Over the recent years, several proteins that make up the mitochondrial calcium uniporter complex (MCUC) mediating Ca²⁺ uptake into the mitochondrial matrix have been identified in mammals, including ...the channel-forming protein MCU. Although six MCU gene homologs are conserved in the model plant Arabidopsis (Arabidopsis thaliana) in which mitochondria can accumulate Ca²⁺, a functional characterization of plant MCU homologs has been lacking. Using electrophysiology, we show that one isoform, AtMCU1, gives rise to a Ca²⁺-permeable channel activity that can be observed even in the absence of accessory proteins implicated in the formation of the active mammalian channel. Furthermore, we provide direct evidence that AtMCU1 activity is sensitive to the mitochondrial calcium uniporter inhibitors Ruthenium Red and Gd³⁺, as well as to the Arabidopsis protein MICU, a regulatory MCUC component. AtMCU1 is prevalently expressed in roots, localizes to mitochondria, and its absence causes mild changes in Ca²⁺ dynamics as assessed by in vivo measurements in Arabidopsis root tips. Plants either lacking or overexpressing AtMCU1 display root mitochondria with altered ultrastructure and show shorter primary roots under restrictive growth conditions. In summary, our work adds evolutionary depth to the investigation of mitochondrial Ca²⁺ transport, indicates that AtMCU1, together with MICU as a regulator, represents a functional configuration of the plant mitochondrial Ca²⁺ uptake complex with differences to the mammalian MCUC, and identifies a new player of the intracellular Ca²⁺ regulation network in plants.
We evaluated the presence and role of internal calcium stores in human uncapacitated spermatozoa by determining the effects of two inhibitors of Ca2+ ATPase of the sarco-endoplasmic reticulum ...(SERCA–ATPase), thapsigargin and cyclopiazonic acid (CPA) on intracellular calcium concentrations, Ca2+i, plasma membrane potential and acrosome reaction. Using a fluorescent conjugate of thapsigargin, we localized internal Ca2+ stores on the acrosome, post-acrosomal region and sperm midpiece. SERCA–ATPase inhibitors induced a rise in Ca2+i both in Ca2+ and Ca2+-free media but under these latter conditions it was reduced with a progressive decline to baseline values; the re-addition of Ca2+-stimulated a rise in Ca2+i. This demonstrated that internal Ca2+ store depletion can evoke the opening of Ca2+-channels on sperm plasma membrane, thus showing the existence of `capacitative' Ca2+ entry into these specialized cells. The addition of thapsigargin to human spematozoa induced a dose-dependent increase in acrosome reaction percentages, but only when Ca2+ was present in the external medium. Plasma membrane potential monitoring showed that these inhibitors induced a depolarization dependent on Ca2+ influx from external medium and that this was preceded by a transient hyperpolarization caused by activation of Ca2+-dependent K+ channels. When K+-dependent plasma membrane hyperpolarization was inhibited, the thapsigargin- and CPA-stimulated rise in Ca2+i plasma membrane depolarization and acrosome reaction were abolished. In conclusion, the present study demonstrates that human spermatozoa possess internal Ca2+ stores and that the capacitative Ca2+ entry pathway present in these cells regulates important biological processes that are fundamental for the acrosome reaction.
The mitochondrial Ca
uniporter machinery is a multiprotein complex composed by the Ca
selective pore-forming subunit, the mitochondrial uniporter (MCU), and accessory proteins, including MICU1, MICU2 ...and EMRE. Their concerted action is required to fine-tune the uptake of Ca
into the mitochondrial matrix which both sustains cell bioenergetics and regulates the apoptotic response. To adequately fulfil such requirements and avoid impairment in mitochondrial Ca
handling, the intracellular turnover of all the MCU components must be tightly regulated. Here we show that the MCU complex regulator MICU1, but not MCU and MICU2, is rapidly and selectively degraded by the Ubiquitin Proteasome System (UPS). Moreover, we show that the multifunctional E3 ubiquitin ligase Parkin (PARK2), whose mutations cause autosomal recessive early-onset Parkinson's disease (PD), is a potential candidate involved in this process since its upregulation strongly decreases the basal level of MICU1. Parkin was found to interact with MICU1 and, interestingly, Parkin Ubl-domain, but not its E3-ubquitin ligase activity, is required for the degradation of MICU1, suggesting that in addition to the well documented role in the control of Parkin basal auto-inhibition, the Ubl-domain might exert important regulatory functions by acting as scaffold for the proteasome-mediated degradation of selected substrates under basal conditions, i.e. to guarantee their turnover. We have found that also MICU2 stability was affected upon Parkin overexpression, probably as a consequence of increased MICU1 degradation. Our findings support a model in which the PD-related E3 ubiquitin ligase Parkin directly participates in the selective regulation of the MCU complex regulator MICU1 and, indirectly, also of the MICU2 gatekeeper, thus indicating that Parkin loss of function could contribute to the impairment of the ability of mitochondria to handle Ca
and consequently to the pathogenesis of PD.
Extracellular agonists mobilize Ca
2+ from SERCA-comprising intracellular Ca
2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca
2+ release from both these compartments ...was studied in HeLa cells stably expressing the luminescent Ca
2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal Ca
2+ as detected by the aequorin measurements were correlated with parallel changes in total Ca
2+ content of the stores. The latencies and initial rates of Ca
2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca
2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca
2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic Ca
2+ rise. Time-course experiments further revealed that the peak of the cytosolic Ca
2+ response occurred before the lumenal Ca
2+ reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic Ca
2+ upon agonist stimulation, but the kinetics of the Ca
2+ release are different.
By pumping calcium from the cytosol to the ER, sarco/endoplasmic reticulum calcium ATPases (SERCAs) play a major role in the control of calcium signaling. We describe two SERCA1 splice variants ...(S1Ts) characterized by exon 4 and/or exon 11 splicing, encoding COOH terminally truncated proteins, having only one of the seven calcium-binding residues, and thus unable to pump calcium. As shown by semiquantitative RT-PCR, S1T transcripts are differentially expressed in several adult and fetal human tissues, but not in skeletal muscle and heart. S1T proteins expression was detected by Western blot in nontransfected cell lines. In transiently transfected cells, S1T homodimers were revealed by Western blot using mildly denaturing conditions. S1T proteins were shown, by confocal scanning microscopy, to colocalize with endogenous SERCA2b into the ER membrane. Using ER-targeted aequorin (erAEQ), we have found that S1T proteins reduce ER calcium and reverse elevation of ER calcium loading induced by SERCA1 and SERCA2b. Our results also show that SERCA1 variants increase ER calcium leakage and are consistent with the hypothesis of a cation channel formed by S1T homodimers. Finally, when overexpressed in liver-derived cells, S1T proteins significantly induce apoptosis. These data reveal a further mechanism modulating Ca2+accumulation into the ER of nonmuscle cells and highlight the relevance of S1T proteins to the control of apoptosis.
. Objectives: Polycystin‐1 (PC1), a signalling receptor regulating Ca2+‐permeable cation channels, is mutated in autosomal dominant polycystic kidney disease, which is typically characterized by ...increased cell proliferation. However, the precise mechanisms by which PC1 functions on Ca2+ homeostasis, signalling and cell proliferation remain unclear. Here, we investigated the possible role of PC1 as a modulator of non‐capacitative Ca2+ entry (NCCE) and Ca2+ oscillations, with downstream effects on cell proliferation. Results and discussion: By employing RNA interference, we show that depletion of endogenous PC1 in HEK293 cells leads to an increase in serum‐induced Ca2+ oscillations, triggering nuclear factor of activated T cell activation and leading to cell cycle progression. Consistently, Ca2+ oscillations and cell proliferation are increased in PC1‐mutated kidney cystic cell lines, but both abnormal features are reduced in cells that exogenously express PC1. Notably, blockers of the NCCE pathway, but not of the CCE, blunt abnormal oscillation and cell proliferation. Our study therefore provides the first demonstration that PC1 modulates Ca2+ oscillations and a molecular mechanism to explain the association between abnormal Ca2+ homeostasis and cell proliferation in autosomal dominant polycystic kidney disease.
Abstract Cadmium, a toxic environmental contaminant, exerts adverse effects on different cellular pathways such as cell proliferation, DNA damage and apoptosis. In particular, the modulation of Ca2+ ...homeostasis seems to have an important role during Cd2+ injury, but the precise assessment of Ca2+ signalling still remains poorly understood. We used aequorin-based probes specifically directed to intracellular organelles to study Ca2+ changes during cadmium injury. We observed that cadmium decreased agonist-evoked endoplasmic reticulum (ER) Ca2+ signals and caused a 40% inhibition of sarcoplasmic–ER calcium ATPases activity. Moreover, time course experiments correlate morphological alterations, processing of xbp-1 mRNA and caspase-12 activation during cadmium administration. Finally, the time response of ER to cadmium injury was compared with that of mitochondria. In conclusion, we highlighted a novel pathway of cadmium-induced cell death triggered by ER stress and involving caspase-12. Mitochondria and ER pathways seemed to share common time courses and a parallel activation of caspase-12 and caspase-9 seemed likely to be involved in acute cadmium toxicity.
Hepatitis C virus (HCV) core, known to be involved in liver carcinogenesis, is processed in the endoplasmic reticulum (ER). We thus investigated the impact of three HCV core isolates on ER stress, ER ...calcium signalling and apoptosis. We show that HCV core constructs trigger hyperexpression of Grp78/BiP, Grp 94, calreticulin and sarco/endoplasmic reticulum calcium ATPase, inducing ER stress. By using the ER-targeted aequorin calcium probe, we found that ER calcium depletion follows ER stress in core-expressing cells. HCV core induces apoptosis through overexpression of the CHOP/GADD153 proapoptotic factor, Bax translocation to mitochondria, mitochondrial membrane depolarization, cytochrome c release, caspase-3 and PARP cleavage. Furthermore, reversion of HCV core-induced ER calcium depletion (by transfection of SERCA2) completely abolished mitochondrial membrane depolarization, suggesting that both ER stress (through CHOP overexpression) and calcium signalling play a major role in the HCV core-mediated control of apoptosis. ER stress and apoptosis were also found in a proportion of HCV-full-length replicon-expressing cells and in the liver of HCV core transgenic mice. In conclusion, our data demonstrate that HCV core deregulates the control of apoptosis by inducing ER stress and ER calcium depletion providing new elements to understand the mechanisms involved in HCV-related liver chronic diseases.
Activation of nuclear transcription factors, breakdown of nuclear envelope and apoptosis represent a group of nuclear events thought to be modulated by changes in nucleoplasmic Ca2+ concentration, ...Ca2+n. Direct evidence for, or against, this possibility has been, however, difficult to obtain because measurements of Ca2+n are hampered by major technical problems. Here we describe a new approach for selectively monitoring Ca2+ concentrations inside the nucleus of living cells, which is based on the construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and a nuclear translocation signal derived from the rat glucocorticoid receptor. This modified aequorin (nuAEQ), stably expressed in HeLa cells, was largely confined to the nucleoplasm and thus utilized for monitoring Ca2+n in intact cells. No significant differences were observed between Ca2+n and cytosolic Ca2+ concentration (Ca2+i) under resting conditions. Upon stimulation of surface receptors linked to inositol‐1,4,5‐trisphosphate (InsP3) generation, and thus to intracellular Ca2+ signalling, the kinetics of Ca2+i and Ca2+n increases were indistinguishable. However, for the same rise in Ca2+i, the amplitude of Ca2+n increase was larger when evoked by Ca2+ mobilization from internal stores than when induced by Ca2+ influx across the plasma membrane. The functional significance of these transient nucleus‐cytosol Ca2+ gradients is discussed.
Although the physiological relevance of mitochondrial Ca2+ homeostasis is widely accepted, no information is yet available on the molecular identity of the proteins involved in this process. Here we ...analyzed the role of the voltage-dependent anion channel (VDAC) of the outer mitochondrial membrane in the transmission of Ca2+ signals between the ER and mitochondria by measuring cytosolic and organelle Ca2+ with targeted aequorins and Ca2+-sensitive GFPs. In HeLa cells and skeletal myotubes, the transient expression of VDAC enhanced the amplitude of the agonist-dependent increases in mitochondrial matrix Ca2+ concentration by allowing the fast diffusion of Ca2+ from ER release sites to the inner mitochondrial membrane. Indeed, high speed imaging of mitochondrial and cytosolic Ca2+ changes showed that the delay between the rises occurring in the two compartments is significantly shorter in VDAC-overexpressing cells. As to the functional consequences, VDAC-overexpressing cells are more susceptible to ceramide-induced cell death, thus confirming that mitochondrial Ca2+ uptake plays a key role in the process of apoptosis. These results reveal a novel function for the widely expressed VDAC channel, identifying it as a molecular component of the routes for Ca2+ transport across the mitochondrial membranes.
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