The Interactive Fly

Zygotically transcribed genes

Calcium binding and calcium dependent enzymes and proteins

THADA regulates the organismal balance between energy storage and heat production
Decoding calcium signaling dynamics during Drosophila wing disc development
ER-Ca2+ sensor STIM regulates neuropeptides required for development under nutrient restriction in Drosophila

Calcium binding and calcium dependent enzymes and proteins

  • Signaling proteins

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    THADA regulates the organismal balance between energy storage and heat production

    Human susceptibility to obesity is mainly genetic, yet the underlying evolutionary drivers causing variation from person to person are not clear. One theory rationalizes that populations that have adapted to warmer climates have reduced their metabolic rates, thereby increasing their propensity to store energy. This study uncovered the function of a gene that supports this theory. THADA is one of the genes most strongly selected during evolution as humans settled in different climates. THADA knockout flies are obese, hyperphagic, have reduced energy production, and are sensitive to the cold. THADA binds the sarco/ER Ca2+ ATPase (SERCA) and acts on it as an uncoupler. Reducing SERCA activity in THADA mutant flies rescues their obesity, pinpointing SERCA as a key effector of THADA function. In sum, this identifies THADA as a regulator of the balance between energy consumption and energy storage, which was selected during human evolution (Moraru, 2017).

    Obesity has reached pandemic proportions, with 13% of adults worldwide being obese. Although the modern diet triggers this phenotype, 60%-70% of an individual's susceptibility to obesity is genetic. The underlying evolutionary drivers that cause susceptibility vary from person to person and are not clear. Since obesity is most prevalent in populations that have adapted to warm climates, an emerging theory proposes that populations in warm climates evolved low metabolic rates to reduce heat production, making them prone to obesity. In contrast, populations in cold climates evolved high energy consumption for thermogenesis, making them more resistant to obesity. This theory predicts the existence of genes that have been selected in the human population by climate adaptation which regulate the balance between heat production and energy storage (Moraru, 2017).

    The gene Thyroid Adenoma Associated (THADA) has played an important role in human evolution. Comparison of the Neanderthal genome with the genomes of current humans reveals that SNPs in THADA were the most strongly positively selected SNPs genome-wide in the evolution of modern humans. Furthermore, as hominins left Africa circa 70,000 years ago, they adapted to colder climates. Genome-wide association studies (GWAS) identified THADA as one of the top genes that was evolutionarily selected in response to cold adaptation, suggesting a link between THADA and energy metabolism. THADA was also identified as one of the top risk loci for type 2 diabetes by GWAS Although follow-up studies could not confirm an association between THADA SNPs and various aspects of insulin release or insulin sensitivity, some studies did find an association between THADA and pancreatic β-cell response or marginal evidence for an association with body mass index. In sum, THADA has been connected to both metabolism and adaptation to climate. Nonetheless, nothing is known about the function of THADA in animal biology, at the physiological or the molecular level. Animals lacking THADA function have not yet been described. An analysis of the amino acid sequence of THADA provides little or no hints regarding its molecular function (Moraru, 2017).

    To study the function of THADA, THADA knockout flies were generated. THADA knockout animals are obese and produce less heat than controls, making them sensitive to the cold. THADA binds the sarco/ER Ca2+ ATPase (SERCA) and regulates organismal metabolism via calcium signaling. In addition to unveiling the physiological role and molecular function of this medically relevant gene, the results also show that one gene that has been strongly selected during human evolution in response to environmental temperature plays a functional role in regulating the balance between heat production and energy storage, affecting the propensity to become obese (Moraru, 2017).

    This study reports the physiological and molecular function of THADA in animals. THADA mutants were found to be obese, sensitive to the cold, and have reduced heat production compared with controls. THADA interacts physically with SERCA and modulates its activity. The combination of improved calcium pumping and cold sensitivity of THADA mutants indicates that THADA acts as an SERCA uncoupler, similar to sarcolipin. This interaction between THADA and SERCA appears to be an important part of THADA function, since the obesity phenotype of THADA mutants can be rescued by mild SERCA knockdown (Moraru, 2017).

    Calcium signaling is increasingly coming into the spotlight as an important regulator of organismal metabolism. In a genome-wide in vivo RNAi screen in Drosophila to search for genes regulating energy homeostasis, calcium signaling was the most enriched gene ontology category among obesity-regulating genes (Baumbach, 2014). Cytosolic calcium levels can alter organismal adiposity by more than 10-fold (from 15% to 250% of control levels) (Baumbach, 2014), indicating that it is an important regulator of organismal metabolism. In line with these numbers, THADAKO flies have 250% the triglyceride levels of control flies. The phenotypes observed for other regulators of calcium signaling all point in the same general direction that high ER calcium leads to hyperphagia and obesity. Likewise, mice heterozygous for a mutation in IP3R are susceptible to developing glucose intolerance on a high-fat diet (Moraru, 2017).

    The molecular mechanisms by which ER calcium regulates organismal metabolism are not yet fully understood, but this important question will surely be the subject of intense research in the near future. Calcium levels are known to regulate activity of tricarboxylic acid cycle enzymes such as α-ketoglutarate dehydrogenase, isocitrate dehydrogenase, and pyruvate dehydrogenase, which could explain part of the effect of calcium on metabolism (Moraru, 2017).

    THADA mutation leads to obesity due to roles of THADA both in the fat body and in neurons. This has also been observed for IP3R mutants. Calcium signaling regulates lipid homeostasis directly and cell-autonomously in the fat body, as observed in seipin mutants (Bi, 2014) or when Stim expression was modulated specifically in fat tissue. In addition, it regulates feeding via the CNS. Interestingly, while THADA mutant females have elevated glycogen levels, THADA mutant males do not. It is not known why this is the case: it could be due to the higher energetic demand in females compared with males, leading to stronger metabolic phenotypes in females, or THADA might regulate glycogen metabolism differently in the two sexes (Moraru, 2017).

    GWAS identified THADA as one of the top risk loci for type 2 diabetes. The data presented in this study indicates that THADA regulates lipid metabolism and feeding, suggesting that the association between THADA and diabetes may be causal in nature. THADA mutant flies develop obesity, but have normal circulating sugar levels under standard laboratory food conditions. Interestingly, mouse mutants for IP3R likewise do not become insulin resistant under a regular diet, but do become insulin resistant on a high-fat diet. Combined, these data suggest that the primary effect of altered THADA activity and calcium signaling is on lipid metabolism, and that a combination with high-fat feeding may be required to lead to type 2 diabetes over time. This could potentially explain why follow-up association studies did not find links between THADA and insulin sensitivity but did find links between THADA and adiposity (Moraru, 2017 and references therein).

    Insects are ectotherms, meaning that their internal physiological sources of heat are not sufficient to control their body temperature. Nonetheless they do produce heat, and the main sources of heat are either of muscular origin due to movement or shivering, or of biochemical origin from futile cycles that consume ATP with no net work. For instance, bumblebees preheat their flight muscles by simultaneously activating phosphofructokinase and fructose 1,6-bisphosphatase, which catalyze opposing enzymatic reactions, leading to the futile hydrolysis of ATP and release of heat. Drosophila also have mitochondrial uncoupling proteins, which potentially generate a futile metabolic cycle by dissipating the mitochondrial membrane potential. It is proposed in this stduy that uncoupled hydrolysis of ATP by SERCA could constitute one additional example of such a futile cycle that produces heat. It cannot be excluded, however, that THADA knockout flies might also have changes in their evaporative heat loss contributing to their reduced thermogenesis. The thermogenic phenotypes in THADA knockout flies are relatively mild, perhaps reflecting the ectothermic nature of flies. Hence it will be of interest to study in the future the metabolic parameters of THADA knockout mice (Moraru, 2017).

    The combination of cold sensitivity and obesity in THADA mutant animals is interesting in terms of the evolutionary origins of the current obesity pandemic. The prevalence of obesity is highest in populations that have adapted to warmer climates, suggesting that people in warm climates evolved reduced metabolic rates to prevent overheating, and in combination with a modern diet this reduced metabolic rate leads to obesity. Interestingly, THADA is a gene that provides support for this theory. SNPs in THADA are among the SNPs genome-wide that have been most strongly selected as humans adapted to climates of different temperatures). Indeed, comparison of the Neanderthal genome with the genomes of current humans reveals that SNPs in THADA were the most strongly positively selected SNPs genome-wide in the evolution of modern humans. The data presented in this study show that THADA simultaneously affects sensitivity to cold and obesity. Uncoupled SERCA ATPase activity is a major contributor to non-shivering thermogenesis. Similar to animals mutant for another SERCA uncoupling protein, sarcolipin, this study found that THADA mutants are sensitive to the cold. This provides a possible explanation for why evolution selected for SNPs in THADA. In addition, THADA, via SERCA, also regulates lipid homeostasis. THADA thereby provides a genetic and molecular link between climate adaptation and obesity (Moraru, 2017).

    Decoding calcium signaling dynamics during Drosophila wing disc development

    The robust specification of organ development depends on coordinated cell-cell communication. This process requires signal integration among multiple pathways, relying on second messengers such as calcium ions. Calcium signaling encodes a significant portion of the cellular state by regulating transcription factors, enzymes, and cytoskeletal proteins. However, the relationships between the inputs specifying cell and organ development, calcium signaling dynamics, and final organ morphology are poorly understood. In this study a quantitative image-analysis pipeline was designed for decoding organ-level calcium signaling. With this pipeline, spatiotemporal features were extracted of calcium signaling dynamics during the development of the Drosophila larval wing disc, a genetic model for organogenesis. Specific classes of wing phenotypes were identified that resulted from calcium signaling pathway perturbations, including defects in gross morphology, vein differentiation, and overall size. Four qualitative classes of calcium signaling activity were found. These classes can be ordered based on agonist stimulation strength Galphaq-mediated signaling. In vivo calcium signaling dynamics depend on both receptor tyrosine kinase/phospholipase C gamma and G protein-coupled receptor/phospholipase C beta activities. Spatially patterned calcium dynamics were found to correlate with known differential growth rates between anterior and posterior compartments. Integrated calcium signaling activity decreases with increasing tissue size, and it responds to morphogenetic perturbations that impact organ growth. Together, these findings define how calcium signaling dynamics integrate upstream inputs to mediate multiple response outputs in developing epithelial organs (Brodskiy, 2019).

    Organ development requires the coordination of many cells to form a structurally integrated tissue. Important properties of the final organ architecture include its shape, size, and spatial distribution of cell types. Notably, the information processing network required for development resembles a 'bow-tie' network structure with many input signals that are funneled through a limited number of second messengers (The wing disc as a model system of signal integration during organogenesis). Signal integration and pathway crosstalk result in many possible downstream outputs that are determined by effector proteins that regulate cellular processes, including cell division, migration, mechanical properties, death, and cell differentiation state. However, how these diverse input signals regulate the dynamics of second messengers is poorly understood. Further, how organ-level properties, such as size and shape, emerge from the integration of second messenger signaling remains to be fully elucidated (Brodskiy, 2019).

    A key second messenger that serves as a central node in the bow-tie structure is the calcium ion (Ca2+). Ca2+ signaling is a ubiquitous transducer of cellular information and plays key roles in regulating cell behaviors, such as cell division, growth, and death. Ca2+ dynamics regulate cellular properties and behavior during animal development, and perturbations to Ca2+ signaling often lead to disease. Cells can encode complex signals into a Ca2+ signaling 'signature,' which includes amplitude, frequency, and integrated intensity of Ca2+ oscillations. Cells decode these signaling signatures by modulating the activities of downstream enzymes and transcription factors (Brodskiy, 2019).

    Intercellular Ca2+ signaling is correlated with many developmental processes. For example, they have been found to regulate scale development in the butterfly. Ca2+ waves are indispensable to activate Drosophila egg development, and Ca2+ spikes are important for development of Drosophila and Xenopus embryos. Ca2+ signaling responds to Hedgehog (Hh) signaling in the frog neural cord, correlates with Decapentaplegic (Dpp) secretion in Drosophila imaginal discs, and is indispensable for human neural rosette development. Ca2+ dynamics also are essential for cell migration and tissue contractility in zebrafish, Japanese newt, and chick embryos. Recently, intercellular Ca2+ transients (ICTs) have been observed in the Drosophila wing disc, both in vivo and ex vivo, and have been implicated as a first response to wounding and robustness in regeneration, tissue homeostasis, and mechanotransduction. Inhibition of Ca2+ significantly also rescues cancerous overgrowth of wings, thus showing its regulatory role in tissue growth. However, a quantitative characterization of Ca2+ dynamics in organ development is lacking, in part because of a lack of image-processing methods and a suitable model system to analyze the stochastic nature of the signals. Consequently, there is a need for a systems-level description of Ca2+ signaling dynamics to decode the role of Ca2+ signaling in organ development (Brodskiy, 2019).

    The Drosophila wing imaginal disc pouch is a premier model system to study how epithelial cells undergo specific morphogenetic steps to form the intricate structure of an adult wing. The wing disc is a powerful model system because of the availability of tools to perturb gene expression in a specific region of a tissue. Multiple conserved regulatory modules for tissue development have been discovered in the wing disc. In the larval organ, morphogens divide the wing disc pouch into regions that define the differentiation state of cells and coordinate morphogenesis. Morphogen signals that are important for wing disc development include Hh and Dpp, which define the anterior/posterior axis. Wg patterns the dorsal/ventral axis. Widely available genetic tools and simple geometry make the Drosophila wing disc a powerful platform to decode Ca2+ signaling at the systems level (Brodskiy, 2019).

    This study has developed an image-processing pipeline to quantitatively investigate the relationships between Ca2+ signaling and organ size. First key components of the core Ca2+ signaling pathway, termed elsewhere as the 'Ca2+ signaling toolkit', were genetically inhibited to define the range of adult wing phenotypes. Next, a dose-response experiment of fly extract (FEX) to order the specific classes of Ca2+ signaling based on the relative concentration of agonist-based stimulation. The term 'Ca2+ signaling activity' is used to collectively refer to these four Ca2+ signaling classes. How these classes of Ca2+ signaling correlate with disc age and size, both in vivo and ex vivo, was investigated. FEX was shown to stimulates Ca2+ through Gαq/phospholipase C (PLC) β signaling through genetic perturbation experiments. Advanced image-analysis tools were developed to handle the large data sets to extract quantitative Ca2+ dynamics measurements. Using this image-analysis pipeline, a negative power-law correlation between integrated Ca2+ signaling activity and wing disc pouch size was identified. How the genetic state of the tissue modulates Ca2+ signaling dynamics was examined through genetic perturbation. Ca2+ signaling activity responds to perturbations that impact the morphogenic state of the tissue, resulting in deviations from the quantitative correlation curve between Ca2+ signaling activity and developmental progression. Together, these trends indicate that Ca2+ signaling provides a biochemical readout of organ size. The results suggest Ca2+ could be involved in modulating cell proliferation activity during larval growth. In sum, this study provides significant evidence that Ca2+ signaling contributes to intercellular consensus-building during organ development. This research paves the road of revealing the quantitative and mechanistic regulation of organ development by Ca2+ signaling in future studies (Brodskiy, 2019).

    This work has established multiple inputs and outputs for the calcium bow-tie network during wing development. Four classes of spontaneous Ca2+ signaling activity during in vivo development in the wing disc were identified: (1) cellular Ca2+ spikes; (2) ICTs; (3) intercellular Ca2+ waves (ICWs), and (4) elevated Ca2+ fluttering. Increasing Gαq-mediated signaling with increasing concentrations of FEX leads to a natural progression from low (class 1 and 2) to higher levels of Ca2+ signaling responses (classes 3 and 4). These four signaling classes occur both ex vivo and in vivo. Importantly, it was found that multiple classes of Ca2+ activity occur and are a regulated phenomenon in vivo. These findings contradict previous suggestions that ICWs may be an ex vivo artifact. Future work is needed to specify the full set of specific RTKs, GPCRs, and morphogens that modulate Ca2+ dynamics in vivo (Brodskiy, 2019).

    A negative correlation was demonstrated between the stimulated Ca2+ signaling responses and the wing disc age and size for third instar larvae. Overall, these observations provide evidence for Ca2+ signaling as a readout for overall organ size in the developing wing and a regulator of cellular processes during larval wing development. Through linear regression analysis, a negative power-law correlation was demonstrated between larval age/pouch size and integrated Ca2+ signaling activity. These findings suggest that Ca2+ signaling decreases during the latter stages of larval wing disc growth. The maximal log-likelihood estimation of the power exponent occurred when the estimate had a value of -0.8 ± 0.5. This is consistent with many allometric scaling relationships observed in biological systems wherein quarter-power scaling frequently occurs. For example, quarter-power scaling has been observed in the organism metabolic rate, lifespan, growth rate, heart rate, and the concentrations of metabolic enzymes. A -0.75-scaling relationship is consistent, near the maximal log-likelihood estimation, and within the 95% confidence interval of the optimal exponent power. This, in turn, may indicate that the underlying metabolic trajectory of organ growth influences the level of agonist-stimulated calcium signaling activity (Brodskiy, 2019).

    Further, anterior-posterior patterning of Ca2+ signaling activity amplitudes was observed in the wing disc. The amplitude is higher in the posterior than in the anterior compartment. As these compartments have been shown to grow at different rates, this result is consistent with the correlation between Ca2+ signaling activity and the growth state of each compartment. There are several possible explanations for why there is an absence of amplitude patterning between anterior and posterior compartments for larger discs in Hh (smoRNAi) or Dpp (dppRNAi) signaling-perturbed discs. First, Hh and Dpp signaling may be directly responsible for patterning the anterior-posterior amplitude difference, perhaps through regulation of cAMP levels. Second, this may be because the sizes of anterior and posterior compartments are similar under those conditions. Identifying the cause of this phenomenon may yield insight into additional patterning roles for Ca2+ signaling in wing development, including the pupal stages when vein differentiation occurs. Recently, Ca2+ signaling has been connected to proper Hh signaling in zebrafish embryo. This work suggests that Ca2+ signaling may generally be involved in modulating morphogenesis mediated by Hh signaling and other morphogen pathways (Brodskiy, 2019).

    Future work is needed to identify specific mechanisms connecting signal transduction inputs to phenotypic outputs. In a recent article, cellular Ca2+ spikes were found to correlate with secretion of Dpp, a key regulator of wing disc size and tissue patterning. It is speculated that local cellular spike activity might be connected to the positive regulation of organ growth. smoRNAi and dppRNAi leads to smaller wing discs and higher integrated Ca2+ intensity when Ca2+ signaling is stimulated by agonists. The data points from growth-reducing perturbation (smoRNAi and dppRNAi) lie above the negative correlation curve of the control wing discs. In contrast, genetic perturbations leading to more growth (tkvCA and PtenRNAi) result in reduced Ca2+ signaling responses when stimulated (Brodskiy, 2019).

    These results imply a common underlying regulatory mechanism. As a launching point for future work, a simple model is proposed that explains the results reported in this study. First, the experiments demonstrate that FEX stimulates Gαq/PLCβ activity, which results in IP3 generation and IP3-regulated Ca2+ release. Sufficient IP3 production may lead to phosphatidylinositol bisphosphate (PIP2) substrate depletion. In other systems, PIP2 is often rate limiting for Ca2+ signaling. PIP2 is also required for phosphatidylinositol trisphosphate generation, which then stimulates cell growth through PI3K/AKT signaling. It follows that reduced PI3K signaling resulting from decreased growth stimulation (indirectly through inhibition of Hh or Dpp signaling in these experiments) will lead to higher PIP2 substrate availability and a stronger Ca2+ response. Conversely, decreased PIP2 availability through the inhibition of PTEN (which converts phosphatidylinositol trisphosphate to PIP2) or through constitutively active Dpp signaling would lead to attenuated Ca2+ signaling responses when stimulated by FEX (Brodskiy, 2019).

    This interpretation of the data provides a generalizable and testable hypothesis for future work: if PIP2 levels are more abundant (reduced PI3K signaling and growth activity), more IP3 can be generated, resulting in more Ca2+ signaling for a given agonist response. If PIP2 substrate levels are limiting (as results when PTEN is inhibited or more growth is stimulated), less IP3-stimulated Ca2+ signaling can occur. This hypothetical model would predict that sufficient overexpression of Gαq could lead to reduced organ growth by depleting PIP2 substrate availability for growth stimulation. Future work may identify such relationships across biological systems because all of these molecular components are present in most eukaryotic cells. This hypothetical model is termed the 'Ca2+ shunt' hypothesis of growth control (Brodskiy, 2019).

    Ca2+ signaling likely modulates other aspects of growth control during larval development. Ca2+ may integrate signals about the availability of nutrients or about mechanical constraints on the tissue. Several known effectors of size control pathways, such as kibra, a regulator of Hippo signaling, have Ca2+ signaling binding domains as annotated by InterPro (Brodskiy, 2019).

    Additionally, this work motivates new questions regarding how gap-junction communication, and by extension, membrane voltage, influences the overall control of organ size. A decrease in cell-cell gap-junction permeability occurs over the course of wing development. As gap junctions become less permeable, Ca2+ and IP3 diffuse a shorter distance before being reabsorbed into the endoplasmic reticulum or decaying, respectively. This would explain the transition from ICWs to ICTs and spikes as well as why amplitude is spatially patterned in large discs as development proceeds. Other studies have also implicated gap-junction communication in organ size control. For example, Inx2RNAi suppresses growth in the developing eye disc. Connexin43 mutants that disrupt gap-junction communication lead to short fin in zebrafish. Gap-junction communication also regulates cell differentiation as Inx2-mediated Ca2+ flux is essential for border cell specification in Drosophila. These results suggest that part of the role of gap-junction communication in regulating size and influencing tissue patterning is through the regulation of Ca2+ transients across the tissue. Taken together, it is therefore likely that the role of Ca2+ signaling in wing growth is conserved in other organs (Brodskiy, 2019).

    This phenotypic analysis provides additional evidence that the Ca2+ signaling module contributes to modulating wing morphogenesis during pupal development and vein cell differentiation. It should be noted that the crossvein defects suggest that these veins are particularly sensitive to levels of morphogen signaling, including Dpp. In particular, Dpp signaling has been linked to Ca2+ signaling in the developing wing. Perturbing Ca2+ signaling may also be enhancing the crossvein defects that can occur in the MS1096-Gal4 line, which impacts Beadex gene function. Future work will need to investigate the mechanisms leading to wing shape and vein differentiation defects, which are specified during pupal development (Brodskiy, 2019).

    Computational modeling is essential for future efforts to decode the regulation and function of Ca2+ signaling. Understanding the specific roles of Ca2+ signaling in organ development will require computational models that couple multiple signals of Ca2+ signaling across multiple spatiotemporal scales. For example, computational models are particularly useful at the systems level to understand mechanisms for the coupled transport of Ca2+ and wound healing. Regarding this study, these findings that the integrated Ca2+ intensity decreases with development is consistent with a model from the neocortex being applied to the wing disc, in which Ca2+ signaling dynamics are weakly coupled with cell-cycle progression and can influence cell-cycle synchrony with neighbors. In sum, this effort demonstrates key roles of Ca2+ signaling as a signal integrator in epithelial growth and morphogenesis (Brodskiy, 2019).

    ER-Ca2+ sensor STIM regulates neuropeptides required for development under nutrient restriction in Drosophila

    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 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).


    REFERENCES

    Brodskiy, P. A., Wu, Q., Soundarrajan, D. K., Huizar, F. J., Chen, J., Liang, P., Narciso, C., Levis, M. K., Arredondo-Walsh, N., Chen, D. Z. and Zartman, J. J. (2019). Decoding calcium signaling dynamics during Drosophila wing disc development. Biophys J. PubMed ID: 30704858

    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

    Moraru, A., Cakan-Akdogan, G., Strassburger, K., Males, M., Mueller, S., Jabs, M., Muelleder, M., Frejno, M., Braeckman, B. P., Ralser, M. and Teleman, A. A. (2017). THADA regulates the organismal balance between energy storage and heat production. Dev Cell 41(1): 72-81. PubMed ID: 28399403

    Zygotically transcribed genes

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