K, Na, Cl, and Mg in false killer whales are generally within the range of other studied species, while Ca levels are the highest reported. Macro-element concentration ranges were established for diverse tissues and organs of the false killer whale as the current best available baseline reference values for assessments of general condition.
... So, increasing of nutrient uptake rate can be as important reason for increasing of growth parameters and yield in SA treated plants. According to Hepler (1994), the effects of different calcium sources on growth parameters of different crops can be related to the fact that calcium ions (Ca 2+ ) appeared to participate in the regulation of different aspects of cell division. Calcium is one of the most important ions in formation of the mitotic spindle which directly affects cell division. ...
p>Eggplant is one of the most popular and vital vegetable crops in the world. Various plant bio-regulators have been used in different crops to increase uptake of nutrients thereby leading to improvement in growth, flowering, fruit quality, storability and yield. The scope of this study was to evaluate the effects of calcium lactate and salicylic acid foliar application on growth parameters, physiological characteristics and shelf-life of eggplant fruit. Obtained results showed that the highest applied concentrations of calcium lactate (4 mM or 0.8 g l-1) and salicylic acid (1.5 mM or 0.2 g l-1) foliar application led to the highest values of measured growth parameters and yield. Applying of calcium lactate and salicylic acid foliar treatments could increase tissue firmness and ascorbic acid content of fruits. Foliar application of calcium lactate 4 mM (0.8 g l-1) and salicylic acid 1 mM (0.13 g l-1) was the best treatment to decrease percentage of fruit decay. In conclusion, our results showed that foliar application of calcium lactate and salicylic acid can be useful and inexpensive treatment to improve growth parameters, physiological characteristics and post-harvest properties of eggplant fruit</p
... The highest amounts of Ca are found in mitochondria. It is involved in cell division and cell elongation (Helper, 1994). It is a messenger in several developmental and environmental changes (Heintz, 1960;Sanders et al., 2002). ...
Calcium (Ca) is an essential plant nutrient responsible for the integrity of cells and plant structure, yet it is generally neglected, because it is available in plenty in most cultivated soils. It is therefore not applied as a fertilizer to crops except in groundnut. In acid soils, where it is limiting, it is applied in large amounts as a soil amendment as lime. In sodic soil, where excess of Na creates problems in crop production Ca is applied as gypsum, another soil amendment. When applied in large amounts as lime or gypsum, Ca nutrition is already taken care of and thus there is no need of annual application as a fertilizer. Nevertheless its importance as a plant nutrient cannot be ignored.
... The stimulatory effect of calcium chloride on lettuce growth may be attributed to the fact that calcium ions (Ca2+) appeared to participate in the regulation of several aspects of cell division. Calcium is a necessary ion in the formation of the mitotic spindle which directly affects cell division (Hepler, 1994). ...
THE PRESENT study was carried out during the summer seasons of 2017 and 2018 in Talkha city, Dakahlia Governorate, Egypt near El-Mansoura-Damietta highway and El Delta Company for Fertilizers and Chemical Industries, where lead (Pb) and copper (Cu) levels in the soil exceed the permissible levels. Therefore, the aim of this study was to alleviate the adverse effects of heavy metals (HMs) on tomato plant (Solanumlycopersicum L.) Super Strain B F1. Twelve treatments were conducted which were the interactions between two silicon (Si) foliar spraying (without or 300 ppm Si) and six soil amendments : 100% chemical fertilizers (CF), 50% farmyard manure (FYM), 100% FYM, 100% CF+ Trichoderma spp. (T.), 50% FYM + T. and 100% FYM + T. Data indicate that Pb and Cu were accumulated in tomato organs to hazardous levels in the following descending order : roots > shoots > fruits and shoots > roots > fruits, respectively which consequently severely reduced tomato plant growth and fruit yield and fruit quality. Results also, showed that the application of Si treatment sole or in combination with soil amendments markedly enhanced plant height, dry weight, total chlorophyll, number of fruits/plant, total yield/fed, Vitamin C, acidity %, TSS and taste index, where as, reduced Pb and Cu concentrations in different plant organs, bio-concentration factor (BCF) and translocation factor (TF). However, Si + FYM + T. treatment was superior in these respects, therefore increased the net return. Also, negative correlations were obtained between fruit yield and BCF of Pb or Cu and between taste index and TF of Pb or Cu.
... Furthermore, various functional experiments manipulating cytoplasmic Ca 2+ during mitosis in these model systems strongly argued for an essential role for Ca 2+ during normal mitotic progression (10,(12)(13)(14). When similar experiments were attempted in somatic mammalian cells the results were inconclusive (15)(16)(17)(18)(19)(20) and, in particular, the presence of mitotic Ca 2+ signals could not be confirmed despite the fact that Ca 2+ -binding proteins including calmodulin are required (21,22). ...
Mitosis defects can lead to premature ageing and cancer. Understanding mitosis regulation therefore has important implications for human disease. Early data suggested that calcium (Ca²⁺) signals could influence mitosis, but these have hitherto not been observed in mammalian cells. Here, we reveal a prolonged yet spatially restricted Ca²⁺ signal at the centrosomes of actively dividing cells. Local buffering of the centrosomal Ca²⁺ signals, by flash photolysis of the caged Ca²⁺ chelator diazo‐2‐acetoxymethyl ester, arrests mitosis. We also provide evidence that this Ca²⁺ signal emanates from the endoplasmic reticulum. In summary, we characterize a unique centrosomal Ca²⁺ signal as a functionally essential input into mitosis.—Helassa, N., Nugues, C., Rajamanoharan, D., Burgoyne, R. D., Haynes, L. P. A centrosome‐localized calcium signal is essential for mammalian cell mitosis. FASEB J. 33, 14602‐14610 (2019). www.fasebj.org
... These values are subject to 48 change, in particular upon cell stimulation when calcium-selective ion channels 49 open and allow for Ca 2+ influx; thus, setting the basis for calcium signaling as 50 an intricate signal transduction network. Furthermore, calcium signaling is involved 51 in muscle contraction, cell growth, cellular motility, synaptic plasticity, but can also 52 impact apoptosis, the permeability of ion channels and the cytoskeleton [10][11][12][13][14][15][16][17][18]. 53 The concept of Ca 2+ signaling and the importance of Ca 2+ as a ubiquitous 54 second messenger became apparent more than 100 years ago (circa 1883) when 55 studies on heart cells demonstrated that the presence of Ca 2+ was necessary for 56 the contraction of cardiomyocytes [18]. ...
The pioneering work of Richard Altman on the presence of mitochondria in cells set in motion a field of research dedicated to uncovering the secrets of the mitochondria. Despite limitations in studying the structure and function of the mitochondria, advances in our understanding of this organelle prompted the development of potential treatments for various diseases, from neurodegenerative conditions to muscular dystrophy and cancer. As the powerhouses of the cell, the mitochondria represent the essence of cellular life and as such, a selective advantage for cancer cells. Much of the function of the mitochondria relies on Ca²⁺ homeostasis and the presence of effective Ca²⁺ signaling to maintain the balance between mitochondrial function and dysfunction and subsequently, cell survival. Ca²⁺ regulates the mitochondrial respiration rate which in turn increases ATP synthesis, but too much Ca²⁺ can also trigger the mitochondrial apoptosis pathway; however, cancer cells have evolved mechanisms to modulate mitochondrial Ca²⁺ influx and efflux in order to sustain their metabolic demand and ensure their survival. Therefore, targeting the mitochondrial Ca²⁺ signaling involved in the bioenergetic and apoptotic pathways could serve as potential approaches to treat cancer patients. This chapter will review the role of Ca²⁺ signaling in mediating the function of the mitochondria and its involvement in health and disease with special focus on the pathophysiology of cancer.
... The increased seedling growth due to the CaCl 2 and KNO 3 pretreatment might be because of the amplified nuclear copying in dividing cells (Mavi et al. 2006). Calcium ions are interconnected with numerous vital mitotic incidents, mainly in the metaphase-anaphase transition and breakdown of nuclear membranes (Hepler 1994). The Ca 2+ level is intimately linked to the inception of cytoplasmic division (Chang and Meng 1995). ...
Intensified salt stress is an acute hindrance to crop cultivation, whereas plant signaling molecules can efficiently prompt salinity tolerance. Therefore, this study was accomplished to explore the potential salinity stress-mitigating effect of different signaling molecules in rice. The rice (cv. BRRI dhan29) seeds were immersed in 20 mM KNO3, 0.15 mM H2O2, 0.8 mM AsA (ascorbic acid) and 10 mM CaCl2 solutions for 24 h. Eventually, primed seeds were exposed to 75 mM NaCl in Petri dishes during germination. Moreover, 14-day-old rice seedlings were pretreated with different agents, viz., KNO3, H2O2, AsA and CaCl2 (concentrations were same as previous), for 2 days. Primed and non-primed seedlings were grown for 4 days under 75 mM NaCl stress condition. The result revealed that salt stress caused reduced germination indices and pre-seedling and seedling growth inhibition and impaired photosynthetic capacity, whereas catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX) activities were decreased in salt-treated plants. However, application of the four signaling molecules promoted the germination indices and growth and resisted chlorosis. Pretreatment with CaCl2 and AsA was observed to be relatively more efficient in conferring salinity tolerance of rice as reflected from the significant enhanced germination and growth in the saline medium by increasing reactive oxygen species (ROS) scavenging capacity, both at germination and seedling stage. All the selected signaling molecules significantly detoxified excess ROS, i.e., H2O2 and \({\text{O}}_{2}^{ \cdot - }\) and reduced lipid peroxidation by up-regulating the enzymes, CAT, APX and POX. Moreover, H2O2 and KNO3 pretreatment also mitigated salt-imposed oxidative stress and enhanced growth performance of rice seedlings. Overall, the study confirms that CaCl2 and AsA pretreatment were more effective than H2O2 and KNO3 priming to improve salt tolerance in rice.
... However, exogenous treatment with Ca 2+ counterattacked the adverse consequences of salinity of growth ( Fig. 1 and Tables 2, 3). Ca 2+ ions are interconnected with several vital mitotic events, including breakdown of nuclear envelope and metaphase-anaphase transition (Hepler 1994). It also activates calmodulins and increases cytokinine production, which can augment cell division (Snedden and Fromm 2001), plant growth, and biomass production. ...
... Calcium ions play an important role in cellular processes such as transcription, motility, exocytosis and apoptosis [68]. Intracellular calcium acts as a secondary messenger [69,70] and its concentration is strictly regulated by the cell [71]. A toxic effect occurs when more cells are dying than are produced by cell division, and is connected to apoptosis, i.e., programmed cell death. ...
Nanomaterials, such as hydroxyapatite nanoparticles show a great promise for medical applications due to their unique properties at the nanoscale. However, there are concerns about the safety of using these materials in biological environments. Despite a great number of published studies of nanoobjects and their aggregates or agglomerates, the impact of their physicochemical properties (such as particle size, surface area, purity, details of structure and degree of agglomeration) on living cells is not yet fully understood. Significant differences in these properties, resulting from different manufacturing methods, are yet another problem to be taken into consideration. The aim of this work was to investigate the correlation between the properties of nanoscale hydroxyapatite from different synthesis methods and biological activity represented by the viability of four cell lines: A549, CHO, BEAS-2B and J774.1 to assess the influence of the nanoparticles on immune, reproductive and respiratory systems.
... Inorganic ions are indispensable for many biological processes in vivo, such as photosynthesis, 1 neuronal signaling, 2 cell division, 3 and controlling the cell volume. 4 Most of these processes are very selective towards inorganic ions and this ion-specificity is believed to originate from ion-protein interactions. ...
We have exploited glycine as zwitterionic model system to experimentally probe the cation specific interaction of protein surface charges in dilute (≤ 0.25 mol/lit) aqueous solutions of four biologically relevant inorganic salts, NaCl, KCl, MgCl2 and CaCl2, via dielectric relaxation spectroscopy (DRS) and Raman spectroscopy. Glycine is the simplest building block of proteins and exposes the same charged groups (carboxylate and ammonium) to the solvent that dominate the protein-water interface. As counter ion we have selected Cl- due to its biological importance. For all systems, we performed simultaneous fitting of the real (ε') and imaginary (ε'') parts of the dielectric functions, assuming a multimodal relaxation model, obtained from concentration dependent dielectric measurements at ~293 K. We observe a reduction of the dielectric amplitude for the glycine relaxation while the corresponding time constant shows only small (<7%) deviations compared to aqueous glycine solutions. We propose that the observed reduction in dielectric amplitude is due to a reduction of the effective dipole moment ( μeff) of zwitterionic glycine by the interaction of the glycine with the ions even at very low (0.05M) salt concentrations. The interaction between divalent metal ions and zwitterionic glycine is increased compared to the monovalent cation-zwitterion interaction; a finding which is also supported by Raman spectroscopy. Our dielectric relaxation and Raman spectroscopic study indicate that ion-glycine interactions are weak and mediated by the solvent. Cation-specificity of protein surface charges is also observed in dilute salt solutions (≤ 0.25 mol/lit), where electrostatic interactions dominate.
... Furthermore, various functional experiments manipulating cytoplasmic Ca 2+ during mitosis in these model systems strongly argued an essential role for Ca 2+ during normal mitotic progression (Groigno & Whitaker, 1998, Steinhardt & Alderton, 1988, Twigg, Patel et al., 1988, Wilding, Wright et al., 1996. When similar experiments were attempted in somatic mammalian cells the results were inconclusive (Hepler, 1994, Kao, Alderton et al., 1990 is the author/funder. All rights reserved. ...
To generate a complex multicellular organism like a human requires enormous expansion in cell numbers and this is achieved predominantly through mitosis. Defects in mitosis can lead to premature ageing and cancer so understanding how it is regulated has important implications for human disease. Early data from plant and invertebrate model systems indicated that calcium (Ca ²⁺ ) could influence mitosis. Here we explore this key question in the cell biology of mammalian cells by targeting high affinity genetically encoded Ca ²⁺ sensors to mitosis specific subcellular locations. We reveal a prolonged yet spatially restricted Ca ²⁺ signal at the centrosomes of mitotic cells using an actin-targeted Ca ²⁺ sensor. Local depletion of Ca ²⁺ at centrosomes using flash-photolysis of the caged Ca ²⁺ chelator diazo-2 arrests mitosis and we provide evidence that this signal emanates from the endoplasmic reticulum. In summary, we characterize a centrosomal Ca ²⁺ signal as a functionally essential input into mitosis. This extends our understanding of the complex regulatory network controlling cell division and pinpoints Ca ²⁺ as an important controller of this fundamental process.
... These ions can have varying effects on the growth and development of bacteria. For example, Ca 2þ and Mg 2þ are both essential in the regulation of several steps of cell division (Hepler 1994;Webb 1949). Somerton et al. (2015) showed that supplementation of a milk formula with as low as 2 mM CaCl 2 or 2 mM MgCl 2 increased the biofilm formation of three Geobacillus spp while 100 mM NaCl significantly decreased biofilm formation showing that high free Na þ ions and low Ca 2þ and Mg 2þ were collectively needed to reduce biofilm formation. ...
Biofilm formation in dairy wastewater system irrigation pipes can reduce treatment capacity, increasing maintenance and cleaning costs. Understanding the effect of different components in the wastewater on growth and yield of bacteria present could help prevent excessive build-up of biofilms. This study investigated, in aerobic and anaerobic conditions, the effect of calcium, sodium and magnesium concentrations on growth rates, yields and saturation constants of four known biofilm forming bacteria associated with the blockage of an irrigation system. The ions tested (Ca²⁺, Mg²⁺, Na⁺) varied growth rates with biofilm growth in the presence of calcium being significantly slower (P < 0.05) than planktonic growth in sodium. Along with the slower growth, the addition of Ca²⁺ (up to 0.1 M) increased biofilm formation while addition over 0.5 M prevented biofilm formation. Knowing the nutritional requirements of the bacteria and the effects of the ions will be useful in predicting the growth, development and strategies in controlling biofilm formation in dairy wastewater.
... Beyond favoring the release of T cells from APCs, we provided evidence that such unresponsiveness is beneficial for effective clonal expansion because we showed that strong TCR signals antagonize cell division. Ca 2+ has been proposed to participate in several processes during mitosis (Hepler, 1994), and it may be important to limit external perturbations and stimuli that would interfere with these processes (Arredouani et al., 2010). Consistent with this idea, SOCE has been shown to be specifically down-regulated in mitotic HeLa cells (Preston et al., 1991;Smyth et al., 2009). ...
T cells are primed in secondary lymphoid organs by establishing stable interactions with antigen-presenting cells (APCs). However, the cellular mechanisms underlying the termination of T cell priming and the initiation of clonal expansion remain largely unknown. Using intravital imaging, we observed that T cells typically divide without being associated to APCs. Supporting these findings, we demonstrate that recently activated T cells have an intrinsic defect in establishing stable contacts with APCs, a feature that was reflected by a blunted capacity to stop upon T cell receptor (TCR) engagement. T cell unresponsiveness was caused, in part, by a general block in extracellular calcium entry. Forcing TCR signals in activated T cells antagonized cell division, suggesting that T cell hyporesponsiveness acts as a safeguard mechanism against signals detrimental to mitosis. We propose that transient unresponsiveness represents an essential phase of T cell priming that promotes T cell disengagement from APCs and favors effective clonal expansion.
... The stimulatory effect of calcium chloride on lettuce growth may be attributed to the fact that calcium ions (Ca2+) appeared to participate in the regulation of several aspects of cell division. Calcium is a necessary ion in the formation of the mitotic spindle which directly affects cell division (Hepler, 1994). ...
Salicylic acid (SA) and Ca2+ regulate the physiological and biochemical mechanisms
in plants. A factorial experiment was designed to evaluate the effect of foliar application
of calcium chloride (0, 10, 20, 30 or 40 mM) and salicylic acid (0, 0.5, 1, 1.5 or 2 mM) on
the vegetative growth of romaine lettuce cv. Balady during 2013/2014 and 2014/2015 seasons
at the Experimental Farm of Horticulture Department, Faculty of Agriculture, Ain Shams
University, Qalubia Governorate, Egypt. Results clearly indicated positive effects of foliar
applications of calcium chloride and salicylic acid either alone or in combination on lettuce
growth, productivity and some physiological parameters. Foliar application of calcium chloride
at 20 mM significantly increased vegetative growth parameters (plant length, head diameter,
fresh and dry weights of head, number of leaves/head, average leaf area and leaf area index),
chlorophyll (a, b, and total), leaf relative water content, leaf membrane stability index, and
macro- and micro-nutrients. Moreover, salicylic acid spraying at 1.5 mM significantly gave the
highest significant values of all aforementioned parameters. Exogenous applications of calcium
chloride and salicylic acid either alone or in combination reduced nitrate accumulation in the
leaves. Spraying of calcium chloride at 20 mM with salicylic acid at 1.5 mM was the most
effective treatment which can be used as an applicable practice in romaine lettuce cv. Balady
cultivation.
... 9 Cytosolic Ca 2+ serves as a universal second messenger that not only control apoptosis but also influences cell division/growth and gene expression. 10,11 Furthermore, abnormal Ca 2+ homeostasis has been linked to several pathologies including lung, prostate, breast, skin and colon cancers. 12 We and others have previously shown that Calu-3 pulmonary epithelial cells show similar, Ca 2+ -dependent inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) as seen in humans, suggesting that they are a good model for tobacco exposure. ...
Addition of flavors reduces the harsh taste of tobacco, facilitating the initiation and maintenance of addiction among youths. Flavored cigarettes (except menthol) are now banned. However, the legislation on little cigars remains unclear and flavored little cigars are currently available for purchase. Since inhaled tobacco smoke directly exerts toxic effects on the lungs, we tested whether non-flavored and flavored little cigar smoke exposure had the potential for harm in cultured pulmonary epithelia. We cultured Calu-3 lung epithelia on both 96-well plates and at the air–liquid interface and exposed them to smoke from non-flavored Swisher Sweets and flavored (sweet cherry, grape, menthol, peach and strawberry) Swisher Sweets little cigars. Irrespective of flavor, acute little cigar smoke exposure (10×35 ml puffs) significantly increased cell death and decreased the percentage of live cells. Chronic exposure (10×35 ml puffs per day for 4 days) of smoke to Calu-3 cultures significantly increased lactate dehydrogenase release, further indicating toxicity. To determine whether this exposure was associated with increased cell death/apoptosis, a protein array was used. Chronic exposure to smoke from all types of little cigars induced the activation of the two major apoptosis pathways, namely the intrinsic (mitochondrial-mediated) and the extrinsic (death receptor-mediated) pathways. Both flavored and non-flavored little cigar smoke caused similar levels of toxicity and activation of apoptosis, suggesting that flavored and non-flavored little cigars are equally harmful. Hence, the manufacture, advertisement, sale and use of both non-flavored and flavored little cigars should be strictly controlled.
... We can hypothesize that the depolarizing effect of glycine would lead to intracellular Ca 21 increase that in turn may modulate neural differentiation. Indeed, Ca 21 is a well-known modulator of the cell cycle (Hepler, 1994) and Ca 21 increase can promote the expression of genes involved in the CNS development and can enhance neurogenesis and interneuron differentiation (Ghosh et al., 1994;Rosen et al., 1995;Bito et al., 1997;Ginty, 1997;Brustein et al., 2013). A recent review about granule cell migration in the cerebellum highlights the role of Ca 21 in this process and there are some similarities with the cortex (Komuro et al., 2015). ...
The development of the neocortex requires the synergic action of several secreted molecules to achieve the right amount of proliferation, differentiation and migration of neural cells. Neurons are well known to release neurotransmitters (NTs) in adult and a growing body of evidences describes the presence of NTs already in the embryonic brain, long before the generation of synapses. NTs are classified as inhibitory or excitatory based on the physiological responses of the target neuron. However, this view is challenged by the fact that glycine and GABA NTs are excitatory during development. Many reviews have described the role of non-hyperpolarizing GABA at this stage. Nevertheless, a global consideration of the inhibitory neurotransmitters and their downstream signalling during the embryonic cortical development is still needed. For example taurine, the most abundant neurotransmitter during development is poorly studied regarding its role during cortical development. In the light of recent discoveries, we will discuss the functions of glycine, GABA and taurine during embryonic cortical development with an emphasis on their downstream signalling. This article is protected by copyright. All rights reserved.
... In addition, depletion of Ca 2+ during mitosis led to a threefold increase in the number of cells with chromosome misalignment (Fig. 1b). In agreement with previous reports 26,27 , these results clearly indicated that Ca 2+ is required for proper mitotic progression. ...
Chromosome condensation is essential for the faithful transmission of genetic information to daughter cells during cell division. The depletion of chromosome scaffold proteins does not prevent chromosome condensation despite structural defects. This suggests that other factors contribute to condensation. Here we investigated the contribution of divalent cations, particularly Ca2+, to chromosome condensation in vitro and in vivo. Ca2+ depletion caused defects in proper mitotic progression, particularly in chromosome condensation after the breakdown of the nuclear envelope. Fluorescence lifetime imaging microscopy-Förster resonance energy transfer and electron microscopy demonstrated that chromosome condensation is influenced by Ca2+. Chromosomes had compact globular structures when exposed to Ca2+ and expanded fibrous structures without Ca2+. Therefore, we have clearly demonstrated a role for Ca2+ in the compaction of chromatin fibres.
... The crystals can represent 6.3% of the leaf dry weight (Zindler-Frank, 1976) and up to 90% of the total Ca in leaves (Fink, 1991). Calcium accumulated in the form of oxalate maintains a low level of free Ca in the cytoplasm (less than 10 −7 M) to prevent interference in the Ca-dependent signals (Bush, 1995) in the energy metabolism of the phosphates (Kretsinger, 1977) and in microskeletal dynamics (Hepler, 1994), but maintains a continuous supply of Ca for the metabolic process in the plant. This process leads to the assumption that the high concentration of leaf Ca, which is optimally 3.73%-5.73% ...
Iron (Fe) chlorosis reduces the concentration of photosynthetic pigments, photosynthates, and crop yield. The effect of Fe chlorosis on leaf composition and cell structure wasevaluatedinMexicanlime(Citrusaurantifolia)withdifferentdegreesofFechlorosis. Iron chlorosis significantly reduced concentrations of chlorophylls a, b, and a+b, andcausedthickeningofleaves,duetotheincreaseinpalisadeandspongyparenchyma cells. The chloroplasts of the chlorotic and albino leaves showed a disorganized ultrastructure; they had an elongated shape with disarrayed thylakoids, underdeveloped grana,scarcestarchgranules,andhole-likefoldsinthethylakoidmembranes.Theaccumulationofcalciumoxalatecrystalsintheupperandlowersidesoftheepidermis,crystal length, and total crystal content increased with Fe chlorosis severity. The green leaves, incontrast,hadchloroplastswithtypicalultrastructure.ThedegreeofFechlorosisinthe leaves significantly affected the concentrations of potassium (K); Fe, manganese (Mn), Fe2+, and the phosphorus (P)/Fe and K/calcium (Ca) ratios.
... In normally dividing cells of angiosperms, ER undergoes a series of morphological changes, coupled with the successive mitotic phases, and is believed to be involved in controlling the local concentration of the cytosolic calcium during cell division (Hepler, 1994; Gunning and Steer, 1996). In untreated roots of A. cephalonica distinct ER systems were organized at each stage of the cell cycle, reflecting those of the correspondent MT arrays and displaying similar features with those described for other gymnosperms (Zachariadis et al., 2001Zachariadis et al., , 2003). ...
... Alternatively, stunted growth and development as observed when FhCaM function was disrupted by both molecular genetic and pharmacological means, does represent a desirable therapeutic outcome. This might reflect either: (i) FhCaM's direct involvement in controlling Ca 2+ dynamics in dividing cells (Ca 2+ fluxes have a well-established role in the cell cycle; see [45,46]) where disrupting CaM function could directly inhibit cell division; or (ii) FhCaMs are important housekeeping proteins required for normal cellular function [47], such that FhCaM's impact on growth occurs via a more general impact on cellular health/function in FhCaM-expressing cells. Although the data presented in this study cannot further delineate between these possibilities, growth suppression could represent an appealing therapeutic outcome following FhCaM-selective drug intervention. ...
Background
Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.
Methods
In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.
Results
Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.
Conclusions
These data indicate that the Ca2+-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.
Electronic supplementary material
The online version of this article (doi:10.1186/s13071-016-1324-9) contains supplementary material, which is available to authorized users.
... CaOx precipitates in plants are believed to regulate the free calcium concentration. [1,4] To keep the free Ca 2 + concentration in the cytosol below the critical level of interference with other cell processes (e.g.,C a-dependent signaling [15] or phosphate-based energy metabolism [16] )e xcess Ca 2 + is stored as crystallinec alcium oxalate that exists in hydrated forms known as monoclinic whewellite (monohydrate, COM), [17] tetragonal weddellite (dihydrate,C OD), [18,19] and triclinic caoxite (trihydrate, COT). [20] The thermodynamically stable form is the monohydrate;i na queous solutionst he dihydrate can only develop under special conditions. ...
The mechanisms by which amorphous intermediates transform into crystalline materials are not well understood. To test the viability and the limits of the classical crystallization, new model systems for crystallization are needed. With a view to elucidating the formation of an amorphous precursor and its subsequent crystallization, the crystallization of calcium oxalate, a biomineral widely occurring in plants, is investigated. Amorphous calcium oxalate (ACO) precipitated from an aqueous solution is described as a hydrated metastable phase, as often observed during low-temperature inorganic synthesis and biomineralization. In the presence of water, ACO rapidly transforms into hydrated whewellite (monohydrate, CaC2 O4 ⋅H2 O, COM). The problem of fast crystallization kinetics is circumvented by synthesizing anhydrous ACO from a pure ionic liquid (IL-ACO) for the first time. IL-ACO is stable in the absence of water at ambient temperature. It is obtained as well-defined, non-agglomerated particles with diameters of 15-20 nm. When exposed to water, it crystallizes to give (hydrated) COM through a dissolution/recrystallization mechanism.
A secondary ion mass spectrometry (SIMS) based isotopic imaging technique of ion microscopy was used for observing calcium influx in single renal epithelial LLC‐PK1 cells. The CAMECA IMS‐3f SIMS instrument, used in the study, is capable of producing isotopic images of single cells at 500 nm spatial resolution. Due to the high vacuum requirements of the instrument the cells were prepared cryogenically with a freeze‐fracture method and frozen freeze‐dried cells were used for SIMS analysis. The influx of calcium was imaged directly by exposure of cells to 44Ca stable isotope in the extracellular buffer for 10 min. The 44Ca influx was measured at mass 44 and the distribution of endogenous calcium at mass 40 (40Ca) in the same cell. A direct comparison of interphase cells to cells undergoing division revealed that calcium influx is restricted in metaphase and post‐metaphase stages of cell division. This restriction is lifted in late cytokinesis. The net influx of 44Ca in 10 min. was approximately half under calcium influx restriction in comparison to interphase cells. Under calcium influx restriction the 44Ca concentration was the same between the mitotic chromosome and the cytoplasm. These observations indicate that the endoplasmic reticulum (ER) calcium uptake is compromised under calcium influx restriction in cells undergoing division. This article is protected by copyright. All rights reserved
The specific mechanisms by which Ca2+ affects vegetative growth of plants in karst area are still unclear. The aim of the current experiment was to study the effects of excessive Ca2+ on physiological characteristics, proteins and metabolites of leaves of two-year old Drynaria roosii for 14 days and provide a theoretical reference for analyzing the mechanisms of the interaction between high concentration Ca2+ and vegetative growth of plants in karst area. We sought to investigate the effect of 0 mM Ca2+, 600 mM Ca2+, and 1200 mM Ca2+ on the properties of leaves of D. roosii. Physiological analysis reveals that excessive Ca2+ might inhibit the growth of D. roosii leaves by osmotic stress. Proteomics analysis suggests that differentially expressed proteins mainly involved in protein metabolism, amino acid metabolism, carbohydrate and energy metabolism, photosynthesis, antioxidant and defense. Metabolomics analysis indicates that differentially expressed metabolites mainly involved in amino acid metabolism, lignin biosynthesis, and flavonoid biosynthesis. These results give insights into the biological processes apparently involved in Ca2+ tolerance in this species.
Cytokinesis in plants is fundamentally different from that in animals and fungi. In plant cells, a cell plate forms through the fusion of cytokinetic vesicles and then develops into the new cell wall, partitioning the cytoplasm of the dividing cell. The formation of the cell plate entails multiple stages that involve highly orchestrated vesicle accumulation, fusion, and membrane maturation, which occur concurrently with the timely deposition of polysaccharides such as callose, cellulose, and cross‐linking glycans. This review summarizes the major stages in cytokinesis, endomembrane components involved in cell plate assembly and its transition to a new cell wall. An animation that can be widely used for educational purposes further summarizes the process. Cytokinesis in plants is fundamentally different from animals and fungi. In plant cells, a cell plate forms through the fusion of cytokinetic vesicles and then develops into the new cell wall, partitioning the cytoplasm of the dividing cell. We discuss the major stages in plant cytokinesis, endomembrane components involved in cell‐plate assembly and the transition to a new cell wall, and provide an animation of cytokinesis for educational purposes.
Ca²⁺ is an essential nutrient for plants and animals which plays an important role in plant signal transduction. Although the function and regulation of mechanism of Ca²⁺ in alleviating various biotic and abiotic stresses in plants have been studied deeply, the molecular mechanism to adapt high Ca²⁺ stress is still unclear in cotton. In this study, 103 cotton accessions were germinated under 200 mM CaCl2 stress, and two extremely Ca²⁺-resistant (Zhong 9807, R) and Ca²⁺-sensitive (CRI 50, S) genotypes were selected from 103 cotton accessions. The two accessions were then germinated for 5 days in 0 mM CaCl2 and 200 mM CaCl2 respectively, after which they were sampled for transcriptome sequencing. Morphological and physiological analyses suggested that PLR2 specifically expressed in R may enhance the ability of cotton to scavenge ROS by promoting the synthesis of SDG. In conclusion, this study proposed the adaptation mechanisms to response to the high Ca²⁺ stress in cotton which can contribute to improve the stress resistance of cotton.
The specificity of anionic phospholipids-calcium ion interaction and lipid demixing has been established as a key regulatory mechanism in several cellular signaling processes. The mechanism and implications of this calcium-assisted demixing have not been elucidated from a microscopic point of view. Here, we present an overview of atomic interactions between calcium and phospholipids that can drive non-ideal mixing of lipid molecules in a model lipid bilayer composed of zwitterionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine: POPC) and anionic (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine: POPS) lipids with computer simulations at multiple resolutions. Lipid nanodomain formation and growth was driven by calcium-enabled lipid bridging of the charged PS head groups, which were favored against inter-POPS dipole interactions. Consistent with several experimental studies of calcium associated membrane sculpting, our analyses also suggest modifications in local membrane curvature and cross-leaflet couplings as a response to such induced lateral heterogeneity. In addition, reverse-mapping to a complementary atomistic description revealed structural insights in presence of anionic nanodomains, at timescales not accessed by previous computational studies. This work bridges information across multiple scales to reveal a mechanistic picture of calcium ion’s impact on membrane biophysics.
Salinity is one of the most brutal abiotic stressors, commencing a great stumbling block in the way of attaining food security in Bangladesh. Cultivation of rice in saline soils can be possible after enhancing its salt tolerance. This study aimed to examine the potential impact of exogenous calcium (3 and 5 mM Ca²⁺ in CaCl2) on conferring salt tolerance in rice (cv. BRRI dhan28). At the germination stage, Ca²⁺-primed seeds were grown under 100 mM NaCl stress conditions for nine days. At the seedling stage, rice seedlings were grown in a sandponic culture with Hoagland′s nutrient solution amended or not amended with 100 mM NaCl for 20 days with or without Ca²⁺ supplementation. Our results revealed that NaCl-stressed rice plants showed highly compromised germination indices and growth parameters, which could be attributed to reduced shoot and root growth, decreased photosynthetic pigments, increased H2O2 accumulation, and elevated levels of lipid peroxidation measured as malondialdehyde (MDA). On the other hand, exogenous Ca²⁺ application noticeably improved germination indices, growth and biomass-related parameters under salt stress. Ca²⁺-treated salt-stressed plants displayed amplified chlorophyll content, as well as suppressed the accumulation of H2O2, contributing to oxidative damage protection. Ca²⁺ supplementation for salt-stressed rice seedlings elevated relative water content without increasing excess proline, indicating the role of Ca²⁺ in maintaining water balance under stressful conditions. Furthermore, exogenous Ca²⁺ decreased membrane injury under NaCl stress, as mirrored by notably diminished levels of MDA in stressed seedlings. The defensive role of Ca²⁺ counter to oxidative stress was connected with the elevated activities of antioxidant enzymes such as catalase, ascorbate peroxidase, and peroxidase. In general, the best results in terms of growth at both germination and seedling stages were obtained in response to 3 mM Ca²⁺ treatment. Finally, Ca²⁺ supplementation can be an effective practice to cultivate rice in saline soils.
The present study deals with the assessment of genotoxic effects of the two most commonly used nitrogen (N) fertilizers, ammonium nitrate and urea, using the Allium cepa L. root mitosis bioassay system. Allium cepa mitosis bioassay is known for its sensitivity and it is a classical test for studying chemical toxicity. A pot experiment was set up, in which ammonium nitrate and urea were added to soil at the rate of 200 mg N kg⁻¹ soil. Roots from onion bulbs were fixed in the first and second week after treatment (WAT). Cytological analysis revealed dividing cells and different types of dividing cell anomalies and interphase nuclear anomalies. Per cent mitotic index and per cent anomalies in dividing cells were significantly higher in ammonium nitrate treated root tip cells. Urea proved to be mitodepressive in both weeks. Anomalies in dividing cells and interphase nuclear anomalies were also observed in the second WAT in both treatments. Per cent interphase nuclear anomalies were higher in case of urea. Therefore, nitrogen fertilizers should be used judiciously, to avoid or minimize the genotoxic risks.
Aluminium toxicity in plants appears to be related to interactions between Al and Ca, including inhibition of Ca uptake by blocking the plasma membrane Ca2+ channels; displacement of apoplasmic Ca2+ and disturbance of intracellular Ca2+ homeostasis. Several lines of evidence have indicated that inhibition of Ca2+ uptake and desorption of apoplasmic Ca2+ may not be primary causes of Al toxicity in plants. The involvement of disturbance of intracellular Ca2+ homeostasis of intact root apical cells in Al toxicity remains to be demonstrated experimentally. With the Ca2+-sensitive fluorescent probe Fluo-3 and the confocal laser scanning microscopy technique, a sustained increase in Fluo-3 fluorescence was observed in intact root apical cells of wheat in the presence of AlC13, suggesting Al elicited an increase in cytosolic Ca2+ activity. The increase in cytosolic Ca2+ activities was greater in Al-sensitive than in Al-tolerant isogenic wheat lines. These findings provide unambiguous evidence in support of disturbance of intracellular Ca2+ homeostasis being involved in Al toxicity in plants. This increase in the cytosolic Ca2+ activities may serve as a trigger to alter a number of biochemical processes by disrupting Ca2+-mediated signal transduction system and/or Ca2+-dependent cytoskeletal dynamics.
Ionized calcium (Ca2+) is the most common signal transduction element in cells ranging from bacteria to specialized neurons. Unlike many other second-messenger molecules, Ca2+ is required for life, yet prolonged high intracellular Ca2+ levels lead to cell death.1
Myosins are a large superfamily of actin- and ATP-dependent motor proteins, which have been divided (on the basis of their conserved head or motor domain) into at least fifteen classes. Three of these classes (class VIII, XI and XIII) are exclusively expressed in plants, and there are now at least 26 plant myosin sequences available. Whereas the myosins in animal and amoeboid cells have been studied in detail, little is known about the function(s) of the myosins in plant cells. Preliminary studies indicate that class XI myosins are involved in the rapid movement of cytoplasm — cytoplasmic streaming — while class VIII myosins are associated with the plasma membrane and plasmodesmata at the post-cytokinetic cell wall. Thus, each of the myosin classes is expected to have distinct functions but within each class the myosins may have overlapping functions. It will be interesting to test this proposal and elucidate the intracellular functions of the different classes of myosins within a single species.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, “quin2”, the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
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.
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.
Increases in intracellular calcium concentration are required for the release of neurotransmitter from presynaptic terminals
in all neurons. However, the mechanism by which calcium exerts its effect is not known. A low-sensitivity calcium-dependent
photoprotein (n-aequorin-J) was injected into the presynaptic terminal of the giant squid synapse to selectively detect high
calcium concentration microdomains. During transmitter release, light emission occurred at specific points or quantum emission
domains that remained in the same place during protracted stimulation. Intracellular calcium concentration microdomains on
the order of 200 to 300 micromolar occur against the cytoplasmic surface of the plasmalemma during transmitter secretion,
supporting the view that the synaptic vesicular fusion responsible for transmitter release is triggered by the activation
of a low-affinity calcium-binding site at the active zone.
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.
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.
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.
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.
Several lines of evidence support the idea that increases in the intracellular free calcium concentration [( Ca2+]i) regulate chromosome motion. To directly test this we have iontophoretically injected Ca2+ or related signaling agents into Tradescantia stamen hair cells during anaphase and measured their effect on chromosome motion and on the Ca2+ levels. Ca2+ at (+)1 nA for 10 s (approximately 1 microM) causes a transient (20 s) twofold increase in the rate of chromosome motion, while at higher levels it slows or completely stops motion. Ca2+ buffers, EGTA, and 5,5'-dibromo-1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, which transiently suppress the ion level, also momentarily stop motion. Injection of K+, Cl-, or Mg2+, as controls, have no effect on motion. The injection of GTP gamma S, and to a lesser extent GTP, enhances motion similarly to a low level of Ca2+. However, inositol 1,4,5-trisphosphate, ATP gamma S, ATP, and GDP beta S have no effect. Measurement of the [Ca2+]i with indo-1 reveals that the direct injections of Ca2+ produce the expected increases. GTP gamma S, on the other hand, causes only a small [Ca2+]i rise, which by itself is insufficient to increase the rate of chromosome motion. Further studies reveal that any negative ion injection, presumably through hyperpolarization of the membrane potential, generates a similar small pulse of Ca2+, yet these agents have no effect on motion. Two major conclusions from these studies are as follows. (a) Increased [Ca2+]i can enhance the rate of motion, if administered in a narrow physiological window around 1 microM; concentrations above 1 microM or below the physiological resting level will slow or stop chromosomes. (b) GTP gamma S enhances motion by a mechanism that does not cause a sustained uniform rise of [Ca2+]i in the spindle; this effect may be mediated through very localized [Ca2+]i changes or Ca2(+)-independent effectors.
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.
Transient elevations in intracellular free Ca2+ are believed to signal the initiation of mitosis. This model predicts that mitosis might be arrested prior to nuclear envelope breakdown (NEB) or anaphase onset if intracellular Ca2+ concentration is buffered or dampened. Microinjection of a discrete dose of Ca2+ into the cell might then release the cell to resume mitotic cycling. Experimentally, one blastomere of two cell sand dollar (Echinaracnius parma) embryos was microinjected with Ca2+ buffers, Ca2+ solutions, or Ca2+ channel antagonists; the uninjected blastomere was the control. Cells were loaded with 10 pl doses of the Ca2+ buffer antipyrylazo III (ApIII) at specific times in the cell cycle to attempt a competitive inhibition of Ca2+-dependent steps in NEB and initiation of mitosis. Injection of 50 microM ApIII 6 min prior to NEB blocked NEB and further cell cycling. Injections of solutions between 0 and 30 microM ApIII were without observable effect. Control injections had no observable effect on the injected cell. Cells injected with 50 microM ApIII 2 min prior to the onset of anaphase in control cells were blocked in metaphase. Cells were sensitive to Ca2+ buffer injections 6 min prior to NEB (with a 40- to 45-sec duration), and 2 min prior to anaphase onset (with a 10- to 20-sec duration). Vital staining of these cells with H33342 demonstrated that they contained only one nucleus that had the same fluorescence intensity as seen prior to microinjection, and thus did not undergo DNA synthesis following the imposition of the Ca2+ buffer block to mitosis. Cells arrested in this fashion did not spontaneously resume mitotic cycling. This Ca2+ buffer-induced mitotic arrest was, however, experimentally reversible. Cells arrested with 50 microM ApIII 6 min prior to NEB could be returned to mitotic activity by injecting 300 microM CaCl2 5 min after the ApIII injection. The double injected cells resumed cycling, NEB, and mitosis after a delay of one cell cycle period, and remained one cell cycle out of phase with the sister (control) cell. Microinjection of antagonists of endomembrane Ca2+ channels inhibited NEB and anaphase onset in a concentration- and time-dependent fashion. The effective doses of compounds tested were 7 micrograms/ml ryanodine and 500 micrograms/ml TMB-8. These results indicate that a transient elevation of intracellular Ca2+ from endomembrane stores is required to initiate mitotic events, namely NEB and anaphase onset.(ABSTRACT TRUNCATED AT 400 WORDS)
To ask what controls the periodic accumulation and destruction of the mitotic across the cell cycle, we have developed a cell-free system from clam embryos that reproduces several aspects of cyclin behavior. One or more rounds of cyclin proteolysis and resynthesis occur in vitro, and the destruction of the cyclins is highly specific. The onset, duration, and extent of cyclin destruction and the appropriately stagered disappearance of cyclin A and cyclin B are correctly regulated during the first cycle in the cell-free system. Just as in intact cells, lysates made from early interphase cells require further protein synthesis to reach the cyclin destruction point, and lysates made from later stages do not. Using the cell-free system we show that cyclin disappearance requires ATP and Mg2+. By combining lysates from different cell cycle stages, we show that (a) interphase lysates do not contain a dominant inhibitor of cyclin destruction and (b) the timing of cyclin destruction is determined by the cell cycle stage of the cytoplasm rather than the cell cycle stage of the substrate cyclins themselves. Among a large variety of agents tested, only a few affect cyclin destruction. Tosyl-lysine chlormethyl ketone (TLCK, a protease inhibitor), 6-dimethylaminopurine (6-DMAP, a kinase inhibitor), certain sulfhydryl-blocking agents, ZnCl2 and EDTA (but not EGTA) completely block cyclin destruction in vitro. Addition of 1 mM Ca2+ to the cell-free system has no effect on cyclin stability, but 5 mM Ca2+ leads to the rapid destruction of cyclins and a small number of other proteins.
Swiss 3T3 fibroblasts and LLC-PK epithelial cells in prometaphase or metaphase were either injected with fura-2 or loaded with the acetoxymethyl ester derivative of fura-2 (fura-2 AM) and monitored by microspectrofluorimetry. With both methods of loading, we observed two aspects of intracellular free calcium (Cai) metabolism. (a) Most fibroblasts and epithelial cells exhibited a gradual rise from 75 nM in metaphase to 185 nM during cleavage, returning to baseline by early G1. (b) Mitotic Swiss 3T3 cells exhibited rapid transient Cai changes, similar to those previously reported [Poenie, M., J. Alderton, R. Y. Tsien, R. A. Steinhardt. 1985. Nature (Lond.). 315:147-149; Poenie, M., J. Alderton, R. Steinhardt, and R. Tsien. 1986. Science (Wash. DC). 233:886-889; Ratan, R., and M. L. Shelanski. 1988. J. Cell Biol. 107:993]. These Cai transients occurred repetitively, often beginning in metaphase and continuing long after daughter cell formation. Eliminating serum or calcium from the medium abolished the transients, but delayed neither the gradual Cai elevation nor anaphase onset. Co-injection of EGTA or 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) with fura-2 in calcium-free medium, but not in calcium containing medium, blocked both anaphase and the sustained Cai elevation in almost all cases. Blocked cells were rescued by returning calcium to the medium, whereupon Cai slowly but steadily rose as the cell entered anaphase. Spindle microtubules persisted through the EGTA block. Depolymerization of spindle microtubules by nocodazole also reversibly blocked anaphase onset and the sustained Cai elevation, but did not block transients. This study has revealed the following: (a) anaphase in mammalian fibroblasts and epithelial cells is not triggered by brief calcium transients; (b) anaphase is a calcium-modulated event, usually accompanied by a sustained elevation of Cai above 50 nM; (c) the elevation of Cai is dependent upon an intact spindle; and (d) fibroblasts progress through mitosis by drawing upon either intracellular or extracellular sources of calcium.
Mini-synthese des problemes de mesure de concentration de calcium intracellulaire dans des cellules en division chez des invertebres (oursin Lytechinus, Hemicentrotus), chez des mammiferes (rat-kangourou, cellules 3T3) et chez les vegetaux (Haemanthus et Tradescantia) par differentes techniques, les unes utilisant l'ester de fura-2, des indicateurs fluorescents comme le quine, l'aequorine, le BAPTA ou des indicateurs colores comme l'arsenazo III, et des mesures par iontophorese
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.
Changes in free calcium concentration [( Ca]) have been detected during anaphase in stamen hair cells of Tradescantia. Cells have been injected iontophoretically with the calcium sensitive metallochromic dye arsenazo III and changes in differential absorbance have been measured using a spinning wheel microspectrophotometer. The results obtained on single cells progressing from midmetaphase through to cytokinesis show that the free [Ca] first begins in increase after the initial separation of the sister chromosomes marking the onset of anaphase. The increase continues for 10-15 min while the chromosomes move to the poles; thereafter the [Ca] declines with the cell plate appearing about the time that the ion returns to its basal level. The close temporal correlation firstly between the rise in [Ca] and the breakdown of spindle microtubules (MTs) during anaphase and secondly, between the subsequent fall in [Ca] and the emergence of the MT-containing phragmoplast provides evidence consistent with the idea that endogenous fluctuations in [Ca] control the disassembly/assembly of MTs during mitosis.
A new family of highly fluorescent indicators has been synthesized for biochemical studies of the physiological role of cytosolic free Ca2+. The compounds combine an 8-coordinate tetracarboxylate chelating site with stilbene chromophores. Incorporation of the ethylenic linkage of the stilbene into a heterocyclic ring enhances the quantum efficiency and photochemical stability of the fluorophore. Compared to their widely used predecessor, "quin2", the new dyes offer up to 30-fold brighter fluorescence, major changes in wavelength not just intensity upon Ca2+ binding, slightly lower affinities for Ca2+, slightly longer wavelengths of excitation, and considerably improved selectivity for Ca2+ over other divalent cations. These properties, particularly the wavelength sensitivity to Ca2+, should make these dyes the preferred fluorescent indicators for many intracellular applications, especially in single cells, adherent cell layers, or bulk tissues.
Although the regulation of events in the cell division cycle by calcium or other cations has been the subject of much interest and speculation, experimental studies have been hampered by the difficulty of measuring submicromolar intracellular free calcium concentrations ([Ca2+]i) over an entire cell cycle. We now describe experiments using a new fluorescent calcium chelator, fura-2 (see Fig. 1c for structure), for continuous measurement of [Ca2+]i from fertilization through the first cleavage of individual eggs of the sea urchin Lytechinus pictus. We also show for comparison the results of parthenogenetic activation by ammonia. In addition to the known transient rise of [Ca2+]i at fertilization, further peaks are now revealed during pronuclear migration, nuclear envelope breakdown, the metaphase/anaphase transition and cleavage. Parthenogenetic activation by ammonia also elicits a sustained rise starting at nuclear envelope breakdown.
We have measured levels of intracellular free calcium ([Ca2+]i) in albino Xenopus laevis embryos using recombinant aequorin and a photon-counting system. We observed sinusoidal oscillations in [Ca2+]i that had the same frequency as cleavage, with cleavage occurring when [Ca2+]i was lowest. An increase in calcium was seen to precede first cleavage. The cyclic changes in calcium were superimposed on a secondary pattern that increased, peaked between third and fifth cleavages and then slowly declined to a level similar to that measured before first cleavage. The amplitude of the oscillations was small during the first few cleavages but became larger with each cycle, with the largest oscillations occurring when the secondary pattern peaked (between third and fifth cleavage). As the secondary pattern declined, the amplitude of the oscillations also became smaller. The oscillations are due to release of calcium from intracellular stores, since the signal was the same in calcium-free solution as in normal medium.
When cleavage was blocked with the microtubule-disrupting drugs colchicine or nocodazole, the [Ca2+]i oscillations persisted. Calcium oscillations of a similar magnitude and frequency were also present in artificially activated eggs. The secondary pattern was different in cleavage-blocked embryos and artificially activated eggs, the baseline increasing until about the third cycle and then remaining elevated for the rest of the recording (>8 hours).
By fixing embryos at various points in the calcium cycle, we determined that mitosis began shortly after calcium levels reached their peak and was complete before the calcium level dropped to its lowest point. The fact that the calcium oscillations persist when nuclear division is not occurring suggests that either they operate independently of any downstream events that they might control or they are related to other cyclic activities in the eggs and embryos such as cycling of pHi, MPF, or surface contraction waves.
When aequorin was injected into individual blastomeres of 64-cell embryos, the shape of the signal was substantially different from that seen in recordings from whole embryos. Compared to the whole-embryo recordings, the signal from a subregion of the embryo rose more quickly and had a slower, biphasic decline. These differences indicate that [Ca2+]i increases are not occurring uniformly across the embryo but are spatially localized, perhaps progressing as waves.
Transient changes in intracellular calcium ([Ca2+]i) have been shown to punctuate the cell cycle in various types of cells in culture and in early embryos. The [Ca2+]i transients are correlated with cell-cycle events: pronuclear migration, nuclear envelope breakdown, the metaphase-anaphase transition of mitosis, and cytokinesis. Mitotic events can be induced by injecting calcium and prevented by injecting calcium chelators into the sea urchin embryo. Cell-cycle calcium transients differ from the transients linked to membrane signal transduction pathways: they are generated by an endogenous mechanism, not by plasma membrane receptor complexes, and their trigger is unknown. We report here that the phosphoinositide messenger system oscillates during the early embryonic cell cycle in the sea urchin, leading to cyclic increases in inositol trisphosphate that trigger cell-cycle [Ca2+]i transients and mitosis by calcium release from intracellular stores.
A slow cortical wave of high calcium accompanies the elongation of cleavage furrows in medaka fish eggs as well as in Xenopus eggs. We explored the role of such waves by injecting calcium buffers into Xenopus eggs at various times before and during first and second cleavage. Injection earlier than about 15 minutes before first cleavage normally starts delays it for hours. Injection between about 15 minutes and a few minutes before cleavage normally starts allows a (short) furrow to form on time but usually yields an eccentric one. This forms away from the injection side, often as far off-center as the egg’s equator, and then regresses. Injection soon after it starts quickly arrests elongation of the furrow and eventually induces its regression; while injection a bit later likewise soon arrests elongation but allows delocalized furrow deepening to continue. The dependence of these inhibitory actions upon the dissociation constants and final cytosolic concentrations of the injected buffers indicates that they act as shuttle buffers to suppress needed zones of high calcium in the micromolar range. We conclude that the high calcium that is found within these furrows is needed to induce them, to extend them and even to maintain them.
Moreover, while short, eccentric furrows often form as far off center as the equator, they somehow always form along a meridian through the animal pole. This seems difficult to explain by the orthodox, diastral model. Rather, it suggests that the cleavage furrows in Xenopus- and perhaps in animal cells quite generally- are directly induced by a diastema or telophase disc rather than by the asters.
Membranes are common components of the mitotic apparatus of dividing cells (Harris 1975; Hepler 1977). Although they have been recognized for over twenty years in ultrastructural studies of the mitotic apparatus, they have received much less attention than the spindle microtubules and have occupied a relatively minor role in our effort to elucidate the mechanism and control of mitosis.
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.
We have previously shown that changes in the intracellular free calcium concentration ([Ca2+]i) modulate the rate of anaphase chromosome motion in stamen hair cells of Tradescantia. Elevations between 0.8-1.0 μM accelerate motion, while increases above 2.0 μM or decreases below resting level inhibit motion. The related signaling agent GTPγS also accelerates motion, however, by a mechanism that does not appear to involve changes in [Ca2+]i. To explore further the mechanism by which Ca2+ and GTPγS regulate chromosome motion we have analyzed the direct effect of these agents on the structure of the spindle microtubules (MTs). First, we injected carboxyfluorescein-derivatized brain tubulin and allowed it to incorporate into spindle MTs. Then, during appropriate times of anaphase, we injected Ca2+ or related agents and monitored their effect on spindle MT fluorescence using a confocal laser scanning microscope. A high level of Ca2+ (10 μM), known to inhibit motion, causes extensive degradation of spindle MT structure. An intermediate level (2 μM), which slows but does not stop movement, produces a distinct decay of fluorescence. A level of Ca2+ (0.8-1 μM) known to accelerate motion, however, generates only a small change in which the kinetochore fibers appear less distinct, and the overall spindle fluorescence is more diffuse. The Ca2+ buffer EGTA, which transiently blocks motion, has no detectable effect on spindle structure. GTPγS, which enhances motion also has no discernible effect on spindle structure. The results with Ca2+ support the idea that the ion facilitates anaphase motion through depolymerization of kinetochore MTs. However, if the depolymerization is extensive then inhibition of motion ensues. GTPγS, on the other hand, accelerates motion without inducing a detectable effect on spindle MT structure.
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.
Fluorescence ratiometric imaging of Lilium pollen tubes loaded with the Ca2+ indicator Fura-2 dextran has revealed a distinct elevation of free intracellular calcium ion concentration ([Ca2+]i) at the extreme tip of actively growing Lilium pollen tubes that declines to a uniform basal level of ∼170 nM throughout the length of the tube. The calcium gradient occurs within the first 10-20 μm proximal to the tip. Experimental inhibition of tip growth, usually achieved through the injection of the Ca2+ buffer 5,5′-dibromo BAPTA, results in the loss of the [Ca2+]i gradient. Occasionally these inhibited cells reinitiate growth, and when they do so ratio imaging reveals that the tip gradient of free [Ca2+]i re-emerges. The results presented here are very different from those previously published by revealing the presence of the [Ca2+]i gradient that is restricted to the 10-20 μm adjacent to the tube tip. Further, these experiments demonstrate a strict correlation between the presence of a [Ca2+]i gradient, and tip growth in Lilium pollen tubes.
Several lines of evidence support the idea that increases in the intracellular free calcium concentration [( Ca2+]i) regulate chromosome motion. To directly test this we have iontophoretically injected Ca2+ or related signaling agents into Tradescantia stamen hair cells during anaphase and measured their effect on chromosome motion and on the Ca2+ levels. Ca2+ at (+)1 nA for 10 s (approximately 1 microM) causes a transient (20 s) twofold increase in the rate of chromosome motion, while at higher levels it slows or completely stops motion. Ca2+ buffers, EGTA, and 5,5'-dibromo-1,2- bis(o-aminophenoxy)ethane- N,N,N',N'-tetraacetic acid, which transiently suppress the ion level, also momentarily stop motion. Injection of K+, Cl-, or Mg2+, as controls, have no effect on motion. The injection of GTP gamma S, and to a lesser extent GTP, enhances motion similarly to a low level of Ca2+. However, inositol 1,4,5-trisphosphate, ATP gamma S, ATP, and GDP beta S have no effect. Measurement of the [Ca2+]i with indo-1 reveals that the direct injections of Ca2+ produce the expected increases. GTP gamma S, on the other hand, causes only a small [Ca2+]i rise, which by itself is insufficient to increase the rate of chromosome motion. Further studies reveal that any negative ion injection, presumably through hyperpolarization of the membrane potential, generates a similar small pulse of Ca2+, yet these agents have no effect on motion. Two major conclusions from these studies are as follows. (a) Increased [Ca2+]i can enhance the rate of motion, if administered in a narrow physiological window around 1 microM; concentrations above 1 microM or below the physiological resting level will slow or stop chromosomes. (b) GTP gamma S enhances motion by a mechanism that does not cause a sustained uniform rise of [Ca2+]i in the spindle; this effect may be mediated through very localized [Ca2+]i changes or Ca2(+)-independent effectors.
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.
Exocytosis is a key event in plant cells, linking the internal and external environments. Cell-wall precursors, membrane material, and soluble and membrane-associated proteins are supplied by exocytosis and allow cells to grow and differentiate. In this review, we describe exocytotic systems in plants and summarize information on how exocytosis is controlled and itself controls cell development. Mechanisms regulating exocytosis in other organisms are outlined and some critical directions for future research on plant exocytosis are suggested.
The development and dynamics of the phragmoplast cytoskeleton have been analyzed in living stamen hair cells of Tradescantia. Microtubules and actin microfilaments have been identified by microinjecting either carboxyfluorescein labeled brain tubulin or rhodamine phalloidin. Examination with the confocal laser scanning microscope has permitted sequential imaging of the fluorescent cytoskeletal elements in single living cells progressing through division. Phragmoplast microtubules initially emerge through the lateral coalescence of preexisting interzone microtubules. As cytokinesis progresses, these tightly clustered microtubules shorten in length and expand centrifugally toward the cell periphery. By contrast, the phragmoplast microfilaments appear to arise de novo in late anaphase in close association with the proximal surfaces of the reconstituting daughter nuclei. The microfilaments are oriented parallel to the microtubules but conspicuously do not occupy the equatorial region where microtubules interdigitate and where the cell plate vesicles aggregate and fuse. As development proceeds the microfilaments shorten in length and expand in girth, similar to microtubules, although they remain excluded from the cell plate region. In terminal phases of cell plate formation, microtubules degrade first in the central regions of the phragmoplast and later toward the edges, whereas microfilaments break down more uniformly throughout the phragmoplast. © 1993 Wiley-Liss, Inc.
Five BAPTA buffers with differential affinities for Ca2+ have been examined for their effects on cell plate formation in stamen hair cells ofTradescantia. The five include 5,5-dimethyl BAPTA (Kd=0.15 M), BAPTA (Kd=0.22 M), 5,5-dibromo BAPTA (Kd=1.5 M), 5-methyl,5-nitro BAPTA (Kd=22 M), and 5-nitro BAPTA (Kd=40 M). At a concentration of 5 mM and 25 mM in the pipette, the buffers were iontophoretically microinjected into dividing stamen hair cells (2 nA for 1 min) prior to or at the onset of cell plate formation. At the lowest concentration (5 mM), only one buffer, 5,5-dibromo BAPTA, inhibits cell plate formation, and is most effective if delivered at the moment of cell plate vesicle aggregation. The inhibitory effects appear as a slowing of cell plate expansion, the formation of distorted plates, or the complete dissolution of plates that might have initiated normally. When the pipette tip concentration is elevated to 25 mM, the effects of 5,5-dibromo BAPTA become more profound. At these levels 5,5-dimethyl BAPTA, BAPTA, and 5-nitro BAPTA also modulate cell plate formation, producing effects similar to that of 5,5-dibromo BAPTA at the lower concentration. Independent studies using fura-2 as a fluorescent analogue of the BAPTA buffers, indicate that the apparent effective concentration for 5,5-dibromo BAPTA is between 1.0–1.4 mM; its threshold concentration is not known but expected to be somewhat lower. For the other buffers the threshold concentration is between 1.5–2.2 mM. The concentration dependence supports the idea that the buffers facilitate diffusion of Ca2+ away from regions of elevated concentration. The results thus provide evidence that local Ca2+ gradients may be present in the vicinity of the cell plate and that they participate in the cytokinetic process.
A series of periodic increases in intracellular free calcium concentration ([Ca2+]i) occurred upon fertilization in golden hamster eggs. The spatial distribution of the Ca2+ transients was investigated in single zona-free, aequorin-injected eggs, inseminated by single sperm. A supersensitive TV camera system for recording Ca2+-aequorin luminescence enabled us to observe the spatial distribution of the Ca2+ rise. In the first response, which usually occurred 10–30 sec after the sperm attachment, the increase in [Ca2+]i began near the sperm attachment site, and the Ca2+ rise spread over the entire egg within 4–7 sec. The Ca2+ rise attained its peak in 5–8 sec, declined with almost even distribution, and ceased in 12–17 sec. The spreading Ca2+ rise was repeated in the second and sometimes the third response, starting from the same focus, but spreading more rapidly (∼2 sec). In succeeding responses [Ca2+]i increased synchronously in the whole cytoplasm within 1 sec. When additional sperm attached to the egg after the occurrence of the first response by the first sperm, the spread of the Ca2+ rise could take place from near the site of additional sperm attachment but only in the second or third response.
The actomyosin contractile-ring mechanism remains the paradigm for cytokinesis after 20 years of experimental testing. Recent evidence suggests that Ca2+ triggers the contraction and that cell-cycle kinases regulate the timing of cytokinesis. New work is required to identify the signals from the mitotic spindle that specify the position of the furrow.
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 role of calcium in cortical granule exocytosis and activation of the cell cycle at fertilization was examined in the mouse egg using the calcium chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) and the fluorescent calcium indicator fluo-3. BAPTA and fluo-3 were introduced into zona-free mouse eggs by a 30-min incubation with 0.01-50 microM BAPTA acetoxymethyl ester (AM) and/or 1-20 microM fluo-3 AM prior to in vitro fertilization. Incubation of eggs in greater than or equal to 5.0 microM BAPTA AM inhibited cortical granule exocytosis in all cases. Introduction of the calcium chelator into the egg blocked second polar body formation at greater than or equal to 1.0 microM BAPTA AM. Sperm entry occurred in all eggs regardless of the BAPTA AM concentration. Sperm induce a large transient increase in calcium lasting 2.3 +/- 0.6 min, followed by repetitive transients lasting 0.5 +/- 0.1 min and occurring at 3.4 +/- 1.4-min intervals. Incubation with greater than or equal to 5.0 microM BAPTA AM inhibited all calcium transients. Introduction of BAPTA also inhibited calcium transients, exocytosis, and the resumption of meiosis following application of the calcium ionophore A23187 or SrCl2, which activate eggs. These results demonstrate that the calcium increase at fertilization is required for cortical granule exocytosis and resumption of the cell cycle in a mammalian egg.
The results of experiments recently reported from this and other laboratories provide firm support for Heilbrunn's thesis that mitotic events are initiated by transient elevation of intracellular Ca2+, derived from intracellular stores. The ATP-dependent MA Ca2(+)-pump working in concert with an endomembrane Ca2+ channel appears to share the responsibility for regulating these Ca2+ signals. Further results demonstrated a limited time window during which the cell is sensitive to agents that impose mitotic arrest by interfering with transient elevations in intracellular "free" Ca2+ concentration. From this it appears that a discrete, timed increase in cytosolic Ca2+ derived from endomembrane stores is a necessary signal for regulating the onset of NEB, AO, and mitosis. Results from the arrest and release experiments provide support for a model in which Ca2+ is used to coordinate the action of parallel independent and interdependent biochemical pathways whose interaction results in the cytologic events of mitosis. These pathways apparently are operating under the influence of a metabolic "clock" that continues to cycle, at least once, in the absence of a Ca2+ transient sufficient to initiate NEB or AO. The discrete and temporal regulation of this Ca2+ transient through the interaction of the endomembrane Ca2+ pump, an endomembrane Ca2+ channel, and intracellular Ca2(+)-dependent reaction pathways suggest a mechanism incorporating a negative feedback loop to limit the size and duration of the Ca2+ transient and prevent the release of excessive amounts of Ca2+. Deeper understanding of the regulatory mechanism that governs the onset of mitosis requires: (1) quantitative imaging of intracellular Ca2+, especially the Ca2+ signal throughout the cell cycle, with high spatial and temporal resolution; and (2) identifying the molecules responsible for regulating the expression and reception of the Ca2+ signal itself. It is clear that Ca2(+)-dependent pathways are necessary elements of the mitotic process. Molecular candidates for the regulators and regulatees have yet to be identified. The upstream controlling molecules of these transmembrane Ca2+ regulatory elements, as well as the initial mitotic "start" signal, await future identification. Downstream regulation is also clearly indicated, perhaps through regulation of cyclin expression, degradation, or both.
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.
The polarity of fucoid eggs is fixed either when tip growth starts or a bit earlier. A steady flow of calcium ions into the incipient tip is thought to establish a high calcium zone that is needed for its localization and formation. To test this hypothesis, we have injected seven different 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-type calcium buffers into Pelvetia eggs many hours before tip growth normally starts. Critical final cell concentrations of each buffer prove to block outgrowth (as well as cell division) for up to 2 weeks. This critical inhibitory concentration is lowest for two buffers with dissociation constants or Kd values of 4-5 x 10(-6) M and increases steadily as the buffers' Kd values shift either below or above this optimal value to ones as low as 4 x 10(-7) M or as high as 9.4 x 10(-5) M. To analyze these results, we have derived an equation (based on the concept of facilitated diffusion) for the effects of diffusable calcium buffers on steady-state calcium gradients. The data fit this equation quite well if it is assumed that cytosolic free calcium at the incipient tip is normally kept at about 7 microM and, thus, far above the general cytosolic level.
We review the recent advances in understanding transitions within the cell cycle. These have come from both genetic and biochemical
approaches. We discuss the phylogenetic conservation of the mechanisms that induce mitosis and their implications for other
transitions in the cell cycle.
Using the calcium-specific, chemiluminescent photoprotein aequorin, we have measured changes in the concentration of free cytosolic calcium at fertilization in single eggs of the ascidians Phallusia mammillata and Ciona intestinalis. Shortly after insemination, the free calcium concentration rises within a minute from a resting level of about 90 nM in the unfertilized egg to a peak level of about 7 microM in Phallusia and about 10 microM in Ciona. The total duration time of this fertilization transient is 2-3 min. It is immediately followed by a series of 12 to 25 briefer calcium transients with peak levels of about 1-4 microM. These postfertilization pulses occur at regular intervals of 1-3 min during the completion of meiosis, and they stop as soon as the second polar body is formed at about 25 min. An interesting exception to this pattern was observed in eggs from Ciona that had been raised at lower temperatures during the winter months. Insemination in the absence of external calcium in Phallusia results in a pulse pattern very similar to the normal pattern. From this result we infer that the bulk (if not all) of the calcium required for both the fertilization pulse and the meiotic oscillations is released from internal sources.
The process of mitosis includes a series of morphological changes in the cell in which the directional movements of chromosomes are the most prominent. The presence of a microtubular array, known as the spindle or mitotic apparatus, provides at least a scaffold upon which these movements take place. The precise mechanism for chromosome movement remains obscure, but new findings suggest that the kinetochore may play a key role in chromosome movement toward the spindle pole, and that sliding interactions between or among adjacent microtubules may provide the mechanochemical basis for spindle elongation. The physiological regulation of the anaphase motors and of spindle operation either before or after anaphase remains equally elusive. Elicitors that may serve as controlling elements in spindle function include shifts in cytosolic calcium activity and perhaps the activation or inactivation of protein kinases, which in turn produce changes in the state of phosphorylation of specific spindle components.
The cycles of DNA synthesis and chromatin condensation in dividing cells are controlled by signals from the cytoplasm. Changes in the concentration of free calcium (Cai) in the cytoplasm control a variety of cellular functions and it has thus been suggested that observed variations in Cai during the cell cycle may be the cytoplasmic signal that co-ordinates nuclear and cytoplasmic division. We show here that increases in Cai induced by the calcium-releasing second messenger inositol 1,4,5-triphosphate (Ins(1,4,5)P3), or by calcium buffers, cause premature chromatin condensation and breakdown of the nuclear envelope in sea urchin (Lytechinus pictus) early embryos. Both natural and induced chromatin condensation are prevented by calcium chelators. The nucleus becomes sensitive to the Cai signal 45 min after fertilization, but remains insensitive if protein synthesis is prevented. Our experiments demonstrate that Cai regulates the behaviour of the nucleus during the cell cycle, suggest that Ins(1,4,5)P3 is a cell cycle messenger and indicate that there is an interaction between the protein and ionic signals that control the state of chromatin during the cell cycle.
Cytosolic free calcium has recently been implicated in the regulation of mitosis in plant and animal cells. We have previously found correlations between increases in the levels of intracellular free calcium [Ca2+]i and visible transitions of structure at nuclear envelope breakdown (NEBD) and the onset of anaphase during mitosis in sea urchin embryos and tissue culture cells. To go beyond correlations it is necessary to manipulate [Ca2+]i, and in sea urchin embryos this requires the injection of calcium-chelator buffer solutions as the changes in free calcium in the cell cycle are dependent on intracellular stores. We report here that blocking the increase in [Ca2+]i which just precedes NEBD prevents this from taking place and halts mitosis. Subsequent injections which momentarily increase [Ca2+]i, or a natural recovery of the higher calcium levels, result in NEBD and the successful continuation of mitosis. Similarly, artificially increasing calcium by early injections results in early NEBD. We conclude that the increase in [Ca2+]i preceding NEBD is an essential regulatory step required for entry into mitosis.
Continuous measurement and imaging of the intracellular free calcium ion concentration ([Ca2+]i) of mitotic and interphase PtK1 cells was accomplished with the new fluorescent Ca2+ indicator fura-2. No statistically significant difference between basal [Ca2+]i of interphase and mitotic cells was detected. However, mitotic cells showed a rapid elevation of [Ca2+]i from basal levels of 130 nM to 500 to 800 nM at the metaphase-anaphase transition. The [Ca2+]i transient was brief, lasting approximately 20 seconds and the elevated [Ca2+]i appeared uniformly distributed over the entire spindle and central region of the cell. The close temporal association of the [Ca2+]i transient with the onset of anaphase suggests that calcium may have a signaling role in this event.