InteractiveFly: GeneBrief

Stromal interaction molecule: Biological Overview | References


Gene name - Stromal interaction molecule

Synonyms -

Cytological map position - 14A1-14A1

Function - signaling protein

Keywords - calcium ion binding activity, ER-Ca2+ sensor required for store-operated Ca2+ entry - dimerizes and undergoes structural rearrangements facilitating binding Orai - regulates the release of neuropeptides - regulates lipid mobilization in response to Adipokinetic hormone (Akh) and insulin signaling in fat body - regulates synaptic release from pupal dopaminergic neurons allowing for sustained flight

Symbol - Stim

FlyBase ID: FBgn0045073

Genetic map position - chrX:15,934,495-15,940,451

NCBI classification - SAM domain of STIM-1,2-like proteins, STIM1 Orai1-activating region

Cellular location - ER-resident transmembrane protein



NCBI links: EntrezGene, Nucleotide, Protein
BIOLOGICAL OVERVIEW

Neuroendocrine cells communicate via neuropeptides to regulate behaviour and physiology. This study examines how STIM (Stromal Interacting Molecule), an ER-Ca2+ sensor required for Store-operated Ca2+ entry, regulates neuropeptides required for Drosophila development under nutrient restriction (NR). Two STIM-regulated peptides, Corazonin and short Neuropeptide F, were found to be required for NR larvae to complete development. Further, a set of secretory DLP (Dorso lateral peptidergic) neurons which co-express both peptides was identified. Partial loss of dSTIM caused peptide accumulation in the DLPs, and reduced systemic Corazonin signalling. Upon NR, larval development correlated with increased peptide levels in the DLPs, which failed to occur when dSTIM was reduced. Comparison of systemic and cellular phenotypes associated with reduced dSTIM, with other cellular perturbations, along with genetic rescue experiments, suggested that dSTIM primarily compromises neuroendocrine function by interfering with neuropeptide release. Under chronic stimulation, dSTIM also appears to regulate neuropeptide synthesis (Megha, 2019).

Metazoan cells commonly use ionic Ca2+ as a second messenger in signal transduction pathways. To do so, levels of cytosolic Ca2+ are dynamically managed. In the resting state, cytosolic Ca2+ concentration is kept low and maintained thus by the active sequestration of Ca2+ into various organelles, the largest of which is the ER. Upon activation, ligand-activated Ca2+ channels on the ER, such as the ryanodine receptor or inositol 1,4,5-trisphosphate receptor (IP3R), release ER-store Ca2+ into the cytosol. Loss of ER-Ca2+ causes STromal Interacting Molecule (STIM), an ER-resident transmembrane protein, to dimerize and undergo structural rearrangements. This facilitates the binding of STIM to Orai, a Ca2+ channel on the plasma membrane, whose pore then opens to allow Ca2+ from the extracellular milieu to flow into the cytosol. This type of capacitative Ca2+ entry is called Store-operated Ca2+ entry (SOCE). Of note, key components of SOCE include the IP3R, STIM and Orai, that are ubiquitously expressed in the animal kingdom, underscoring the importance of SOCE to cellular functioning. Depending on cell type and context, SOCE can regulate an array of cellular processes (Megha, 2019).

Neuronal function in particular is fundamentally reliant on the elevation of cytosolic Ca2+. By tuning the frequency and amplitude of cytosolic Ca2+ signals that are generated, distinct stimuli can make the same neuron produce outcomes of different strengths. The source of the Ca2+ influx itself contributes to such modulation as it can either be from internal ER-stores or from the external milieu, through various activity-dependent voltage gated Ca2+ channels (VGCCs) and receptor-activated Ca2+ channels or a combination of the two. Although the contributions of internal ER-Ca2+ stores to neuronal Ca2+ dynamics are well recognized, the study of how STIM and subsequently, SOCE-mediated by it, influences neuronal functioning, is as yet a nascent field (Megha, 2019).

Mammals have two isoforms of STIM, STIM1 and STIM2, both which are widely expressed in the brain. As mammalian neurons also express multiple isoforms of Orai and IP3R, it follows that STIM-mediated SOCE might occur in them. Support for this comes from studies in mice, where STIM1-mediated SOCE has been reported for cerebellar granule neurons and isolated Purkinje neurons, while STIM2-mediated SOCE has been shown in cortical and hippocampal neurons. STIM can also have SOCE-independent roles in excitable cells, that are in contrast to its role via SOCE. In rat cortical neurons and vascular smooth muscle cells, Ca2+ release from ER-stores prompts the translocation of STIM1 to ER-plasma membrane junctions, and binding to the L-type VGCC, CaV1.2. Here STIM1 inhibits CaV1.2 directly and causes it to be internalized, reducing the long-term excitability of these cells. In cardiomyocyte-derived HL1 cells, STIM1 binds to a T-type VGCC, CaV1.3, to manage Ca2+ oscillations during contractions. These studies indicate that STIM regulates cytosolic Ca2+ dynamics in excitable cells, including neurons and that an array of other proteins determines if STIM regulation results in activation or inhibition of neurons. Despite knowledge of the expression of STIM1 and STIM2 in the hypothalamus, the major neuroendocrine centre in vertebrates, studies on STIM in neuroendocrine cells are scarce. This study therefore used Drosophila melanogaster to address this gap (Megha, 2019).

Neuroendocrine cells possess elaborate machinery for the production, processing and secretion of neuropeptides (NPs), which perhaps form the largest group of evolutionarily conserved signalling agents. Inside the brain, NPs typically modulate neuronal activity and consequently, circuits; when released systemically, they act as hormones. Drosophila is typical in having a vast repertoire of NPs that together play a role in almost every aspect of its behaviour and physiology. Consequently, NP synthesis and release are highly regulated processes. As elevation in cytosolic Ca2+ is required for NP release, a contribution for STIM-mediated SOCE to NE function was hypothesized (Megha, 2019).

Drosophila possess a single gene for STIM, IP3R and Orai, and all three interact to regulate SOCE in Drosophila neurons. In dopaminergic neurons, dSTIM is important for flight circuit maturation, with dSTIM-mediated SOCE regulating expression of a number of genes, including Ral, which controls neuronal vesicle exocytosis. In glutamatergic neurons, dSTIM is required for development under nutritional stress and its' loss results in down-regulation of several ion channel genes which ultimately control neuronal excitability. Further, dSTIM over-expression in insulin-producing NE neurons could restore Ca2+ homeostasis in a non-autonomous manner in other neurons of an IP3R mutant, indicating an important role for dSTIM in NE cell output, as well as compensatory interplay between IP3R and dSTIM. At a cellular level, partial loss of dSTIM impairs SOCE in Drosophila neurons as well as mammalian neural precursor cells. Additionally, reducing dSTIM in Drosophila dopaminergic neurons attenuates KCl-evoked depolarisation and as well as vesicle release. Because loss of dSTIM specifically in dimm+ NE cells results in a pupariation defect on nutrient restricted (NR) media, this study used the NR paradigm as a physiologically relevant context in which to investigate STIM's role in NE cells from the cellular as well as systemic perspective (Megha, 2019).

This study employed an in vivo approach coupled to a functional outcome, in order to broaden understanding of how STIM regulates neuropeptides. A role for dSTIM-mediated SOCE in Drosophila neuroendocrine cells for survival on NR was previously established. The previous study offered the opportunity to identify SOCE-regulated peptides, produced in these neuroendocrine cells, that could be investigated in a physiologically relevant context (Megha, 2019).

In Drosophila, both Crz and sNPF have previously been attributed roles in many different behaviours. Crz has roles in adult metabolism and stress responses, sperm transfer and copulation, and regulation of ethanol sedation. While, sNPF has been implicated in various processes including insulin regulation circadian behaviour, sleeping and feeding. Thus, the identification of Crz and sNPF in coping with nutritional stress is perhaps not surprising, but a role for them in coordinating the larval to pupal transition under NR is novel (Megha, 2019).

A role for Crz in conveying nutritional status information is supported by this study. In larvae, Crz+ DLPs are known to play a role in sugar sensing and in adults, they express the fructose receptor Gr43a. Additionally, they express receptors for neuropeptides DH31, DH44 and AstA, which are made in the gut as well as larval CNS. Together, these observations and are strongly indicative of a role for Crz+ DLPs in directly or indirectly sensing nutrients, with a functional role in larval survival and development in nutrient restricted conditions (Megha, 2019).

Several neuropeptides and their associated signalling systems are evolutionarily conserved. The similarities between Crz and GnRH (gonadotrophin-releasing hormone), and sNPF and PrRP (Prolactin-releasing peptide), at the structural, developmental and receptor level therefore, is intriguing. Structural similarity of course does not imply functional conservation, but notably, like sNPF, PrRP has roles in stress response and appetite regulation. This leads to the conjecture that GnRH and PrRP might play a role in mammalian development during nutrient restriction (Megha, 2019).

dSTIM regulates Crz and sNPF at the levels of peptide release and likely, peptide synthesis upon NR. It is speculated that neuroendocrine cells can use these functions of STIM, to fine tune the amount and timing of peptide release, especially under chronic stimulation (such as 24hrs NR), which requires peptide release over a longer timeframe. Temporal regulation of peptide release by dSTIM may also be important in neuroendocrine cells that co-express peptides with multifunctional roles, as is the case for Crz and sNPF. It is conceivable that such different functional outcomes may require distinct bouts of NP release, varying from fast quantile release to slow secretion. As elevation in cytosolic Ca2+ drives NP vesicle release, neurons utilise various combinations of Ca2+ influx mechanisms to tune NP release. For example, in Drosophila neuromuscular junction, octopamine elicits NP release by a combination of cAMP signalling and ER-store Ca2+, and the release is independent of activity-dependent Ca2+ influx. In the mammalian dorsal root ganglion, VGCC activation causes a fast and complete release of NP vesicles, while activation of TRPV1 causes a pulsed and prolonged release. dSTIM-mediated SOCE adds to the repertoire of mechanisms that can regulate cytosolic Ca2+ levels and therefore, vesicle release. This has already been shown for Drosophila dopaminergic neurons (Richhariya, 2018) and this study extends the scope of release to peptides. Notably, dSTIM regulates exocytosis via Ral in neuroendocrine cells, like in dopaminergic neurons (Megha, 2019).

In Drosophila larval Crz+ DLPs, dSTIM appears to have a role in both fed, as well as NR conditions. On normal food, not only do Crz+ DLPs exhibit small but significant levels of neuronal activity but also, loss of dSTIM in these neurons reduced Crz signalling. Thus, dSTIM regulates Ca2+ dynamics and therefore, neuroendocrine activity, under basal as well as stimulated conditions. This is consistent with observations that basal SOCE contributes to spinogenesis, ER-Ca2+ dynamics as well as transcription. This regulation appears to have functional significance only in NR conditions as pupariation of larvae, with reduced levels of dSTIM in Crz+ neurons, is not affected on normal food. In a broader context, STIM is a critical regulator of cellular Ca2+ homeostasis as well as SOCE, and a role for it in the hypothalamus has been poorly explored. Because STIM is highly conserved across the metazoan phyla, this study predicts a role for STIM and STIM-mediated SOCE in peptidergic neurons of the hypothalamus. There is growing evidence that SOCE is dysregulated in neurodegenerative diseases. In neurons derived from mouse models of familial Alzheimer's disease and early onset Parkinson's, reduced SOCE has been reported. How genetic mutations responsible for these diseases manifest in neuroendocrine cells is unclear. If they were to also reduce SOCE in peptidergic neurons, it's possible that physiological and behavioural symptoms associated with these diseases, may in part stem from compromised SOCE-mediated NP synthesis and release (Megha, 2019).

Chronic dysfunction of Stromal interaction molecule by pulsed RNAi induction in fat tissue impairs organismal energy homeostasis in Drosophila

Obesity is a progressive, chronic disease, which can be caused by long-term miscommunication between organs. It remains challenging to understand how chronic dysfunction in a particular tissue remotely impairs other organs to eventually imbalance organismal energy homeostasis. This study introduced RNAi Pulse Induction (RiPI) mediated by short hairpin RNA (shRiPI) or double-stranded RNA (dsRiPI) to generate chronic, organ-specific gene knockdown in the adult Drosophila fat tissue. Organ-restricted RiPI targeting Stromal interaction molecule (Stim), an essential factor of store-operated calcium entry (SOCE), results in progressive fat accumulation in fly adipose tissue. Chronic SOCE-dependent adipose tissue dysfunction manifests in considerable changes of the fat cell transcriptome profile, and in resistance to the glucagon-like Adipokinetic hormone (Akh) signaling. Remotely, the adipose tissue dysfunction promotes hyperphagia likely via increased secretion of Akh from the neuroendocrine system. Collectively, this study presents a novel in vivo paradigm in the fly, which is widely applicable to model and functionally analyze inter-organ communication processes in chronic diseases (Xu, 2019).

Much like mammals, flies have an energy storage tissue called fat body, which functions similar to liver and white adipose tissue. Importantly, the majority of extra energy in mammals and flies is deposited as triacylglycerols (TAGs) in adipose tissue intracellular organelles called Lipid Droplets (LDs). Notably, a number of lipid metabolism effectors and regulators has been found to be conserved from fly to man. One of these regulatory pathways is the store-operated calcium entry (SOCE), a major determinant of the versatile second messenger intracellular Ca2+ (iCa2+). One core component of SOCE is the endoplasmic reticulum (ER) calcium sensor Stromal interaction molecule [abbreviated Stim in fly (Pathak, 2017), STIM1 in mammals] (Xu, 2019).

In non-excitable cells, the canonical G protein-coupled receptor (GPCR) signaling generates inositol 1,4,5- trisphosphate (IP3) in the cytosol, which stimulates a first wave of Ca2+ release from ER stores to the cytoplasm by activating the calcium channel-receptor IP3R. Stim/STIM1 senses the ER calcium depletion and re-localizes to ER-plasma membrane junctions, where it interacts with plasma membrane calcium channel proteins (called olf186-F in flies and Calcium release-activated calcium channel protein 1 (ORAI1) in mammals) to form active Ca2+ release-activated Ca2+ channels (CRACs), thereby ultimately facilitating the extracellular calcium entry. With this iCa2+ increase, SOCE regulates a plethora of cellular processes over a wide temporal spectrum. Previously work used acute knockdown of Stim in the fat body to identify SOCE as an adiposity regulator in flies (Baumbach, 2014; Pathak, 2017). A recent study confirmed that also mammalian STIM1 regulates lipid metabolism in liver and muscle (Maus, 2017). Moreover, the plasma membrane translocation of STIM1 required for normal SOCE is impaired in hepatocytes of obese mice, which causes further metabolic dysfunction (Arruda, 2017). While fat accumulation in response to acute SOCE interference is well established, the adverse consequences of chronic iCa2+ malfunction are unknown as Stim/STIM loss-of-function is lethal in mice and in flies (Pathak, 2017), while extended tissue-specific Stim knockdown using a temperature-controlled expression system suffers from technical limitations (Xu, 2019).

Therefore, in this study a novel strategy was developed called short-hairpin or double-stranded RNAi Pulse Induction (RiPI) to achieve tissue-specific, chronic knockdown of genes of interest exemplified by Stim in adult fly fat body. The siRNAs generated by a short-term RiPI are long-lasting, which chronically reduces the expression of Stim mRNA in adult fly fat tissue and consequently leads to progressive severe obesity in flies. Evidence is provided that chronic knockdown of Stim not only causes the tissue-autonomous dysfunction of lipid mobilization in response to Adipokinetic hormone (Akh) and reduced insulin signaling in fly fat body, but also remotely controls hyperphagia. The results suggest that chronic dysfunction of Stim/SOCE in the energy storage tissue can dramatically modulate the systemic regulatory network, which drives the organismal energy imbalance (Xu, 2019).

This study presents in vivo evidence for chronic targeted gene knockdown following RiPI in the adult Drosophila fat body. A short pulse induction of shRNA targeting the AkhR gene generates persisting siRNAs, which causes significant down-regulation of AkhR for at least 10 days. Persistence of RNAi has been associated with RNA-dependent RNA polymerase (RdRP)-mediated siRNA amplification in C. elegans and in human cells. In Drosophila, however, it remains controversial whether the genome encodes a functional RdRP. Therefore, slow degradation of the transgene-derived siRNAs might confer the chronic gene knockdown. In fact, RNAi effector double-stranded siRNAs (21nt and 24nt) are more stable than the 18nt double-stranded RNAs in the human cytosolic extract. Moreover, in human HEK293T cells, the anti-sense strand of siRNA is more resistant to intracellular nucleases compared to the sense strand of the siRNA duplex, which is likely due to the incorporation of anti-sense siRNAs into the activated RNA induced silencing complex (RISC). Therefore, the involvement of RISC might allow the slow degradation of siRNAs in adult Drosophila fat body cells. The slow decline of the siRNA level is apparently sufficient to chronically knockdown the endogenous gene expression of AkhR and Stim, which causes progressive body fat increase. This mode of action is further supported by the fact that pulsed overexpression of RNAi resistant Stim-mRNA only transiently rescues the fat content increase due to Stim-TRiPI. Consistently, long-term gene silencing (at least 11 days) is also observed in adult flies after injection of low concentrations of dsRNAs. Similarly, in an EGFP-transgenic mouse model, the inhibition of the reporter expression lasts as long as two months after siRNA injection. In summary, this study shows that in vivo RiPI generates long-lasting RNAi, which allows chronic knockdown of target genes in a tissue-specific manner. It is proposed that RiPI is a versatile tool to study causative relationships and temporal sequences in inter-organ communication processes (Xu, 2019).

Using RiPI, this study has established a Drosophila obesity model based on chronic, adipose tissue-directed knockdown of Stim, which shares remarkable similarity to characteristics of human obesity. First, the visibly enlarged abdomen of the obese flies corresponds to increased waist circumference, which gains importance as meaningful parameter to assess android adiposity. Similarly, body fat accumulation causes significant weight gain, another readout to quantify obesity in rodents and human. Second, the excessive fat accumulation correlates with climbing deficits of the obese flies, with physical fitness reduction being another hallmark of human adiposity. Moreover, obese Stim-TRiPI flies have reduced life span, which is reminiscent of the higher mortality rates in human obesity patients. Third, this study demonstrates that early-onset hyperphagia drives the positive energy balance in Stim-TRiPI flies. Consistently, increased food intake is the major driver of human obesity. Hyperphagia is linked to increased dietary glucose conversion into storage fat in obese Stim-TRiPI flies. Notably, increased food intake and elevated glucose conversion into storage lipids has also been reported after silencing obesity blocking neurons in the fly central brain. With hyperphagia being an important contributor, obesity development in Stim-RiPI flies is not monocausal. It is noteworthy that the rise in fat storage in Stim-DRiPI substantially exceeds the food intake increase. Moreover, matching the food intake of Stim-TRiPI On and Off flies still results in body fat accumulation. Importantly, there is a significantly reduced metabolic rate of Stim-DRiPI flies. Finally, the observed hyperglycemia at day 10, physical fitness reduction at day 24 and shortened life span of Stim-TRiPI On flies are associated with obesity development, similar to type 2 diabetes (T2D), exercise intolerance and mortality, which are also highly correlated with human obesity. In summary, chronic knockdown of Stim in the adult fat body causes fly obesity by a number of physiological factors culminating in organismal energy imbalance similar to mammalian adiposity (Xu, 2019).

This study highlights the critical roles played by Stim in interaction with Akh/AkhR signaling and insulin signaling in the fly fat body tissue. Reduced expression of Mdh1 and Gprk2 suggests impaired Akh/AkhR signaling in the fat body of Stim-TRiPI flies. Mammalian MDH1 has been linked to glycolysis in cells with mitochondrial dysfunction, obese Stim-TRiPI On flies display normal glycogen storage and mobilization during starvation. Similar findings are also observed in AkhA, AkhAP, and AkhR1 mutant fly larvae and adult flies, albeit their capability to mobilize glycogen is weakly impaired. A possible explanation is that flies employ corazonin, a starvation-responsive pathway complementary to Akh, to utilize glycogen. In addition to storage glycogen, the reduced expression of genes involved in lipolysis predicts an impairment of starvation-induced storage lipid mobilization. Indeed, obese Stim-TRiPI flies display an abnormal lipid mobilization profile under starvation and die with residual fat resources. Similarly, impaired lipid mobilization is also observed in flies with loss-of-function mutation in the TAG lipase gene bmm or in flies lacking either InsP3R or AkhR. Consistently, loss-of-function of STIM1/2 in mammalian cells, also impairs lipolysis via down-regulation of cAMP. Moreover, decreased catecholamine-stimulated lipolysis has been identified in human obese individuals. Collectively, thw results show that fat body tissue of obese Stim-RiPI On flies is resistant in response to Akh signaling, which drives the obesity development (Xu, 2019).

Moreover, this study supports the possibility to model T2D in adult flies. Obese Stim-TRiPI flies show reduced expression of the glucose clearance gene Hex-C, whose mammalian homolog was also suppressed in T2D patients. Besides, evidence is provided to support that obese Stim-TRiPI flies have hyperglycemia, impairment of insulin signaling in fat body tissue, and larger lipid droplets. Similar features were also described in fly larvae reared on high sugar diet36,87, which resemble mammalian insulin resistance88. Regarding unchanged circulating dIlp-2 level in obese Stim-DRiPI flies, insulin-like peptide secretion might be interfered by the knockdown of Stim in the insulin producing cells of Stim-DRiPI flies mediated by ubiquitous driver daGS, more investigation on circulating insulin levels of obese Stim-DRiPI flies by specific driver needs to be done in future. Interestingly, the indicators of insulin signaling impairment mentioned above occur at later stage of Stim-RiPI obesity development, and accordingly are possibly the consequence of Stim-TRiPI On mediated-fat gain, which also supports the concept that obesity compromises insulin signaling (Xu, 2019).

Apart from the specific role of the fat body in storage lipid handling and glucose clearance, this study shows that chronic Knockdown of Stim in this organ remotely promotes Akh secretion from the fly CC neuroendocrine cells, which leads to hyperphagia. RNAseq and gene expression analysis indicate a list of genes encoding candidate hormone or secreted proteins. Among them, CCHa2, daw, and Lst has been shown to function as hormones to regulate insulin-like peptide secretion. In addition, CCHa2, daw, Lst are also regulated by Akh overexpression in opposite direction. Whether differential expression of these genes mentioned above mediate the (mis)communication between the fat body and the CC cells is currently unknown. Nevertheless, the communication between the fat body and the CC cells is essential for the food intake increase as well as further obesity development induced by long-term knockdown of Stim. Interestingly, a study provided evidence that muscle tissue in flies communicates with the CC cells to control Akh secretion via the myokine Unpaired2 (Upd2). Upd2 had been previously shown to act as adipokine, which signals the fed state from the fat body. Unlike mammalian leptin, Upd2 remotely acts on insulin-producing cells in the central brain to regulate insulin secretion but not food intake. Recently, Akh mRNA expression was shown to be regulated by a gut-neuronal relay via midgut-secreted peptide Buriscon α in response to nutrients. Given the fact that the transcription of Akh is unaffected in Stim-RiPI On flies, identification of the adipokine, which regulates the Akh release directly or indirectly to affect food intake in the Stim-RiPI fly obesity model requires future research efforts (Xu, 2019).

In conclusion, this work introduces RNAi Pulse Induction as a novel in vivo paradigm for chronic, tissue-specific gene interference. RiPI makes essential genes accessible to long-term functional analysis in the adult fly, as exemplified in this study by establishing a Drosophila obesity model caused by chronic knockdown of Stim in the adult fat body. Moreover, this study reveals, that the fat body integrates the tissue-autonomous and the systemic branches of Akh signalling: by regulation of lipid mobilization via SOCE in the fat body, and possibly by remote-control of Akh secretion from the CC cells. Recently, the evolutionarily conserved role of SOCE in controlling energy metabolism has attracted the interest of mammalian studies. While Akh is structurally not conserved to humans, there is a growing number of remotely-controlled orexigenic peptide hormones in mammals with asprosin being one of the latest additions. Collectively, these findings in the fly add further evidence to the existence of conserved regulatory principles in animal energy homeostasis control emanating from SOCE signalling in fat storage tissues (Xu, 2019).

dSTIM- and Ral/exocyst-mediated synaptic release from pupal dopaminergic neurons sustains Drosophila flight

Manifestation of appropriate behavior in adult animals requires developmental mechanisms that help in the formation of correctly wired neural circuits. Flight circuit development in Drosophila requires store-operated calcium entry (SOCE) through the STIM/Orai pathway. SOCE-associated flight deficits in adult Drosophila derive extensively from regulation of gene expression in pupal neurons, and one such SOCE-regulated gene encodes the small GTPase Ral. The cellular mechanism by which Ral helps in maturation of the flight circuit was not understood. This study shows that knockdown of components of a Ral effector, the exocyst complex, in pupal neurons also leads to reduced flight bout durations, and this phenotype derives primarily from dopaminergic neurons. Importantly, synaptic release from pupal dopaminergic neurons is abrogated upon knockdown of dSTIM, Ral, or exocyst components. Ral overexpression restores the diminished synaptic release of dStim knockdown neurons as well as flight deficits associated with dSTIM knockdown in dopaminergic neurons. These results identify Ral-mediated vesicular release as an effector mechanism of neuronal SOCE in pupal dopaminergic neurons with functional consequences on flight behavior (Richhariya, 2018).

CRISPR-Cas-induced mutants identify a requirement for dSTIM in larval dopaminergic cells of Drosophila melanogaster

Molecular components of store-operated calcium entry have been identified in the recent past and consist of the endoplasmic reticulum (ER) membrane-resident calcium sensor STIM and the plasma membrane-localized calcium channel Orai. The physiological function of STIM and Orai is best defined in vertebrate immune cells. However, genetic studies with RNAi strains in Drosophila suggest a role in neuronal development and function. This study generated a CRISPR-Cas-mediated deletion for the gene encoding STIM in Drosophila (dSTIM), which was demonstrate as a larval lethal. To study STIM function in neurons, the CRISPR-Cas9 method was merged with the UAS-GAL4 system to generate either tissue- or cell type-specific inducible STIM knockouts (KOs). The data identify an essential role for STIM in larval dopaminergic cells. The molecular basis for this cell-specific requirement needs further investigation (Pathak, 2017).

The two major components of SOCE, STIM and Orai, have been implicated in both vertebrate and invertebrate development. In this study, a complete KO for the dSTIM gene was generated, as well as a modified inducible version, so as to understand the role of dSTIM in the development and viability of Drosophila. To generate STIMko animals, the CRISPR-Cas9 technique was adopted and putative heterozygous STIMko founders were screened by PCR. A comparison of the phenotypes of STIMko organisms with an existing orai hypomorphic allele established that SOCE is required during second and early third instar for viability. Results from a combination of rescue experiments, plus an inducible strain designed for generating dSTIM KOs, demonstrate that a major focus of SOCE requirement are dopaminergic neurons in the CNS. This is significant because dopaminergic neurons are known to regulate multiple aspects of neuronal physiology and behavior in mammals and Drosophila. In addition, dSTIM function may be required in nonneuronal cells for growth and viability. It remains to be established if all phenotypes associated with the KO of dSTIM arise as a consequence of loss of SOCE through Orai or from the ability of STIM to regulate other channels including the voltage-gated Ca2+ channels. A role for SOCE in the larval CNS agrees with previous findings of IP3R mutants, and more recent studies demonstrating that the IP3R regulates SOCE in Drosophila neurons. The precise target(s) of SOCE in the larval nervous system and in nonneuronal cells needs further investigation (Pathak, 2017).

Interestingly, complete lethality, developmental delays, and growth deficits of STIMko larvae and pupae was replicated closely by specific targeting of the dSTIM locus in dopaminergic cells. Indeed, pan-neuronal targeting of the dSTIM locus resulted in only 40-45% lethality, suggesting that dopaminergic cells are especially susceptible to loss of SOCE. An alternate explanation for differences between the extent of lethality observed in organisms when STIMdual; cas9 is driven by either ElavC155GAL4 or THGAL4 could be a low level of differential leaky GAL4 expression in nonneuronal tissues, and therefore 'nonspecific' expression of the UAScas9 construct in the two strains. At present, this issue cannot be resolved, but based on the stronger phenotype of TH > STIMdual vs. ElavC155 > STIMdual this seems unlikely, because visible nonspecific expression of THGAL4 (as viewed by driving UASmGFP in early third instar larvae) is considerably more restricted in the whole animal as compared to expression of ElavC155GAL4. It should be possible to address this more rigorously in future by generating fluorescently-marked dSTIM alleles, allowing for visualization of loss of one or both alleles in any tissue of interest (Pathak, 2017).

The difference in lethality observed between ElavC155GAL4- and THGAL4-driven STIMdual could in part also arise due to differential efficiency of tissue-specific mutagenesis in the two GAL4 strains. It should be possible to address this issue in future by using a strain that creates dSTIM KOs at a higher efficiency. The STIMdual strain used in this study targets two sites at the ends of the dSTIM open reading frame with the idea that they should create a complete KO. The detectable presence of dSTIM transcripts and protein in larval brain lysates of ElavC155GAL4 > STIMdual;cas9 suggests that a complete KO of both alleles may not occur in all neuronal cells marked by ElavC155GAL4, though some of the residual transcripts and protein could arise from nonneuronal cells, such as glia that do not express ElavC155GAL4. Recent studies suggest that increasing the target sites to three or more within a locus is a more dependable strategy for obtaining tissue-specific KOs (Pathak, 2017).

Nevertheless, a critical requirement for SOCE in dopaminergic cells is supported by earlier results with cell- and tissue-specific knockdown of SOCE components. Whereas the KO data strongly implicate dopaminergic cells as the focus of SOCE requirement in larvae, the rescue experiments also support a pan-neuronal requirement for dSTIM and dOrai. Pan-neuronal (ElavC155GAL4) overexpression of dOrai rescued lethality of orai3 homozygotes to a greater extent than overexpression from dopaminergic cells alone, though developmental delays and size were rescued to similar extents. However, in STIMko organisms, both pan-neuronal and dopaminergic overexpression led to a partial and comparable level of rescue of lethalityThese differences are attributed to the hypomorphic nature of the orai3 allele, as compared to dSTIMko which is a null allele. Differential expression patterns of ElavC155GAL4 and nSybGAL4 may also contribute to the difference in rescue of orai3 and STIMko organisms. Rescue of STIMko was attempted with nSybGAL4 because the ElavC155GAL4 transgene and STIMko are both on the X chromosome. Despite the near complete lethality of TH > cas9; STIMdual larvae, TH-driven rescue of STIMko organisms remained at 30%. dSTIM expression in dopaminergic cells is, thus, not sufficient for complete viability of STIMko animals, and indicates a requirement in other neuronal subdomains and tissues. Previously, it was demonstrated that SOCE is required for the regulation of TH gene transcription in pupae. The larval requirement for SOCE in dopaminergic cells may be similar, though effects of SOCE on cellular processes other than gene regulation remain a possibility (Pathak, 2017).

Inositol 1,4,5-trisphosphate receptor and dSTIM function in Drosophila insulin-producing neurons regulates systemic intracellular calcium homeostasis and flight

Calcium (Ca(2+)) signaling is known to regulate the development, maintenance and modulation of activity in neuronal circuits that underlie organismal behavior. In Drosophila, intracellular Ca(2+) signaling by the inositol 1,4,5-trisphosphate receptor and the store-operated channel (dOrai) regulates the formation and function of neuronal circuits that control flight. This study shows that restoring InsP(3)R activity in insulin-producing neurons of flightless InsP(3)R mutants (itpr) during pupal development can rescue systemic flight ability. Expression of the store operated Ca(2+) entry (SOCE) regulator dSTIM in insulin-producing neurons also suppresses compromised flight ability of InsP(3)R mutants suggesting that SOCE can compensate for impaired InsP(3)R function. Despite restricted expression of wild-type InsP(3)R and dSTIM in insulin-producing neurons, a global restoration of SOCE and store Ca(2+) is observed in primary neuronal cultures from the itpr mutant. These results suggest that restoring InsP(3)R-mediated Ca(2+) release and SOCE in a limited subset of neuromodulatory cells can influence systemic behaviors such as flight by regulating intracellular Ca(2+) homeostasis in a large population of neurons through a non-cell-autonomous mechanism (Agrawal, 2010).

Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight

Neuronal Ca2+ signals can affect excitability and neural circuit formation. Ca2+ signals are modified by Ca2+ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca2+ stores and their release to neuronal processes is poorly understood. This study shows, by neuron-specific siRNA depletion, that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca2+ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP3R) can partially compensate for their loss of flight. Ca2+ measurements show that Orai gain-of-function contributes to the quanta of Ca2+-release through mutant InsP3Rs and elevates store-operated Ca2+ entry in Drosophila neurons. These data show that replenishment of intracellular store Ca2+ in neurons is required for Drosophila flight (Venkiteswaran, 2009).

Several aspects of neuronal function are regulated by ionic calcium (Ca2+). Specific attributes of a Ca2+ 'signature' such as amplitude, duration, and frequency of the signal can determine the morphology of a neural circuit by affecting the outcome of cell migration, the direction taken by a growth-cone, dendritic development, and synaptogenesis. Ca2+ signals also determine the nature and strength of neural connections in a circuit by specifying neurotransmitters and receptors. Most of these Ca2+ signals have been attributed to the entry of extracellular Ca2+ through voltage-operated channels or ionotropic receptors. However, other components of the 'Ca2+ tool-kit' coupled to Ca2+ release from intracellular Ca2+ stores are also present in neurons. These molecules include the store-operated Ca2+ (SOC) channel, encoded by the Orai gene, identified in siRNA screens for molecules that reduce or abolish Ca2+ influx from the extracellular milieu after intracellular Ca2+ store depletion. Several reports have confirmed its identity as the pore forming subunit of the Ca2+-release activated Ca2+ (CRAC) channel. Activation of this CRAC channel is mediated by the endoplasmic reticulum (ER) resident protein STIM (stromal interaction molecule), also identified in an RNAi screen for molecules that regulate SOC influx. STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. STIM senses the drop in ER Ca2+ levels, and interacts with Orai by a mechanism which is only just being understood. Orai and STIM function in conjunction with the sarco-endoplasmic reticular Ca2+-ATPase pump (SERCA) to maintain ER store Ca2+ and basal Ca2+. The importance of intracellular Ca2+ homeostasis and SOC entry (SOCE) in neural circuit formation and in neuronal function and physiology remains to be elucidated (Venkiteswaran, 2009).

This study reports how Orai and STIM mediated Ca2+ influx and Ca2+ homeostasis in Drosophila neurons contribute to cellular and systemic phenotypes. Reduced SOCE, measured in primary neuronal cultures, is accompanied by a range of defects in adults, including altered wing posture, increased spontaneous firing, and loss of rhythmic flight patterns. These phenotypes mirror the spontaneous hyperexcitability of flight neuro-muscular junctions and loss of rhythmic flight patterns observed in Drosophila mutants of the inositol 1,4,5-trisphosphate receptor (InsP3R, itpr gene). The InsP3R is a ligand gated Ca2+-channel present on the membranes of intracellular Ca2+ stores. It is thought to be critical for various aspects of neuronal function. Mutants in the gene coding for the mouse InsP3R1 are ataxic. Cerebellar slices from InsP3R1 knockout mice show reduced long-term depression, indicating that altered synaptic plasticity of the cognate neural circuits could underlie the observed ataxia (Venkiteswaran, 2009).

To understand the temporal and spatial nature of intracellular Ca2+ signals required during flight circuit development and function, dOrai (CG11430) and dSERCA (encoded by CaP-60A gene, CG3725) function was modulated by genetic means in itpr mutants [using a dominant mutant allele (Kum170) for the gene (Ca-P60A) encoding the SERCA]. This modulation can restore flight to flightless adults, by altering several parameters of intracellular Ca2+ homeostasis including SOCE. These results suggest that components of the central pattern generator (CPG) required for maintenance of normal rhythmic flight in adults have a stringent requirement for SOCE after InsP3R stimulation (Venkiteswaran, 2009).

This study shows that SOC entry through the Orai/STIM pathway and the rate of clearance of cytoplasmic Ca2+ by SERCA together shape intracellular Ca2+ response curves in Drosophila larval neurons. The phenotypic changes associated with altering Orai/STIM function on their own and in itpr mutant combinations suggest that these Ca2+ dynamics are conserved through development among neurons in pupae and adults. The development and function of the flight circuit appears most sensitive to these cellular Ca2+ dynamics, changes in which have a profound effect on its physiological and behavioral outputs. Direct measurements of Ca2+ in flight circuit neurons are necessary in future to understand why these cells are more sensitive to changes in intracellular Ca2+ signaling. Other circuits such as those required for walking, climbing and jumping remain unaffected. Possible effects of altering intracellular Ca2+ homeostasis on higher order neural functions have yet to be determined (Venkiteswaran, 2009).

The flow of information in a neural circuit goes through multiple steps within and between cells. Suppression experiments, such as the ones described in this study, present a powerful genetic tool for understanding the mechanisms underlying both the formation of such circuits and their function. The correlation observed between adult phenotypes and Ca2+ dynamics in populations of larval neurons from the various genotypes supports the following conclusions. Out-spread wings, higher spontaneous firing, and initiation of rhythmic firing on air-puff delivery in itprku (a heteroallelic mutant combination of itpr) are suppressed by either increasing the quanta (through hypermorphic alleles of dOrai and by dOrai+ overexpression) or by increasing the perdurance (through mutant Kum170) of the intracellular Ca2+ signal. Flight ability and maintenance of flight patterns requires SOCE in addition to increased quanta and perdurance of the Ca2+ signals, suggesting that SOCE in neurons contributes to recurring Ca2+ signals essential for flight maintenance (Venkiteswaran, 2009).

The signals that trigger InsP3 generation in Drosophila neurons and the nature of the downstream cellular response remain to be investigated. Previous work has shown that rescue of flight and related physiological phenotypes in itpr mutants require UASitpr+ expression in early to midpupal stages, indicating the InsP3R activity is necessary during development of the flight circuit (Banerjee, 2004). Due to perdurance of the InsP3R, its requirement in adults was not established. This study found that a basal level of dOrai+ expression through development followed by ubiquitous overexpression in adults can help initiate flight in itprku, indicating a requirement for SOCE in adult neurons that probably constitute the CPG for flight. The precise neuronal circuit and neurons in the flight CPG are under investigation (Frye, 2004). Aminergic, glutamatergic, and insulin producing neurons could assist in development and/or directly constitute the circuit. Similar patterns of neuronal activity in the flight circuit of itpr mutants, either by generating different combinations of Ca2+ fluxes (as shown in this study), or by UASitpr+ expression in nonoverlapping neuronal domains supports the idea that different aspects of neuronal activity can compensate for each other to maintain constant network output (Venkiteswaran, 2009).

Precisely how hypermorphic dOrai alleles modify itprku function to increase the quanta of Ca2+ release remains to be investigated. The ability of itprku to maintain elevated [Ca2+]ER at 25 °C suggests a possible interaction between this heteroallelic combination and Orai/STIM. The mutated residue in itprka1091 (Gly to Ser at 1891) lies in the modulatory domain, whereas in itprug3, it lies in the ligand binding domain (Ser to Phe at 224); both residues are conserved in mammalian InsP3R isoforms (Srikanth, 2004). The mutant residues could directly affect InsP3R interactions with a store Ca2+ regulating molecule like STIM (Taylor, 2006). Recent reports (Redondo, 2006) also demonstrate the formation of macromolecular assemblies of InsP3R, SERCA, and SOC channels, suggesting specific functional interactions between them (Venkiteswaran, 2009).

Last, these results suggest new ways of treating diseases where altered intracellular Ca2+ signaling or homeostasis has been suggested as a causative agent. Perhaps, the best documented of these diseases are spino-cerebellar ataxia 15, which arises by heterozygosity of the mammalian IP3R1 gene , severe combined immunodeficiency due to a mutation in Orai1, and Darier's disease from a mutation in SERCA2. Based on the underlying changes in intracellular Ca2+ properties in these genetic diseases, this study suggests ways of deciding appropriate combination of drugs that might target the causative gene products and their functionally interacting partners (Venkiteswaran, 2009).

The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers.

Ca2+-release-activated Ca2+ (CRAC) channels underlie sustained Ca2+ signalling in lymphocytes and numerous other cells after Ca(2+) liberation from the endoplasmic reticulum (ER). RNA interference screening approaches identified two proteins, Stim and Orai, that together form the molecular basis for CRAC channel activity. Stim senses depletion of the ER Ca2+ store and physically relays this information by translocating from the ER to junctions adjacent to the plasma membrane, and Orai embodies the pore of the plasma membrane calcium channel. A close interaction between Stim and Orai, identified by co-immunoprecipitation and by Förster resonance energy transfer, is involved in the opening of the Ca2+ channel formed by Orai subunits. Most ion channels are multimers of pore-forming subunits surrounding a central channel, which are preassembled in the ER and transported in their final stoichiometry to the plasma membrane. This study shows, by biochemical analysis after cross-linking in cell lysates and intact cells and by using non-denaturing gel electrophoresis without cross-linking, that Orai is predominantly a dimer in the plasma membrane under resting conditions. Moreover, single-molecule imaging of green fluorescent protein (GFP)-tagged Orai expressed in Xenopus oocytes showed predominantly two-step photobleaching, again consistent with a dimeric basal state. In contrast, co-expression of GFP-tagged Orai with the carboxy terminus of Stim as a cytosolic protein to activate the Orai channel without inducing Ca2+ store depletion or clustering of Orai into punctae yielded mostly four-step photobleaching, consistent with a tetrameric stoichiometry of the active Orai channel. Interaction with the C terminus of Stim thus induces Orai dimers to dimerize, forming tetramers that constitute the Ca2+-selective pore. This represents a new mechanism in which assembly and activation of the functional ion channel are mediated by the same triggering molecule (Penna, 2008).

Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis

Phagocytosis is important during development and in the immune response for the removal of apoptotic cells and pathogens, yet its molecular mechanisms are poorly understood. In C. elegans, the CED2/5/10/12 pathway regulates actin during phagocytosis of apoptotic cells, whereas the role of the CED1/6/7 pathway in phagocytosis is unclear. This study reports that Undertaker (UTA), a Drosophila Junctophilin protein, is required for Draper (CED-1 homolog)-mediated phagocytosis. Junctophilins couple Ca2+ channels at the plasma membrane to those of the endoplasmic reticulum (ER), the Ryanodine receptors. This study places Draper, its adaptor drCed-6, UTA, the Ryanodine receptor Rya-r44F, the ER Ca2+ sensor dSTIM (Stromal interaction molecule), and the Ca2+-release-activated Ca2+ channel dOrai (olf186-F) in the same pathway that promotes calcium homeostasis and phagocytosis. Thus, these results implicate a Junctophilin in phagocytosis and link Draper-mediated phagocytosis to Ca2+ homeostasis, highlighting a previously uncharacterized role for the CED1/6/7 pathway (Cuttell, 2008).

Phagocytosis is a crucial process during development and in innate immunity of all multicellular organisms. It allows for rapid engulfment of dying cells and pathogens by specialized phagocytes, such as macrophages and neutrophils in mammals. Phagocytosis is also an essential function of dendritic cells that present processed antigens to lymphocytes, thus linking innate and adaptive immunity. In C. elegans, the death genes ced-2, 5, 10, and 12 activate the small GTPase CED-10 that triggers actin cytoskeleton rearrangement during phagocytosis; the parallel CED1/6/7 pathway also converges on CED-10, but its precise role in phagocytosis remains elusive (Cuttell, 2008 and references therein).

During Drosophila embryogenesis, two macrophage receptors, Croquemort (CRQ), a CD36 homolog, and Draper (DRPR), a CED-1 homolog, play a role in apoptotic cell clearance, much like their counterparts in mammals or C. elegans. The Drosophila homolog of CED-6, Dmel/Ced-6 (hereafter called drCed-6), and DRPR are also required in glial cells for axon pruning and the engulfment of degenerating neurons (Cuttell, 2008 and references therein).

In a deficiency screen, a mutant was characterized in which embryonic macrophages poorly engulfed apoptotic cells. In an RNAi screen using S2 cells, undertaker/retinophilin (uta) was identified as being responsible for this phenotype. uta encodes a membrane occupational and recognition nexus (MORN) repeat-containing protein with homology to mammalian Junctophilins (JPs). JPs form junctional complexes between the plasma membrane (PM) and the endoplasmic/sarcoplasmic reticulum (ER/SR) Ca2+ storage compartment. These complexes allow for crosstalk between Ca2+ channels at the PM and the ER/SR Ca2+ channels, or Ryanodine receptors (RyRs), thus regulating Ca2+ homeostasis and functions of excitable cells. Although a role for Ca2+ in phagocytosis of various particles by mammalian phagocytes has been previously described, the molecular mechanisms underlying Ca2+ fluxes associated with these events are not known (Cuttell, 2008).

This study reports that, as for UTA, the Drosophila Ryanodine receptor, Rya-r44F (Xu, 2000), plays a role in phagocytosis of apoptotic cells in vivo. A requirement in phagocytosis was found for store-operated Ca2+ entry (SOCE) via dSTIM, a Ca2+ sensor of the ER/SR lumen, and CRACM1/dOrai, a Ca2+-release-activated Ca2+ channel (CRAC). uta and rya-r44F genetically interact with drced-6 and drpr, and uta, drced6, and drpr are required for SOCE in S2 cells. Thus, these genes act in the same pathway that plays a general role in phagocytosis, as uta, dstim, dorai, drced-6, and drpr are also required for efficient phagocytosis of bacteria. These results provide a link between SOCE and phagocytosis, imply that UTA plays a similar role in macrophages to that of JPs in excitable cells, and shed light on a role for the CED1/6/7 pathway in Ca2+ homeostasis during phagocytosis (Cuttell, 2008).

Binding of various particles induces a rise in [Ca2+]i in mammalian phagocytes. In dendritic cells, [Ca2+]i increases upon apoptotic cell binding via integrin, and inhibition studies have suggested that both Ca2+ release from the ER/SR storage pool and extracellular Ca2+ entry into the cytosol are required for this process. Neutrophils also rely on such changes to promote particle engulfment. Yet, the molecular mechanisms underlying this rise in [Ca2+]i and what role it plays in phagocytes are poorly understood (Cuttell, 2008).

This study found that uta, a Drosophila gene encoding a JP-related protein, is required for phagocytosis of apoptotic cells. Genetic evidence is provided of a role for a Ryanodine receptor, Rya-r44F, and uta and rya-44F were genetically linked. SOCE via dstim and dorai promotes efficient apoptotic cell clearance. uta and rya-44F were genetically linked to drpr and drced-6, and a role was found for uta, drpr, and drced-6 in SOCE, thus demonstrating a functional link between the DRPR/drCed-6 pathway and SOCE during phagocytosis (Cuttell, 2008).

A model is proposed whereby apoptotic cell binding via DRPR (and possibly other receptors, such as CRQ) leads to ER Ca2+ release via Rya-r44F. DRPR, which bears an immunoreceptor tyrosine-based activation motif (ITAM) that is phosphorylated via Src and Syk family kinase-mediated signaling, appears to behave like an immunoreceptor. In B and T lymphocytes, engagement of Fc immunoreceptors (the signaling of which also relies on phosphorylation on ITAMs) leads to a rise in [Ca2+]i. Thus, DRPR might play a similar role to that of Fc receptors in the signaling, leading to a rise in [Ca2+]i in macrophages (Cuttell, 2008).

UTA is localized in the ER and at the PM. Thus, it is proposed that, like JPs, UTA forms junctional complexes that link the PM events to the ER and trigger Ca2+ release from ER stores. The current studies, however, did not address whether the formation of UTA junctional complexes is required to trigger ER Ca2+ release via Rya-r44F, nor what triggers ER Ca2+ release. The resting membrane potential of mammalian phagocytes is depolarized upon contact with apoptotic cells. As in mammalian muscle cells, such changes in fly phagocytes might initiate ER Ca2+ release (Cuttell, 2008).

In S2 cells, Ca2+ imaging results with drpr and drced-6 RNAi and that of others with drced-6 RNAi (Vig, 2006) suggest that drpr and drced-6 are required for dOrai-mediated Ca2+ entry upon TG treatment (which bypasses the need for particle binding to the receptor). It is proposed that ER Ca2+ release feeds back onto DRPR and drCed-6 to activate their downstream signaling cascade. Although further studies will be required to test the validity of this proposal, several reports already support it: DRPR-mediated phagocytosis depends on Src and Syk family kinase signaling, and the activity of such kinases can be Ca2+ dependent in mammalian cells (Cuttell, 2008).

It is then proposed that signaling downstream of DRPR and drCed-6 promotes and/or maintains the formation of UTA junctional complexes, thereby linking ER Ca2+ release to SOCE. dSTIM is indeed likely to act as an ER Ca2+ sensor that oligomerizes and redistributes to ER-PM junctions upon ER Ca2+ depletion, as for its mammalian counterparts. It is proposed that UTA junctional complexes are needed to maintain a close proximity between the ER Ca2+ stores and the PM and to juxtapose dSTIM oligomers and dOrai, thereby promoting conformational changes and opening of dOrai. DRPR- and drCed-6-dependent signaling and/or UTA may also be required for dSTIM oligomerization. The resulting increase in [Ca2+]i then promotes engulfment of the particle (Cuttell, 2008).

Ca2+ may promote phagocytosis via several ways. It can enhance scavenger receptor (SR) activity: adhesion of mouse macrophages to a fibronectin-coated surface via integrin binding results in an increase in the number of SRs at their PM, which enhances their binding activity. This enrichment in SRs is dependent on extracellular Ca2+ influx, arguing in favor of a role for Ca2+ in SR trafficking and/or recycling. Several SRs or related receptors play a role in phagocytosis of apoptotic corpses, including the mammalian CD36 and its Drosophila homolog CRQ. Although no change was seen in CRQ expression in uta mutant macrophages, CRQ and UTA colocalize and genetically interact. One possible model is that CRQ is recruited to the phagocytic cup upon apoptotic cell binding after SOCE that depends on UTA, DRPR, and drCed-6, and that this might strengthen the binding and uptake of the corpse (Cuttell, 2008).

In C. elegans, CED-1 (DRPR homolog) is related to the endothelial scavenger receptor SREC. Its recruitment to the phagocytic cup depends on functional CED-7, and may occur by exocytosis. Components of the exocyst were implicated in phagocytosis. Moreover, Orai1 is required for degranulation of mast cells, which occurs by exocytosis. Thus, like Orai1, dOrai may be required for exocytosis and, whereas DRPR appears to always be present at the PM, CRQ may be recruited from its intracellular vesicular pool to the phagocytic cup by exocytosis, as previously proposed for CED-1, to promote apoptotic cell uptake (Cuttell, 2008).

A rise in Ca2+ was observed in mammalian neutrophils upon particle binding, and Ca2+ participates in phagocytosis by promoting F-actin breakdown and phagosome maturation. Mycobacterium tuberculosis is able to invade human macrophages without triggering an increase in [Ca2+]i: in the absence of Ca2+ signaling, phagosomes containing M. tuberculosis fail to mature, perhaps explaining the survival of this bacterium in the cell. A role for Ca2+ in particle binding and phagosome maturation in macrophages, however, was once discounted. uta, dstim, dorai, drCed-6, and drpr are required to trigger SOCE. Yet, although they are poorly phagocytic, macrophages in drced-6 hypomorphs and drpr null mutants engulf bacteria into fully matured phagosomes, arguing against Ca2+ being involved in phagosome maturation. This maturation, however, might still occur with lower efficiency when SOCE fails, as RNAi-treated S2 cells for all genes in this pathway poorly engulfed bacteria (Cuttell, 2008).

The findings that UTA links DRPR-mediated phagocytosis and Ca2+ homeostasis provide the opportunity to pursue the dissection of the DRPR pathway in Drosophila. DRPR is homologous to CED-1, which belongs to the CED1/6/7 pathway where CED-7 is an ABC transporter. Interestingly, an ABC transporter can modulate Ca2+ channel activity in plants, further supporting a link between the CED1/6/7-like pathways and Ca2+ homeostasis, which appears to have been conserved throughout evolution. Furthermore, a mutation in human Orai1 was found in some patients with severe combined immune deficiency (Feske, 2006). Thus, pursuing such studies might be relevant to mammalian systems and to human health (Cuttell, 2008).

Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai

Recent RNA interference screens have identified several proteins that are essential for store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity in Drosophila and in mammals, including the transmembrane proteins Stim (stromal interaction molecule) and Orai. Stim probably functions as a sensor of luminal Ca2+ content and triggers activation of CRAC channels in the surface membrane after Ca2+ store depletion. Among three human homologues of Orai (also known as olf186-F), ORAI1 on chromosome 12 was found to be mutated in patients with severe combined immunodeficiency disease, and expression of wild-type Orai1 restored Ca2+ influx and CRAC channel activity in patient T cells. The overexpression of Stim and Orai together markedly increases CRAC current. However, it is not yet clear whether Stim or Orai actually forms the CRAC channel, or whether their expression simply limits CRAC channel activity mediated by a different channel-forming subunit. This study shows that interaction between wild-type Stim and Orai, assessed by co-immunoprecipitation, is greatly enhanced after treatment with thapsigargin to induce Ca2+ store depletion. By site-directed mutagenesis, it was shown that a point mutation from glutamate to aspartate at position 180 in the conserved S1-S2 loop of Orai transforms the ion selectivity properties of CRAC current from being Ca2+-selective with inward rectification to being selective for monovalent cations and outwardly rectifying. A charge-neutralizing mutation at the same position (glutamate to alanine) acts as a dominant-negative non-conducting subunit. Other charge-neutralizing mutants in the same loop express large inwardly rectifying CRAC current, and two of these exhibit reduced sensitivity to the channel blocker Gd3+. These results indicate that Orai itself forms the Ca2+-selectivity filter of the CRAC channel (Yeromin, 2006).

The results demonstrate that thapsigargin-triggered store depletion dynamically strengthens an interaction between Stim and Orai, supporting a model for CRAC channel activation in which Stim serves as the Ca2+ sensor to detect store depletion and as the messenger to activate CRAC channels in the plasma membrane. More importantly, it is concluded that Orai is a bona fide ion channel, based on the following: (1) RNA-interference-mediated knockdown of Orai expression suppresses thapsigargin-dependent Ca2+ influx and CRAC channel activity; (2) overexpression of Orai with or without Stim augments CRAC currents that exhibit biophysical properties identical to native CRAC current, and (3) mutations of negatively charged residues within the putative pore region of Orai significantly alter ion selectivity, current rectification and affinity to a charged channel blocker without altering channel activation kinetics. The marked alteration of these properties by a targeted point mutation provides definitive evidence that Orai embodies the pore-forming subunit of the CRAC channel (Yeromin, 2006).

The consensus sequence within the S1-S2 loop, 179VEVQLDxD186, contains the critical glutamate (bold) shown in this study to control ion selectivity properties of the CRAC channel, and two aspartates (underlined) that may help to attract Gd3+ (and Ca2+) towards the pore. It is not similar to pore sequences found in other channels. Unlike the pore regions of voltage-gated Ca2+ (CaV) channels, that contain a relatively long loop and a ring of critical glutamates from different domains that form a high-affinity Ca2+-binding site, the putative pore sequence of Orai is very short, and the key residue for ion selectivity (E180) is adjacent to the putative S1 segment. The corresponding residue is 178 in the Drosophila genome database (accession number AY071273), and 106 in the human Orai1 homologue (accession number BC015369). Because withdrawal of external divalent ions reveals permeability to monovalent cations in both CaV and CRAC channels, it is possible that the CRAC channel ion-selectivity filter is also formed by a ring of glutamates and that the mechanism of Ca2+ permeation is similar, although the single-channel conductance and maximum permeant ion size of the CRAC channel selectivity filter are smaller than that of the CaV channel. Negatively charged side chains also contribute to Ca2+ selectivity of TRPV6; in this instance, aspartate (at position 541) is proposed to coordinate with Ca2+ ions and line the selectivity filter in a ring structure formed by four subunits. The CRAC channel may be a multimer that includes several identical Orai subunits, as a non-conducting pore mutant (E180A) exerts a strong dominant-negative action on native CRAC current. Biochemical approaches and cysteine-scanning mutagenesis should be useful to elucidate better the unique pore architecture of the CRAC channel (Yeromin, 2006).

Genome-wide RNAi screen of Ca2+ influx identifies genes that regulate Ca2+ release-activated Ca2+ channel activity

Recent studies have demonstrated a required and conserved role of Stim in store-operated Ca2+ influx and Ca2+ release-activated Ca2+ (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, 75 hits were identified that strongly inhibited Ca2+ influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim, and one, olf186-F, that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca2+ entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim. olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca2+ pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N-ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca2+ depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity (Zhang, 2006).

The genome-wide screen, based on direct Ca2+ influx measurements, validated Stim and identified several additional genes that are required for CRAC channel activity. olf186-F (Orai) was identified as essential for Ca2+ signaling and activation of CRAC current in S2 cells, confirming two recent reports (Feske, 2006; Vig, 2006). In addition, evidence is provided, based on overexpression, that Orai may form an essential part of the CRAC channel. In mammalian cells overexpression of STIM1 increases Ca2+ influx rates and CRAC currents by ~2-fold, but in S2 cells this study showed that overexpression of Stim alone does not increase CRAC current, consistent with Stim serving as a channel activator rather than the channel itself. In contrast, transfection of olf186-F by itself increased CRAC current densities 3-fold, and cotransfection of olf186-F with Stim resulted in an 8-fold enhancement and the largest CRAC currents ever recorded. These results support the hypothesis that olf186-F constitutes part of the CRAC channel and that Stim serves as the messenger for its activation. Consistent with this hypothesis, the CRAC channel activation kinetics during passive Ca2+ store depletion were significantly faster with cotransfected Stim. Many fundamental aspects of the mechanism of CRAC channel activation remain to be clarified, including the protein-protein interactions that underlie trafficking and channel activation. Site-directed mutagenesis in a heterologous expression system may help to define the putative pore-forming region (Zhang, 2006).

Similar to Stim, knockdown of olf186-F did not produce a severe cell growth phenotype. It was neither a hit in a previous screen of cell survival nor in any other published Drosophila whole-genome RNAi screen. The olf186-F gene is a member of a highly conserved gene family that contains three homologs in mammals, two in chicken, three in zebrafish, and one member only in fly and worm. C09F5.2, the only homolog in Caenorhabditis elegans, is expressed in intestine, hypodermis, and reproductive system as well as some neuron-like cells in the head and tail regions. Worms under RNAi treatment against C09F5.2 are sterile. Analysis of hydrophobic regions of the predicted protein from the fly gene and the three mammalian homologs suggested the presence of four conserved transmembrane segments. Cytoplasmic C termini are suggested by the presence of coiled-coil motifs in each sequence. Sequence alignment between members from human, chicken, and fly revealed strong sequence conservation in putative transmembrane regions and conserved negatively charged residues in loops between transmembrane segments. All three human members are expressed in the immune system. Mutation of a human homolog of Drosophila olf186-F, ORAI1 on chromosome 12, appears to be the cause of defective CRAC channel activity in severe combined immune deficiency patient T cells, consistent with a requirement for functional CRAC channels in the immune response. Interestingly, microarray data from public databases combined with tissue-specific EST counts show that all three human members are expressed in a variety of nonexcitable tissues including thymus, lymph node, intestine, dermis, and many other tissues including the brain, although expression patterns and levels are different among the three members (Zhang, 2006).

Ca-P60A has been proposed to be the only Drosophila SERCA gene. This study validated its ER pump function by showing that ionomycin did not induce significant store release from S2 cells pretreated with dsRNA against Ca-P60A. The elevation in resting [Ca2+]i and rapidly changing Ca2+ transients during changes in external Ca2+ before addition of TG may indicate a low level of constitutive CRAC channel activity induced by store depletion. In addition, SERCA knockdown inhibited CRAC channel activity after passive store depletion in whole-cell patch recordings. These results are consistent with the SERCA pump being required for normal activity of CRAC channels but do not rule out indirect inhibition of CRAC current as a consequence of residual high resting [Ca2+]i or store depletion. The role of SERCA in CRAC channel function merits further study (Zhang, 2006).


Functions of Stim orthologs in other species

Defective STIM-mediated store operated Ca(2+) entry in hepatocytes leads to metabolic dysfunction in obesity

Defective Ca(2+) handling is a key mechanism underlying hepatic endoplasmic reticulum (ER) dysfunction in obesity. ER Ca(2+) level is in part monitored by the store-operated Ca(2+) entry (SOCE) system, an adaptive mechanism that senses ER luminal Ca(2+) concentrations through the STIM proteins and facilitates import of the ion from the extracellular space. This study shows that hepatocytes from obese mice displayed significantly diminished SOCE as a result of impaired STIM1 translocation, which was associated with aberrant STIM1 O-GlycNAcylation. Primary hepatocytes deficient in STIM1 exhibited elevated cellular stress as well as impaired insulin action, increased glucose production and lipid droplet accumulation. Additionally, mice with acute liver deletion of STIM1 displayed systemic glucose intolerance. Conversely, over-expression of STIM1 in obese mice led to increased SOCE, which was sufficient to improve systemic glucose tolerance. These findings demonstrate that SOCE is an important mechanism for healthy hepatic Ca(2+) balance and systemic metabolic control (Arruda, 2017).

Store-operated Ca(2+) entry controls induction of lipolysis and the transcriptional reprogramming to lipid metabolism

Ca(2+) signals were reported to control lipid homeostasis, but the Ca(2+) channels and pathways involved are largely unknown. Store-operated Ca(2+) entry (SOCE) is a ubiquitous Ca(2+) influx pathway regulated by stromal interaction molecule 1 (STIM1), STIM2, and the Ca(2+) channel ORAI1. This study shows that SOCE-deficient mice accumulate pathological amounts of lipid droplets in the liver, heart, and skeletal muscle. Cells from patients with loss-of-function mutations in STIM1 or ORAI1 show a similar phenotype, suggesting a cell-intrinsic role for SOCE in the regulation of lipid metabolism. SOCE is crucial to induce mobilization of fatty acids from lipid droplets, lipolysis, and mitochondrial fatty acid oxidation. SOCE regulates cyclic AMP production and the expression of neutral lipases as well as the transcriptional regulators of lipid metabolism, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1alpha), and peroxisome proliferator-activated receptor alpha (PPARalpha). SOCE-deficient cells upregulate lipophagy, which protects them from lipotoxicity. These data provide evidence for an important role of SOCE in lipid metabolism (Maus, 2017).

Stable STIM1 knockdown in self-renewing human neural precursors promotes premature neural differentiation

Ca(2+) signaling plays a significant role in the development of the vertebrate nervous system where it regulates neurite growth as well as synapse and neurotransmitter specification. Elucidating the role of Ca(2+) signaling in mammalian neuronal development has been largely restricted to either small animal models or primary cultures. This study derived human neural precursor cells (NPCs) from human embryonic stem cells to understand the functional significance of a less understood arm of calcium signaling, Store-operated Ca(2+) entry or SOCE, in neuronal development. Human NPCs exhibited robust SOCE, which was significantly attenuated by expression of a stable shRNA-miR targeted toward the SOCE molecule, STIM1. Along with the plasma membrane channel Orai, STIM is an essential component of SOCE in many cell types, where it regulates gene expression. Therefore, this study measured global gene expression in human NPCs with and without STIM1 knockdown. Interestingly, pathways down-regulated through STIM1 knockdown were related to cell proliferation and DNA replication processes, whereas post-synaptic signaling was identified as an up-regulated process. To understand the functional significance of these gene expression changes the self-renewal capacity was measured of NPCs with STIM1 knockdown. The STIM1 knockdown NPCs demonstrated significantly reduced neurosphere size and number as well as precocious spontaneous differentiation toward the neuronal lineage, as compared to control cells. These findings demonstrate that STIM1 mediated SOCE in human NPCs regulates gene expression changes, that in vivo are likely to physiologically modulate the self-renewal and differentiation of NPCs (Gopurappilly, 2018).


REFERENCES

Search PubMed for articles about Drosophila Stim

Agrawal, N., Venkiteswaran, G., Sadaf, S., Padmanabhan, N., Banerjee, S. and Hasan, G. (2010). Inositol 1,4,5-trisphosphate receptor and dSTIM function in Drosophila insulin-producing neurons regulates systemic intracellular calcium homeostasis and flight. J Neurosci 30(4): 1301-1313. PubMed ID: 20107057

Arruda, A. P., Pers, B. M., Parlakgul, G., Guney, E., Goh, T., Cagampan, E., Lee, G. Y., Goncalves, R. L. and Hotamisligil, G. S. (2017). Defective STIM-mediated store operated Ca(2+) entry in hepatocytes leads to metabolic dysfunction in obesity. Elife 6. PubMed ID: 29243589

Baumbach, J., Xu, Y., Hehlert, P. and Kuhnlein, R. P. (2014). Galphaq, Ggamma1 and Plc21C control Drosophila body fat storage. J Genet Genomics 41(5): 283-292. PubMed ID: 24894355

Cuttell, L., et al. (2008). Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis. Cell 135(3): 524-34. PubMed ID: 18984163

Gopurappilly, R., Deb, B. K., Chakraborty, P. and Hasan, G. (2018). Stable STIM1 knockdown in self-renewing human neural precursors promotes premature neural differentiation. Front Mol Neurosci 11: 178. PubMed ID: 29942250

Maus, M., Cuk, M., Patel, B., Lian, J., Ouimet, M., Kaufmann, U., Yang, J., Horvath, R., Hornig-Do, H. T., Chrzanowska-Lightowlers, Z. M., Moore, K. J., Cuervo, A. M. and Feske, S. (2017). Store-operated Ca(2+) entry controls induction of lipolysis and the transcriptional reprogramming to lipid metabolism. Cell Metab 25(3): 698-712. PubMed ID: 28132808

Megha, Wegener, C. and Hasan, G. (2019). ER-Ca2+ sensor STIM regulates neuropeptides required for development under nutrient restriction in Drosophila. PLoS One 14(7): e0219719. PubMed ID: 31295329

Pathak, T., Trivedi, D. and Hasan, G. (2017). CRISPR-Cas-induced mutants identify a requirement for dSTIM in larval dopaminergic cells of Drosophila melanogaster. G3 (Bethesda) 7(3): 923-933. PubMed ID: 28131984

Penna, A., et al. (2008). The CRAC channel consists of a tetramer formed by Stim-induced dimerization of Orai dimers. Nature 456(7218): 116-20. PubMed ID: 18820677

Richhariya, S., Jayakumar, S., Kumar Sukumar, S. and Hasan, G. (2018). dSTIM- and Ral/exocyst-mediated synaptic release from pupal dopaminergic neurons sustains Drosophila flight. eNeuro 5(3). PubMed ID: 29938216

Venkiteswaran, G. and Hasan, G. (2009). Intracellular Ca2+ signaling and store-operated Ca2+ entry are required in Drosophila neurons for flight. Proc. Natl. Acad. Sci. 106(25): 10326-10331. PubMed ID: 19515818

Xu, Y., Borcherding, A. F., Heier, C., Tian, G., Roeder, T. and Kuhnlein, R. P. (2019). Chronic dysfunction of Stromal interaction molecule by pulsed RNAi induction in fat tissue impairs organismal energy homeostasis in Drosophila. Sci Rep 9(1): 6989. PubMed ID: 31061470

Yeromin, A. V., et al. (2006) Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature 443: 226-229. PubMed ID: 16921385

Zhang, S. L., et al. (2006). Genome-wide RNAi screen of Ca2+ influx identifies genes that regulate Ca2+ release-activated Ca2+ channel activity. Proc. Natl. Acad. Sci. 103: 9357-9362. PubMed ID: 16751269


Biological Overview

date revised: 5 August 2019

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