Ca2+ homeostasis regulates Xenopus oocyte maturation

In contrast to the well-defined role of Ca2+ signals during mitosis, the contribution of Ca2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca2+ deprivation. These findings argue that Ca2+ homeostasis is important in establishing the oocyte's competence to undergo maturation in preparation for fertilization and embryonic development.     

Selective modulation of subtype III IP3R by Akt regulates ER Ca2+ release and apoptosis

Ca²⁺ transfer from endoplasmic reticulum (ER) to mitochondria can trigger apoptotic pathways by inducing release of mitochondrial pro-apoptotic factors. Three different types of inositol 1,4,5-trisphosphate receptor (IP₃R) serve to discharge Ca²⁺ from ER, but possess some peculiarities, especially in apoptosis induction. The anti-apoptotic protein Akt can phosphorylate all IP₃R isoforms and protect cells from apoptosis, reducing ER Ca²⁺ release. However, it has not been elucidated which IP₃R subtypes mediate these effects. Here, we show that Akt activation in COS7 cells, which lack of IP₃R I, strongly suppresses IP₃-mediated Ca²⁺ release and apoptosis. Conversely, in SH-SY 5Y cells, which are type III-deficient, Akt is unable to modulate ER Ca²⁺ flux, losing its anti-apoptotic activity. In SH-SY 5Y-expressing subtype III, Akt recovers its protective function on cell death, by reduction of Ca²⁺ release. Moreover, regulating Ca²⁺ flux to mitochondria, Akt maintains the mitochondrial integrity and delays the trigger of apoptosis, in a type III-dependent mechanism. These results demonstrate a specific activity of Akt on IP₃R III, leading to diminished Ca²⁺ transfer to mitochondria and protection from apoptosis, suggesting an additional level of cell death regulation mediated by Akt.     

The presenilin-2 loop peptide perturbs intracellular Ca2+ homeostasis and accelerates apoptosis

In cells undergoing apoptosis, a 22-amino-acid presenilin-2-loop peptide (PS2-LP, amino acids 308-329 in presenilin-2) is generated through cleavage of the carboxyl-terminal fragment of presenilin-2 by caspase-3. The impact of PS2-LP on the progression of apoptosis, however, is not known. Here we show that PS2-LP is a potent inducer of the mitochondrial-dependent cell death pathway when transduced as a fusion protein with HIV-TAT. Biochemical and functional studies demonstrate that TAT-PS2-LP can interact with the inositol 1,4,5-trisphosphate receptor and activate Ca(2+) release from the endoplasmic reticulum. These results indicate that PS2-LP-mediated alteration of intracellular Ca(2+) homeostasis may be linked to the acceleration of apoptosis. Therefore, targeting the function of PS2-LP could provide a useful therapeutic tool for the treatment of cancer and degenerative diseases.     

Annexin-mediated Ca2+ influx regulates growth plate chondrocyte maturation and apoptosis

Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca(2+) influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca(2+), whereas K-201, a specific annexin Ca(2+) channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca(2+) influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca(2+) influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes.     

Simultaneous imaging of ER and cytosolic Ca2+ dynamics reveals long-distance ER Ca2+ waves in plants

Calcium ions (Ca²⁺) play a key role in cell signaling across organisms. In plants, a plethora of environmental and developmental stimuli induce specific Ca²⁺ increases in the cytosol as well as in different cellular compartments including the endoplasmic reticulum (ER). The ER represents an intracellular Ca²⁺ store that actively accumulates Ca²⁺ taken up from the cytosol. By exploiting state-of-the-art genetically encoded Ca²⁺ indicators, specifically the ER-GCaMP6-210 and R-GECO1, we report the generation and characterization of an Arabidopsis (Arabidopsis thaliana) line that allows for simultaneous imaging of Ca²⁺ dynamics in both the ER and cytosol at different spatial scales. By performing analyses in single cells, we precisely quantified (1) the time required by the ER to import Ca²⁺ from the cytosol into the lumen and (2) the time required to observe a cytosolic Ca²⁺ increase upon the pharmacological inhibition of the ER-localized P-Type IIA Ca²⁺-ATPases. Furthermore, live imaging of mature, soil-grown plants revealed the existence of a wounding-induced, long-distance ER Ca²⁺ wave propagating in injured and systemic rosette leaves. This technology enhances high-resolution analyses of intracellular Ca²⁺ dynamics at the cellular level and in adult organisms and paves the way to develop new methodologies aimed at defining the contribution of subcellular compartments in Ca²⁺ homeostasis and signaling.

Dual color imaging allows the simultaneous analysis of calcium dynamics in the endoplasmic reticulum and cytosol from single cells to adult entire plants.     

BI-1 regulates endoplasmic reticulum Ca2+ homeostasis downstream of Bcl-2 family proteins

BI-1 (Bax inhibitor-1) is an evolutionarily conserved multitransmembrane protein that resides in the endoplasmic reticulum (ER) and that has documented cytoprotective functions in both animals and plants. Recent studies indicate that BI-1 shares in common with Bcl-2/Bax family proteins the ability to regulate the amounts of Ca(2+) that can be released from the ER by agents, such as the ER-Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (TG). Using an ER-targeted, Ca(2+) indicator (cameleon), with characteristics optimized for measuring ER Ca(2+) ([Ca(2+)](er)), we studied the effects of BI-1 on [Ca(2+)](er) in resting and TG-treated cells. Similar to cells overexpressing antiapoptotic Bcl-2 or Bcl-X(L), overexpression of BI-1 resulted in lower resting [Ca(2+)](er), with concomitantly less Ca(2+) released into the cytosol upon stimulation by TG and with a higher rate of Ca(2+) leakage from the ER. Co-expression of SERCA restored levels of [Ca(2+)](er) to normal, showing opposing actions of the ER-Ca(2+)ATPase and BI-1 on ER Ca(2+) homeostasis. Conversely, cells with deficient BI-1 have increased [Ca(2+)](er), and release more Ca(2+) into the cytosol when challenged with TG. In BI-1-deficient cells, Bcl-X(L) fails to reduce [Ca(2+)](er), indicating that BI-1 functions downstream of Bcl-X(L). In bax(-/-)bak(-/-) double knock-out cells, both BI-1 and Bcl-X(L) retained their ability to reduce [Ca(2+)](er), suggesting that BI-1 and Bcl-X(L) operate downstream of or parallel to Bax/Bak. The findings reveal a hierarchy of functional interactions of BI-1 with Bcl-2/Bax family proteins in regulating ER Ca(2+) homeostasis.     

Inhibition of ER stress attenuates kidney injury and apoptosis induced by 3-MCPD via regulating mitochondrial fission/fusion and Ca2+ homeostasis

Cell Biology and Toxicology - 3-Chloro-1, 2-propanediol (3-MCPD) is a food-borne toxic substance well-known for more than 40 years that is mainly associated with nephrotoxicity. A better...     

Distinct roles of mitochondria- and ER-localized Bcl-xL in apoptosis resistance and Ca2+ homeostasis

Bcl-2 proteins are major regulators of cellular responses to intrinsic and extrinsic apoptotic stimuli. Among them, overexpression of the antiapoptotic protein Bcl-x(L) modulates intracellular Ca(2+) homeostasis and organelle-specific apoptotic signaling pathways. However, the specific activities of Bcl-x(L) at mitochondria and the endoplasmic reticulum (ER) have not been fully defined. To further explore this, we generated mouse embryonic fibroblast (MEF) cell lines deficient in Bcl-x(L) expression (Bcl-x-KO). Deficiency in Bcl-x(L) expression did not induce compensatory changes in the expression of other Bcl-2 proteins, and Bcl-x-KO MEF cells showed increased sensitivity to various apoptotic stimuli compared with wild-type MEF cells. Targeting Bcl-x(L) at mitochondria but not at the ER restored apoptosis protection in Bcl-x-KO MEF cells to the degree observed in wild-type MEF cells. However, expression of ER-targeted Bcl-x(L) but not mitochondrially targeted Bcl-x(L) was required to restore Ca(2+) homeostasis in Bcl-x-KO MEF cells. Of importance, ER-targeted Bcl-x(L) was able to protect cells against death stimuli in the presence of endogenous Bcl-x(L). These data indicate that mitochondrial Bcl-x(L) can regulate apoptosis independently of ER Bcl-x(L) and that when localized exclusively at the ER, Bcl-x(L) impinges on Ca(2+) homeostasis but does not affect apoptosis unless Bcl-x(L) is present in additional cellular compartments.     

Function of a STIM1 homologue in C. elegans: evidence that store-operated Ca2+ entry is not essential for oscillatory Ca2+ signaling and ER Ca2+ homeostasis

1,4,5-trisphosphate (IP(3))-dependent Ca(2+) signaling regulates gonad function, fertility, and rhythmic posterior body wall muscle contraction (pBoc) required for defecation in Caenorhabditis elegans. Store-operated Ca(2+) entry (SOCE) is activated during endoplasmic reticulum (ER) Ca(2+) store depletion and is believed to be an essential and ubiquitous component of Ca(2+) signaling pathways. SOCE is thought to function to refill Ca(2+) stores and modulate Ca(2+) signals. Recently, stromal interaction molecule 1 (STIM1) was identified as a putative ER Ca(2+) sensor that regulates SOCE. We cloned a full-length C. elegans stim-1 cDNA that encodes a 530-amino acid protein with approximately 21% sequence identity to human STIM1. Green fluorescent protein (GFP)-tagged STIM-1 is expressed in the intestine, gonad sheath cells, and spermatheca. Knockdown of stim-1 expression by RNA interference (RNAi) causes sterility due to loss of sheath cell and spermatheca contractile activity required for ovulation. Transgenic worms expressing a STIM-1 EF-hand mutant that constitutively activates SOCE in Drosophila and mammalian cells are sterile and exhibit severe pBoc arrhythmia. stim-1 RNAi dramatically reduces STIM-1GFP expression, suppresses the EF-hand mutation-induced pBoc arrhythmia, and inhibits intestinal store-operated Ca(2+) (SOC) channels. However, stim-1 RNAi surprisingly has no effect on pBoc rhythm, which is controlled by intestinal oscillatory Ca(2+) signaling, in wild type and IP(3) signaling mutant worms, and has no effect on intestinal Ca(2+) oscillations and waves. Depletion of intestinal Ca(2+) stores by RNAi knockdown of the ER Ca(2+) pump triggers the ER unfolded protein response (UPR). In contrast, stim-1 RNAi fails to induce the UPR. Our studies provide the first detailed characterization of STIM-1 function in an intact animal and suggest that SOCE is not essential for certain oscillatory Ca(2+) signaling processes and for maintenance of store Ca(2+) levels in C. elegans. These findings raise interesting and important questions regarding the function of SOCE and SOC channels under normal and pathophysiological conditions.     

Ca2+ homeostasis in unstimulated platelets

Unstimulated platelets maintain a low cytosolic free Ca2+ concentration and a steep plasma membrane Ca2+ gradient. The mechanisms that are required have not been completely defined. In the present studies, 45Ca2+ was used to examine the kinetics of Ca2+ exchange in intact unstimulated platelets. Quin2 was used to measure the cytosolic free Ca2+ concentration. Under steady-state conditions, the maximum rate of Ca2+ exchange across the platelet plasma membrane, 2 pmol/10(8) platelets/min, was observed at extracellular free Ca2+ concentrations 20-fold less than in plasma. Two intracellular exchangeable Ca2+ pools were identified. The size of the more rapidly exchanging pool (t 1/2, 17 min) and the cytosolic free Ca2+ concentration were relatively unaffected by large changes in the extracellular Ca2+ concentration. In contrast, the size of the more slowly exchanging Ca2+ pool (t 1/2, 300 min) varied with the extracellular Ca2+ concentration, which suggests that it is physically as well as kinetically distinct from the rapidly exchangeable Ca2+ pool. The locations of the Ca2+ pools were determined by differential permeabilization of 45Ca2+-loaded platelets with digitonin. 45Ca2+ in the rapidly exchanging pool was released with lactate dehydrogenase, which suggests that it is located in the cytosol. 45Ca2+ in the slowly exchanging pool was released with markers for both the dense tubular system and mitochondria, but inhibition of mitochondrial Ca2+ uptake with carbonyl cyanide m-chlorophenylhydrazone had no effect on the size of the slowly exchangeable Ca2+ pool or the cytosolic free Ca2+ concentration. In contrast, addition of metabolic inhibitors (KCN plus carbonyl cyanide m-chlorophenylhydrazone plus deoxyglucose) or trifluoperazine caused a decrease in the size of the slowly exchangeable Ca2+ pool and an increase in the cytosolic free Ca2+ concentration. These observations suggest that Ca2+ homeostasis in unstimulated platelets is maintained by limiting Ca2+ influx from plasma, actively promoting Ca2+ efflux, and sequestering Ca2+ within an internal site, which is most likely the dense tubular system and not mitochondria.     

Ca2+ homeostasis defects and hereditary hearing loss

Ca(2+) acts as a fundamental signal transduction element in inner ear, delivering information about sound, acceleration and gravity through a small number of mechanotransduction channels in the hair cell stereocilia and voltage activated Ca(2+) channels at the ribbon synapse, where it drives neurotransmission. The mechanotransduction process relies on the endocochlear potential, an electrical potential difference between endolymph and perilymph, the two fluids bathing respectively the apical and basolateral membrane of the cells in the organ of Corti. In mouse models, deafness and lack or reduction of the endocochlear potential correlate with ablation of connexin (Cx) 26 or 30. These Cxs form heteromeric channels assembled in a network of gap junction plaques connecting the supporting and epithelial cells of the organ of Corti presumably for K(+) recycle and transfer of key metabolites, for example, the Ca(2+) -mobilizing second messenger IP(3) . Ca(2+) signaling in these cells could play a crucial role in regulating Cx expression and function. Another district where Ca(2+) signaling alterations link to hearing loss is hair cell apex, where ablation or missense mutations of the PMCA2 Ca(2+) -pump of the stereocilia cause deafness and loss of balance. If less Ca(2+) is exported from the stereocilia, as in the PMCA2 mouse mutants, Ca(2+) concentration in endolymph is expected to fall causing an alteration of the mechanotransduction process. This may provide a clue as to why, in some cases, PMCA2 mutations potentiated the deafness phenotype induced by coexisting mutations of cadherin-23 (Usher syndrome type 1D), a single pass membrane Ca(2+) binding protein that is abundantly expressed in the stereocilia.     

Mitochondrial Ca2+ homeostasis in health and disease

Although it has long been known that mitochondria possess a complex molecular repertoire for accumulating and releasing Ca2+, only in recent years has a large body of data demonstrated that these organelles promptly respond to Ca(2+)-mediated cell stimulations. In this contribution, we will review the principles of mitochondrial Ca2+ homeostasis and its signaling role in different physiological and pathological conditions.     

Mitochondrial Ca2+ homeostasis in intact cells

Ca2+ is a key regulator not only of multiple cytosolic enzymes, but also of a variety of metabolic pathways occurring within the lumen of intracellular organelles. Until recently, no technique to selectively monitor the Ca2+ concentration within defined cellular compartments was available. We have recently proposed the use of molecularly engineered Ca(2+)-sensitive photoproteins to obtain such a result and demonstrated the application of this methodology to the study of mitochondrial and nuclear Ca2+ dynamics. We here describe in more detail the use of chimeric recombinant aequorin targeted to the mitochondria. The technique can be applied with equivalent results to different cell models, transiently or permanently transfected. In all the cell types we analyzed, mitochondrial Ca2+ concentration ([Ca2+]m) increases rapidly and transiently upon stimulation with agonists coupled to InsP3 generation. We confirm that the high speed of mitochondrial Ca2+ accumulation with this type of stimuli depends on the generation of local gradients of Ca2+ in the cytosol, close to the channels sensitive to InsP3. In fact, only activation of these channels, but not the simple release from internal stores, as that elicited by blocking the intracellular Ca2+ ATPases, results in a fast mitochondrial Ca2+ accumulation. We also provide evidence in favor of a microheterogeneity among mitochondria of the same cells, about 30% of them apparently sensing the microdomains of high cytosolic Ca2+ concentration ([Ca2+]c). The changes in [Ca2+]m appear sufficiently large to induce a rapid activation of mitochondrial dehydrogenases, which can be followed by monitoring the level of NAD(P)H fluorescence. A general scheme can thus be envisaged by which the triggering of a plasma membrane receptor coupled to InsP3 generation raises the Ca2+ concentration both in the cytoplasm (thereby triggering energy-consuming processes, such as cell proliferation, motility, secretion, etc.) and in the mitochondria, where it activates the metabolic activity according to the increased cell needs.     

Polypeptide N-acetylgalactosaminyltransferase 18 non-catalytically regulates the ER homeostasis and O-glycosylation

Mucin-type O-glycosylation plays important roles in various biological processes. It is initiated by a family of 20 conserved UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). Unlike most ppGalNAc-Ts localized to the Golgi apparatus, ppGalNAc-T18 is predominantly distributed on the endoplasmic reticulum (ER) and exhibits no ppGalNAc-T catalytic activity in vitro. Herein, we found that ppGalNAc-T18 silencing in cells decreased O-glycosylation levels and activated ER stress leading to apoptosis. After treatment with chemical chaperone 4-phenylbutyric acid (PBA) or forced expression of ppGalNAc-T18 in the ppGalNAc-T18 knockdown cell, these defects could be significantly alleviated, suggesting that ppGalNAc-T18 is important for ER homeostasis and protein O-glycosylation. Furthermore, we found that ppGalNAc-T18 exerts its functions in O-glycosylation and ER stress via a non-catalytic mechanism. These results reveal a novel molecular role of ppGalNAc-Ts that the ER-localized ppGalNAc-T18 could regulate the O-glycosylation and ER homeostasis in a non-catalytic manner.