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Li Y, Liu Y, Tan H, Wang X, Li X, Zheng Q, Wang F, Yang Y, Wang QC, Zhang B, Zhou A, Wang H, Chai H, Sun Z, Wang JQ, Tang TS, Zhu S, Yu J, Yang M, Chen Q, Guo C
[
Cell,
2016]
Maintaining homeostasis of Ca(2+) stores in the endoplasmic reticulum (ER) is crucial for proper Ca(2+) signaling and key cellular functions. The Ca(2+)-release-activated Ca(2+) (CRAC) channel is responsible for Ca(2+) influx and refilling after store depletion, but how cells cope with excess Ca(2+) when ER stores are overloaded is unclear. We show that TMCO1 is an ER transmembrane protein that actively prevents Ca(2+) stores from overfilling, acting as what we term a "Ca(2+) load-activated Ca(2+) channel" or "CLAC" channel. TMCO1 undergoes reversible homotetramerization in response to ER Ca(2+) overloading anddisassembly upon Ca(2+) depletion and forms a Ca(2+)-selective ion channel on giant liposomes. TMCO1 knockout mice reproduce the main clinical features of human cerebrofaciothoracic (CFT) dysplasia spectrum, a developmental disorder linked to TMCO1 dysfunction, and exhibit severe mishandling of ER Ca(2+) in cells. Our findings indicate that TMCO1 provides a protective mechanism to prevent overfilling of ER stores with Ca(2+) ions.
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[
Biochem Biophys Res Commun,
2016]
Calcium (Ca(2+)) is a versatile intracellular second messenger that operates in various signaling pathways leading to multiple biological outputs. The diversity of spatiotemporal patterns of Ca(2+) signals, generated by the coordination of Ca(2+) influx from the extracellular space and Ca(2+) release from the intracellular Ca(2+) store the endoplasmic reticulum (ER), is considered to underlie the diversity of biological outputs caused by a single signaling molecule. However, such Ca(2+) signaling diversity has not been well described because of technical limitations. Here, we describe a new method to report Ca(2+) signals at subcellular resolution. We report that OER-GCaMP6f, a genetically encoded Ca(2+) indicator (GECI) targeted to the outer ER membrane, can monitor Ca(2+) release from the ER at higher spatiotemporal resolution than conventional GCaMP6f. OER-GCaMP6f was used for in vivo Ca(2+) imaging of C. elegans. We also found that the spontaneous Ca(2+) elevation in cultured astrocytes reported by OER-GCaMP6f showed a distinct spatiotemporal pattern from that monitored by plasma membrane-targeted GCaMP6f (Lck-GCaMP6f); less frequent Ca(2+) signal was detected by OER-GCaMP6f, in spite of the fact that Ca(2+) release from the ER plays important roles in astrocytes. These findings suggest that targeting of GECIs to the ER outer membrane enables sensitive detection of Ca(2+) release from the ER at subcellular resolution, avoiding the diffusion of GECI and Ca(2+). Our results indicate that Ca(2+) imaging with OER-GCaMP6f in combination with Lck-GCaMP6f can contribute to describing the diversity of Ca(2+) signals, by enabling dissection of Ca(2+) signals at subcellular resolution.
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[
Nat Methods,
2018]
It is extremely challenging to quantitate lumenal Ca<sup>2+</sup> in acidic Ca<sup>2+</sup> stores of the cell because all Ca<sup>2+</sup> indicators are pH sensitive, and Ca<sup>2+</sup> transport is coupled to pH in acidic organelles. We have developed a fluorescent DNA-based reporter, CalipHluor, that is targetable to specific organelles. By ratiometrically reporting lumenal pH and Ca<sup>2+</sup> simultaneously, CalipHluor functions as a pH-correctable Ca<sup>2+</sup> reporter. By targeting CalipHluor to the endolysosomal pathway, we mapped lumenal Ca<sup>2+</sup> changes during endosomal maturation and found a surge in lumenal Ca<sup>2+</sup> specifically in lysosomes. Using lysosomal proteomics and genetic analysis, we found that
catp-6, a Caenorhabditis elegans homolog of ATP13A2, was responsible for lysosomal Ca<sup>2+</sup> accumulation-an example of a lysosome-specific Ca<sup>2+</sup> importer in animals. By enabling the facile quantification of compartmentalized Ca<sup>2+</sup>, CalipHluor can expand the understanding of subcellular Ca<sup>2+</sup> importers.
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[
J Environ Sci (China),
2010]
We used toxicity identification evaluation (TIE) method to confirm the combinational effects of identified toxic metals in a paper recycling mill effluent in inducing the decreased lifespan in nematode Caenorhabditis elegans. Exposure to Ca + Al caused more severely decreased lifespan than that exposed to Ca, or Al; and exposure to Ca + Fe induced more severely decreased lifespan than that exposed to Ca, or Fe. Exposure to Ca+Al+Fe caused more severely decreased lifespan than that exposed to Ca, or Ca+Fe. Moreover, the baseline toxicity on lifespan was doubled by doubling the concentration of combined metals (Ca+Al+Fe) in spiking test in original effluent (oe), and lifespan defects in oe+Ca+Al+Fe exposed nematodes were more severe than that in Ca+Al+Fe exposed nematode. Therefore, Ca+Al+Fe exposure may largely explain the formation of decreased lifespan induced by the examined industrial effluent. Furthermore, the observed reduction of lifespan induced by the combination of high level of Ca with other metals may be at least partially independent of the insulin-like pathway.
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[
Cell Rep,
2016]
Sperm induce Ca(2+) waves in the fertilized egg by introducing soluble factors or by surface interactions, which activate egg Ca(2+) channels. Involvement of sperm Ca(2+) channels is predicted by the conduit model; however, this model has not been validated. In Caenorhabditis elegans, the sperm-specific TRP family Ca(2+) channel TRP-3 mediates sperm-oocyte fusion. Here, using high-speed invivo imaging and image analyses, we show that sperm induce an immediate local Ca(2+) rise followed by a Ca(2+) wave in fertilized C.elegans oocytes. Oocytes fertilized by rare
trp-3 escaper sperm showed a lack of local rise and a delay in onset of the Ca(2+) wave. Sperm Ca(2+) imaging suggests that the local rise is not due to the bolus introduction of stored Ca(2+). These results suggest that, along with its primary function in sperm-oocyte fusion, TRP-3 induces Ca(2+) waves in fertilized oocytes, consistent with the conduit model.
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Zhang, X., Ronan, E.A., He, F., Liu, J., Guo, Y., Xu, X.Z.S., Sartbaeva, E.
[
International Worm Meeting,
2019]
Cinnamaldehyde (CA), the essential oil in cinnamon, is shown to have systemic metabolic benefits in both mice and humans, however the underlying molecular mechanisms are not understood. While the only known receptor for CA is transient receptor potential (TRP)A1, the thermogenic response of adipocytes to CA are TRPA1-independent, indicating unknown CA-sensing mechanisms exist. Here we report that CA acts on sensory neurons in C. elegans to evoke complex behavioral responses. In particular, we found that CA can excite the polymodal nociceptive neuron ASH shown by calcium imaging and electrophysiology recordings. We found these effects to be mediated by GPCR signaling, suggesting that the underlying CA receptor is a GPCR. We performed genetic screens to identify the genes mediating CA-avoidance in the ASH neuron. Our results show that C. elegans sense CA through a previously uncharacterized neural and genetic pathway which may be evolutionarily conserved and have physiological significance in higher organisms.
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[
J Physiol,
2005]
Inositol-1,4,5-trisphosphate (IP3)-dependent Ca(2+) oscillations in Caenorhabditis elegans intestinal epithelial cells regulate the nematode defecation cycle. The role of plasma membrane ion channels in intestinal cell oscillatory Ca(2+) signaling is unknown. We have shown previously that cultured intestinal cells express a Ca(2+) selective conductance, IORCa, that is biophysically similar to TRPM7 currents. IORCa activates slowly and stabilizes when cells are patch clamped with pipette solutions containing 10 mM BAPTA and free Ca(2+) concentrations of ~17 nM. However, when BAPTA concentration is lowered to 1 mM, IORCa oscillates. Oscillations in channel activity induced simultaneous oscillations in cytoplasmic Ca(2+) levels. Removal of extracellular Ca(2+) inhibited IORCa oscillations whereas readdition of Ca(2+) to the bath caused a rapid and transient reactivation of the current. Experimental maneuvers that elevated intracellular Ca(2+) blocked current oscillations. Elevation of intracellular Ca(2+) in the presence of 10 mM BAPTA to block IORCa oscillations led to a dose-dependent increase in the rate of current activation. At intracellular Ca(2+) concentrations of 250 nM, current activation was transient. Patch pipette solutions buffered with 1-4 mM of either BAPTA or EGTA gave rise to similar patterns of IORCa oscillations. We conclude that changes in Ca(2+) concentration close to the intracellular opening of the channel pore regulate channel activity. Low concentrations of Ca(2+) activate ORCa. As Ca(2+) enters and accumulates near the pore mouth, channel activity is inhibited. Oscillating plasma membrane Ca(2+) entry may play a role in generating intracellular Ca(2+) oscillations that regulate the C. elegans defecation rhythm.
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[
J Exp Zool,
1999]
Crustaceans serve as an ideal model for the study of calcium homeostasis due to their natural molting cycle. Demineralization and remineralization of the calcified cuticle is accompanied by bidirectional Ca transfer across the primary Ca transporting epithelia: gills, antennal gland (kidney), digestive system, and cuticular hypodermis. The review will demonstrate how a continuum of crustaceans can be used as a paradigm for the evolution of Ca transport mechanisms. Generally speaking, aquatic crustaceans rely primarily on branchial Ca uptake and accordingly are affected by water Ca content; terrestrial crustaceans rely on intake of dietary Ca across the digestive epithelium. Synchrony of mineralization at the cuticle vs. storage sites will be presented Physiological and behavioral adaptations have evolved to optimize Ca balance during the molting cycle in different Ca environments. Intracellular Ca regulation reveals common mechanisms of apical and basolateral membrane transport as well as intracellular sequestration. Regulation of cell Ca concentration will be discussed in intermolt and during periods of the molting cycle when transepithelial Ca flux is significantly elevated. Molecular characterization of the sarco-/endoplasmic reticular Ca pump in aquatic species reveals the presence of two isoforms that originate from a single gene. This gene is differentially expressed during the molting cycle. Gene expression may be regulated by a suite of hormones including ecdysone, calcitonin, and vitamin D. Perspectives for future research are presented.
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[
International Worm Meeting,
2003]
The free cytosolic calcium concentration ([Ca]i) regulates various cellular functions and is tightly regulated in the parameters of time, space, and amplitude. When a cell is given a single dose of stimulus within the physiological range, the cell often generates a repetitive increase/fall cycle of intracellular Ca concentration (Ca oscillation). Also, the [Ca]i increase in a single cell travels as a Ca wave through neighboring cells to coordinate their activities. These [Ca]i regulations are ubiquitously observed in various cell types of many organisms. The C. elegans defecation is a periodic behavior and its activity can be used to monitor the [Ca]i regulation in intestine: the worm defecates every 45 seconds and this periodicity is regulated by the activity of the inositol 1,4,5-trisphosphate receptor that is expressed in the intestine (Dal Santo et al. 1999). To visualize cytoplasmic Ca dynamics in the intestine, we expressed the Ca-indicating protein Cameleon. A Ca spike coincides with the first step of the defecation motor program (pBoc), therefore, the [Ca]i oscillates every 45 seconds. The [Ca]i change was between 100 to 600 nM during the pBoc. This [Ca]i oscillation is initiated at the posterior end and is propagated toward the anterior end. Since the C. elegans intestine consists of 20 cells, this system can also be a model to study the intercellular Ca wave propagation. To make this assay system more useful for physiological study, we have developed a new culture system of the C. elegans adult intestine. Christensen et al. (2002) developed C. elegans embryonic cells recently, but our system is distinct and has unique advantages for the study of intestinal physiology. In this system, we can reproduce a variety of phenomena observed in the intact adult intestine, such as execution of pBoc, the intracellular Ca oscillation, and the intercellular Ca wave propagation. The Ca oscillation was also observed using the chemical Ca indicator Fluo-4 and electrophysiological measurements. (The intestinal membrane potential oscillation was first observed by A. Wei.) Therefore, this new system can combine genetic, molecular-biological, and physiological approaches to study a Ca signaling mechanism. Using this assay system, we have investigated function of the Transient Receptor Potential (TRP) Channels that are proposed to be involved in intracellular Ca dynamics, and will discuss their function and the usefulness of this new system for physiological study.
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[
Elife,
2016]
Mitochondrial Ca(2+) uptake, a process crucial for bioenergetics and Ca(2+) signaling, is catalyzed by the mitochondrial calcium uniporter. The uniporter is a multi-subunit Ca(2+)-activated Ca(2+) channel, with the Ca(2+) pore formed by the MCU protein and Ca(2+)-dependent activation mediated by MICU subunits. Recently, a mitochondrial inner membrane protein EMRE was identified as a uniporter subunit absolutely required for Ca(2+) permeation. However, the molecular mechanism and regulatory purpose of EMRE remain largely unexplored. Here, we determine the transmembrane orientation of EMRE, and show that its known MCU-activating function is mediated by the interaction of transmembrane helices from both proteins. We also reveal a second function of EMRE: to maintain tight MICU regulation of the MCU pore, a role that requires EMRE to bind MICU1 using its conserved C-terminal polyaspartate tail. This dual functionality of EMRE ensures that all transport-competent uniporters are tightly regulated, responding appropriately to a dynamic intracellular Ca(2+) landscape.