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 signalling in Drosophila photoreceptors measured with GCaMP6f
Na+/Ca2+ exchanger mediates cold Ca2+ signaling conserved for temperature-compensated circadian rhythms
From spikes to intercellular waves: Tuning intercellular calcium signaling dynamics modulates organ size control
A mathematical model of calcium signals around laser-induced epithelial wounds
A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo
Independently paced calcium oscillations in progenitor and differentiated cells in an ex vivo epithelial organ
Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure
Insect nephrocyte function is regulated by a store operated calcium entry mechanism controlling endocytosis and Amnionless turnover
Visceral organ morphogenesis via calcium-patterned muscle constrictions
CBP-Mediated Acetylation of Importin α Mediates Calcium-Dependent Nucleocytoplasmic Transport of Selective Proteins in Drosophila Neurons
Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling
The multimodal action of G alpha q in coordinating growth and homeostasis in the Drosophila wing imaginal disc
Circadian pacemaker neurons display cophasic rhythms in basal calcium level and in fast calcium fluctuations

Calcium binding and calcium dependent enzymes and proteins

  • Signaling proteins

  • Others

    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).

    Calcium signalling in Drosophila photoreceptors measured with GCaMP6f

    Phototransduction in Drosophila is mediated by phospholipase C (PLC) and Ca2+-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca2+ stores in this important model for Ca2+ signaling remains obscure. A low affinity Ca2+ indicator (ER-GCaMP6-150) was expressed in the ER, and its fluorescence was measured both in dissociated ommatidia and in vivo from intact flies of both sexes. Blue excitation light induced a rapid (tau approximately 0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca2+ stores, followed by a slower decay, typically reaching approximately 50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100-200 s. Both rapid and slow store depletion were largely unaffected in InsP3 receptor mutants, but were much reduced in trp mutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na+ and in mutants of the Na+/Ca2+ exchanger, CalX, which was immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression of calx greatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na+/Ca2+ exchange across the ER membrane induced by Na+ influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na+/Ca2+ exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors exposed to Ca2+-free solutions (Liu, 2020).

    Phototransduction in microvillar photoreceptors is mediated by a G-protein-coupled phospholipase C (PLC), which hydrolyzes phosphatidyl inositol (4,5) bisphosphate (PIP2) to generate diacylglycerol and inositol (1,4,5) trisphosphate (InsP3). In Drosophila photoreceptors, activation of PLC leads to opening of two related Ca2+-permeable nonselective cation channels: TRP (transient receptor potential) and TRP-like (TRPL) in the microvillar membrane. TRP is the founding member of the TRP ion channel superfamily, so named because the light response in trp mutants is transient, decaying rapidly to baseline during maintained illumination. Because the most familiar product of PLC activity is InsP3, it was initially thought that activation of the TRP/TRPL channels required release of Ca2+ from endoplasmic reticulum (ER) stores via InsP3 receptors (InsP3Rs) and that in the absence of Ca2+ influx via TRP channels the stores depleted leading to the response decay. However, it was subsequently found that phototransduction was intact in InsP3R mutants, whereas response decay in trp mutants was associated with severe depletion of PIP2. This suggested an alternative explanation of the trp decay phenotype, namely failure of Ca2+-dependent inhibition of PLC and the consequent runaway consumption of its substrate, PIP2. Nevertheless, a role for InsP3 and Ca2+ stores in Drosophila phototransduction remains debated. For example, a recent study reported that sensitivity to light was attenuated by RNAi knockdown of InsP3R , although this study was unable to confirm this using either RNAi or null InsP3R mutants (Bollepalli, 2017; Liu, 2020).

    Relevant to this debate, Ca2+ imaging reveals a small, but significant light-induced rise in cytosolic Ca2+ in photoreceptors bathed in Ca2+-free solutions. Although some have attributed this to InsP3-induced Ca2+ release from the ER, it was found that the rise was unaffected in InsP3R mutants but was dependent on Na+/Ca2+ exchange (Hardie, 1996; Asteriti, 2017; Bollepalli, 2017). This suggested that the Ca2+ rise was due to Na+/Ca2+exchange following Na+ influx associated with the light response. However, it is difficult to understand how such a Ca2+ rise could be achieved by Na+/Ca2+ exchange across the plasma membrane when extracellular Ca2+ was buffered to low nanomolar levels. The source of the Ca2+ rise in Ca2+-free bath thus remains unresolved, and to date there have been no measurements of ER store Ca2+ levels in Drosophila photoreceptors. To address this, flies were generated expressing a low-affinity GCaMP6 variant in the ER lumen. Using this probe, a rapid light-induced depletion of ER Ca2+ was demonstrated and characterized, which, like the cytosolic Ca2+ signal in Ca2+-free bath, was unaffected by InsP3R mutations, but dependent on Na+ influx and the CalX Na+/Ca2+ exchanger. These results indicate that the exchanger is also expressed on the ER membrane, that the Na+ influx associated with the light-induced current leads to Ca2+ extrusion from the ER by Na+/Ca2+exchange and that this accounts for the rise in cytosolic Ca2+ observed in Ca2+-free solutions (Liu, 2020).

    This study measured ER Ca2+ levels using a low affinity GCaMP6 variant targeted to the photoreceptor ER lumen, where it generated bright fluorescence throughout the ER network. The probe (ER-GCaMP6-150), originally developed and expressed in mammalian neurons, has a 45-fold dynamic range, which was confirmed in situ, and allows measurements of ER luminal [Ca2+] with excellent signal-to-noise ratio. Not only could ER Ca2+ levels be monitored in dissociated ommatidia, it was also straightforward to make in vivo measurements from the eyes of completely intact flies. The results demonstrate rapid light-induced, PLC-dependent depletion of the ER Ca2+ stores, which refilled in the dark over a time course of 100-200 s (Liu, 2020).

    Strikingly the results indicate that the rapid light-induced store depletion was mediated by Na+/Ca2+ exchange. Drosophila CalX belongs to the NCX family of Na+/Ca2+ exchangers, which are generally considered to act only at the plasma membrane. Although Drosophila CalX clearly does function at the plasma membrane, the results now provide compelling evidence that it also operates across the ER membrane. NCX activity has not previously been reported on the ER; however, Na+/Ca2+ exchange on internal membranes is not without precedent: for example NCX has been reported on the inner nuclear membrane providing a route for Ca2+ transfer between nucleoplasm and the nuclear envelope and hence ultimately the ER network with which it is continuous. In addition a dedicated mitochondrial Na+/Ca2+ exchanger (NCLX) plays important roles in uptake and release of mitochondrial Ca2+ (Liu, 2020).

    The time course of the Na+/Ca2+-dependent rapid store depletion in Ca2+-free solutions appeared very similar to the rise in cytosolic Ca2+ reported from dissociated ommatidia in Ca2+-free bath, the source of which has been a subject of debate for over 20 years. It had recently been claimed that this 'Ca2+-free rise' was due to InsP3-mediated release from ER Ca2+ stores; however, it was found that it was unaffected in null mutants of the InsP3R. Instead, it was found that the Ca2+-free cytosolic rise was dependent on Na+/Ca2+ exchange, but it was difficult to understand how this could be mediated by a plasma membrane exchanger when extracellular Ca2+ was buffered with EGTA to low nanomolar levels. The demonstration of rapid Na+/Ca2+-dependent release of Ca2+ from ER with a very similar time course now provides an obvious mechanism for this Ca2+-free rise and seems finally to have resolved this long-standing enigma. Interestingly the Na+/Ca2+-dependent rapid store depletion signal was most pronounced in very young flies around the time of eclosion. Also of note, it was found that trp mutants were very resistant to depletion, both in vivo and in dissociated ommatidia. This argues strongly and directly against the hypothesis that the trp decay phenotype reflects depletion of the ER Ca2+ stores (Liu, 2020).

    Although up to ~80% rapid store depletion could be observed in newly eclosed adults, even in 1-d-old flies the rapid store depletion signal in vivo was much reduced (to ~10%). However, a much slower depletion was observed in mature adults in vivo, and in dissociated ommatidia after Na+/Ca2+ exchange was blocked. The origin of this slow phase depletion remains uncertain: in dissociated ommatidia from young flies this slower depletion was ~50% attenuated, but not blocked in null InsP3R mutants (itpr), whereas in vivo measurements of the slow depletion phase in adult itpr mutants appeared similar to wild-type. This suggests that although Ca2+ release via InsP3 receptors may contribute to the slow depletion in young flies, some other mechanism(s), such as Ca2+ release via ryanodine receptors, is largely responsible (Liu, 2020).

    This evidence strongly suggests a novel role for NCX exchangers in mediating Na+/Ca2+ exchange across the ER membrane, but its physiological significance is unclear. Although rapid store depletion was routinely observed under experimental conditions used in this study, the Ca2+ released into the cytosol from the ER seems unlikely to play a direct role in phototransduction. First, it has a latency of ~100 ms (cf. ~10 ms for the light-induced current), and second it will in any case be swamped by the much more rapid Ca2+ influx via the light-sensitive channels. Thus measurements of cytosolic Ca2+ in 0 Ca2+ bath indicated a rise to only ~200-300 nm. This compares with much faster rises in the high micromolar range due to direct Ca2+ influx via the light-sensitive TRP channels. One possible role for an ER Na+/Ca2+ exchanger would be that it normally operates as a Ca2+ uptake mechanism and only briefly giving Ca2+ extrusion (and store depletion) following the extreme, and unnatural conditions of many of the current experiments. This the sudden onset of bright illumination from a dark-adapted state, which results in a massive transient surge of Na+ influx. Rapid Ca2+ uptake (store refilling), presumably via re-equilibration of the exchanger as the initial Na+ level subsided during the peak-to-plateau transition, was in fact routinely observed during maintained blue illumination. Furthermore, it is perhaps significant, that despite lacking the rapid depletion phase, the final level of store Ca2+ (i.e., after 30 s illumination) in calxA mutants was if anything lower than that in wild-type backgrounds, although the cytosolic Ca2+ levels experienced in calxA mutants are much higher because of the failure to extrude Ca2+ across the plasma membrane (Liu, 2020).

    Although store depletion seems unlikely to contribute to activation of the phototransduction cascade, the possibility cannot be excluded that it may play some role in long-term light adaptation. Maintenance of ER Ca2+ levels is also important for many other cellular functions including protein folding and maturation in which Ca2+ is a cofactor for optimal chaperone activity. With conspicuously high cytosolic Ca2+ levels in the presence of light, photoreceptors face unusual homeostatic challenges and Na+/Ca2+ exchange across the ER may provide an important additional mechanism. In principle the balance between forward and reverse Na+/Ca2+ exchange (i.e., uptake vs release) by an ER Na+/Ca2+ exchanger will depend on the Na+ gradient across the ER membrane and whether this is actively regulated. There is no information on ER Na+ levels, although luminal Na+ in the nuclear envelope (which is continuous with the ER) has been reported to be concentrated (84 mm) in nuclei from hepatocytes by Na/K-ATPase expressed on nuclear membranes. The possibility that Na+/Ca2+ exchange across the ER might play only a minor physiological role cannot be excluded, but is an unavoidable consequence of the presence of functional CalX protein in ER membranes during protein synthesis and targeting. At least this may account for the enhanced depletion signal measured around the time of eclosion when there may be a rapid final phase of protein synthesis for the developing rhabdomere (Liu, 2020).

    These results provide unique insight into ER Ca2+ stores in Drosophila photoreceptors. The ER-GCaMP6-150 probe lights up an extensive ER network and indicates a high luminal Ca2+ concentration probably in excess of 0.5 mm. The results reveal a rapid, and uniform light-induced depletion of the ER stores mediated by the CalX Na+/Ca2+ exchanger expressed on the ER membrane. The resulting extrusion of Ca2+ into the cytosol can readily account for the rise in cytosolic Ca2+ observed in dissociated ommatidia in Ca2+-free solutions), thus resolving this decades old mystery. In addition to the rapid depletion, a much slower depletion was also resolved that appears to be independent of Na+/Ca2+ exchange and also largely independent of InsP3-induced Ca2+ release. The physiological significance of the ER Na+/Ca2+ exchange activity remains uncertain. It is perhaps more likely that it serves as a low affinity Ca2+ uptake mechanism supplementing the SERCA pump, and that rapid depletion is only seen during unnatural abrupt bright stimulation from dark-adapted backgrounds leading to massive Na+ influx and reverse exchange. Ultimately, to resolve the physiological significance of Na+/Ca2+ exchange across the ER membrane it will probably be necessary to selectively disrupt Na+/Ca2+ exchange on the ER without affecting the exchanger on the plasma membrane, which is known to play very important roles in Ca2+ homeostasis in the photoreceptors with direct consequences for channel activation and adaptation (Liu, 2020).

    Drosophila phototransduction is mediated by phospholipase C leading to activation of cation channels (TRP and TRPL) in the 30000 microvilli forming the light-absorbing rhabdomere. The channels mediate massive Ca2+ influx in response to light, but whether Ca2+ is released from internal stores remains controversial. This study generated flies expressing GCaMP6f in their photoreceptors and measured Ca2+ signals from dissociated cells, as well as in vivo by imaging rhabdomeres in intact flies. In response to brief flashes, GCaMP6f signals had latencies of 10-25ms, reached 50% Fmax with approximately 1200 effectively absorbed photons and saturated (DeltaF/F0 approximately 10-20) with 10000-30000 photons. In Ca2+ free bath, smaller (DeltaF/F0 approximately 4), long latency (~ 200ms) light-induced Ca2+ rises were still detectable. These were unaffected in InsP3 receptor mutants, but virtually eliminated when Na+ was also omitted from the bath, or in trpl;trp mutants lacking light-sensitive channels. Ca2+ free rises were also eliminated in Na+/Ca2+ exchanger mutants, but greatly accelerated in flies over-expressing the exchanger. These results show that Ca2+ free rises are strictly dependent on Na+ influx and activity of the exchanger, suggesting they reflect re-equilibration of Na+/Ca2+ exchange across plasma or intracellular membranes following massive Na+ influx. Any tiny Ca2+ free rise remaining without exchanger activity was equivalent to <10nM (DeltaF/F0 approximately 0.1), and unlikely to play any role in phototransduction (Asteriti, 2017).

    Although Ca2+ signals in Drosophila photoreceptors were first studied over 20 years ago using Ca2+ indicator dyes, only one, recent study had used genetically encoded Ca2+ indicators. That study measured signals from dissociated ommatidia using the Gal4-UAS system, combining UAS-GCaMP6f with GMR-Gal4, which drives expression throughout the retina including all photoreceptor classes as well as accessory cells such as pigment and cone cells. GMR-Gal4 expression also causes significant abnormalities in photoreceptor structure and physiology. In the present study, flies were generated in which GCaMP6f expression was driven directly via the Rh1 (ninaE) promoter ensuring exclusive expression in R1-6 photoreceptors with wild-type morphology and physiology. The excellent signal-to-noise ratio of recordings in ninaE-GCaMP6f flies was distinctly superior to that in GMR-Gal4/UAS-GCaMP6f flies, and in many cases the maximum Δ/F0 ratio approached or exceeded 20 (cf ~3 using GMR-Gal4/UAS-GCaMP6f). This is close to the maximum value (23.5) determined by in situ calibrations or in vitro. Although the blue excitation light used for measuring GCaMP6f fluorescence is a super-saturating stimulus, 2-pulse paradigms allowed sensitive and accurate measurements of intensity and time dependence of signals in response to stimuli in the physiological range. Recordings in vivo from the deep pseudopupil (DPP) of intact flies are simple to perform and can be readily maintained over many hours, making this approach a valuable, and completely non-invasive tool for assessing in vivo photoreceptor performance. Even in the more vulnerable dissociated ommatidia preparation, multiple repeatable measurements could be made for up to at least an hour from the same ommatidium as long as metarhodopsin was reconverted to rhodopsin by long wavelength light after each measurement (Asteriti, 2017).

    In vivo (DPP) or in dissociated ommatidia bathed in physiological solutions, the GCaMP6f signal reached 50% Fmax at intensities equivalent to ~1000-2500 effectively absorbed photons. It is believed that the elementary single photon response (quantum bump) is generated by activation of Ca2+ permeable channels (TRP and TRPL) within a single microvillus and that the consequent Ca2+ rise in the affected microvillus reaches near mM levels. Because such levels inevitably saturate GCaMP6f (Kd 290 nM, saturating at 1-2 μM), to a first approximation the Δ/F0 values under physiological conditions are probably best interpreted as the proportion of microvilli 'flooded' with Ca2+. In total, the rhabdomere contains ~30000 microvilli, meaning that 50% Fmax is reached when only ~3-8% of the microvilli have been activated by a photon. This implies that the Ca2+ influx into a single microvillus must spread to at least the immediately neighbouring microvilli within the timeframe of the response. In ninaE-calx flies over-expressing the Na+/Ca2+ exchanger, or in trp mutants lacking the major Ca2+ permeable channel, 50% Fmax was only obtained with flashes containing ~12000-15000 effective photons. This should activate ~50% of the microvilli, suggesting that in these flies Ca2+ is largely prevented from spreading to neighbouring microvilli under the same conditions (Asteriti, 2017).

    The dark-adapted 'pedestal' level can be used to gain an estimate of the resting Ca2+ concentration in dissociated ommatidia (in physiological solutions) assuming in vitro calibration data. With reference to F0 measured in Ca2+ free solution in the same ommatidia, the mean dark-adapted value in normal bath was 0.77 ± 0.14 (mean ± S.E.M. n = 11). This would be equivalent to ~80 nM (assuming Kd = 290 nM and Fmax 23.5). This value was significantly lower in ninaE-calx flies over-expressing the exchanger (0.19 ± 0.04 n = 11 equivalent to ~50 nM) and higher in calx1 mutants (1.94 ± 0.24 n = 14 equivalent to ~120 nM) (Asteriti, 2017).

    The recovery of GCaMP6f fluorescence to baseline is likely to be a reasonably accurate reflection of the falling Ca2+ levels during response recovery, although the initial decrease (from initial ~mM levels to low μM levels) will still be subject to saturation effects. With relatively dim flashes (up to ~1000 effectively absorbed photons) the GCaMP6f signal in wild-type backgrounds fell to near baseline within ~2-3 s with a half time (t 1/2) of ~1 s. This is slower than the GCaMP6f off-rate (~200 ms), and thus likely to approximate the true time-course of Ca2+ recovery. The recovery was significantly accelerated in ninaE-calx flies (~500 ms), and slowed in calx1 mutants (~2 s increasing to >10 s following brighter flashes), consistent with a dominant role of the Na+/Ca2+ exchanger in Ca2+ extrusion. Nevertheless, even after bright flashes, given sufficient dark-adaptation time (~30-60 s), resting [Ca2+] in calx1 mutants fell to levels close to those in dark-adapted wild-type photoreceptors, reflecting either residual function of the exchanger in this hypomorphic mutant and/or alternative extrusion mechanism(s) (Asteriti, 2017).

    The smaller signals recorded in Ca2+ free bath fall within the dynamic range of GCaMP6f and allow estimates of the absolute Ca2+ levels reached under these conditions (e.g., Δ/F0 of 6 corresponds to ~200 nM). These signals were used to investigate the long disputed origin of the light-induced rise in cytosolic Ca2+ in Ca2+ free solutions. Originally, using INDO-1, it was found that this Ca2+ free rise was dependent upon extracellular Na+ and suggested that the rise might be due to re-equilibration of Na+/Ca2+ exchange in response to the massive light-induced Na+ influx that persists under these conditions. This was challenged by by a study that confirmed the requirement of external Na+ for a significant Ca2+ rise in Ca2+ free solutions, Na2+, but reported that a rise still occurred in Ca2+ free bath in the presence of Na+ when the photoreceptors were voltage clamped at the Na+ equilibrium potential to prevent Na+ influx. It was concluded that a Na+ gradient − but not influx − was required, that the Ca2+ free rise reflected release from internal stores, and that the requirement of extracellular Na+ reflected involvement of some other Na+ dependent process, such as Na/H transport. But how this might affect release of Ca2+ from intracellular stores is far from clear. A more recent study reported that the Ca2+ free rise was attenuated following RNAi knockdown of the IP3R. However, this is difficult to reconcile with an earlier study using INDO-1, where the rise was found to be unaffected in null IP3R mosaic eyes and confirmed again in this study using GCaMP6f (Asteriti, 2017).

    This study used a variety of approaches to investigate the source of this Ca2+ free signal further. It was first confirmed that it was all but abolished in the absence of external Na+, whether substituted for Li+, Cs+, K+ or NMDG+. Importantly, it was found that the rise was also effectively eliminated in trpl;trp double mutants both in vivo and in dissociated ommatidia despite the presence of normal extracellular solutions containing both Na+ and Ca2+. Although it might be argued that, for some reason, PLC activity (and hence InsP3 generation) was compromised in trpl;trp mutants, convincing evidence indicates that net PLC activity is in fact greatly enhanced in trpl;trp due to the lack of Ca2+ and PKC dependent inhibition of PLC. Thus the rate and intensity dependence of PIP2 hydrolysis, measured using GFP-tagged PIP2 binding probes are greatly enhanced in trpl;trp mutants, as are the PLC-induced photomechanical contractions, and the acidification due to the protons released by the PLC reaction. Overall, therefore these results strongly suggest that Na+ influx is indeed required for the Ca2+ free rise. Crucially, the involvement of the Na+/Ca2+ exchanger in this rise was confirmed by finding that it was essentially eliminated in an exchanger mutant (calx1), but greatly accelerated in ninaE-calx photoreceptors over-expressing the exchanger (Asteriti, 2017).

    The question remains, how Na+/Ca2+ exchanger activity could generate such a sizeable Ca2+ signal (~100-200 nM) in cells perfused with EGTA buffered solutions, when free Ca2+ in the bath should be reduced to low nM levels. There is no unequivocal answer to this, and assuming the standard equation for the Na+/Ca2+ exchange equilibrium it would seem difficult for reverse Na+/Ca2+ exchange to raise Ca2+ into the range that was observed. However, at least three, not mutually exclusive factors might result in higher cytosolic Cai levels than predicted. Firstly, external Ca2+ might be relatively resistant to buffering in the intra-ommatidial space, and specifically the extremely narrow spaces between the microvilli or their bases, where the exchanger is believed to be localised. For example, with 500 nM Cao remaining, it is predicted that 130 nM Cai would be reached with 70 mM Nai, 110 mM Nao and the cell depolarised to 0 mV (values that could realistically be reached with the huge inward Na+ currents flowing under these conditions). Although one might also expect Ca2+ influx via the light-sensitive channels at such Cao concentrations, experiments buffering external Ca2+ at different concentrations with EGTA showed that direct Ca2+ influx signals could only be detected once external Ca2+ was raised above ~400 nM. Secondly, resting cytosolic Ca2+ concentration is determined not only by the exchanger, but also by any other Ca2+ fluxes, which might include tonic leakage from intracellular compartments such as endoplasmic reticulum (ER) or mitochondria. Massive Na+ influx would compromise the ability of the exchanger to counter any such fluxes. A third possibility is that, contrary to conventional dogma, the exchanger might also be expressed on intracellular membranes of endoplasmic reticulum or other Ca2+ containing compartments and that Na+ influx leads to re-equilibration of Na+/Ca2+ exchange across these (Asteriti, 2017).

    Whatever the exact mechanism, the results indicate that the Ca2+ rise in Ca2+ free bath is strictly dependent upon both Na+ influx and the activity level of the Na+/Ca2+ exchanger, but unaffected in null IP3R mutants. Its time-course, with no detectable rise for ~200 ms, also appears much too slow to play any role in initiating the light response, which has a latency of ~10 ms and peaks within ~100-200 ms in response to bright illumination even under Ca2+ free conditions. The residual GCaMP6f signal remaining in the absence of Na+ influx and/or in the absence of Na+/Ca2+ exchanger activity − whether achieved by Na+ substitution, trpl;trp or calx mutants − was also still observed in IP3R mutants and was so small that it is questionable whether it reflects a Ca2+ signal. Because of the rapid inhibition of PLC by Ca2+ influx under physiological conditions any presumptive PLC-mediated Ca2+ release under physiological conditions would be even less. Together with a study in which no phototransduction defects were found in null IP3R mutants, these results suggest that InsP3-induced Ca2+ release plays no significant role in Drosophila phototransduction (Asteriti, 2017).

    Na+/Ca2+ exchanger mediates cold Ca2+ signaling conserved for temperature-compensated circadian rhythms

    Historically, before the discovery of the clock genes, a feedback system involving ions and ion regulators in plasma membranes was proposed as the oscillation mechanism of the circadian clock. This 'membrane model' is based on the observation that the circadian rhythms are notably affected by manipulating ion concentrations or ion regulator activities in various eukaryotes. To date, several ions, especially Ca2+, have been shown to play an essential role for oscillation of the transcription-translation feedback loops (TTFLs) in mammals, insects, and plants. In mice and Drosophila, intracellular Ca2+ levels were shown to exhibit robust circadian oscillations, which elicit rhythmic activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII phosphorylates CLOCK to activate CLOCK-BMAL1 heterodimer, a key transcriptional activator in the animal TTFLs. The upstream regulator of the Ca2+-dependent phosphorylation signaling has been a missing link between the TTFL and the membrane model (Kon, 2021).

    Circadian TTFLs are an elaborate system that drives a wide range of overt rhythms with various phase angles and amplitudes. The oscillation speed of the TTFLs is temperature compensated, although many of the biochemical reactions in TTFLs are slowed down by decreasing temperature. This study demonstrates that the temperature compensation of the TTFL in mammalian cells was compromised when Ca2+-dependent phosphorylation signaling was inhibited. An important role was found of NCX-CaMKII activity as the state variable of the circadian oscillator. This present study and a series of preceding works demonstrate that the Ca2+ oscillator plays essential roles in the circadian oscillation mechanism. Functional studies clearly demonstrated essential roles of NCX-dependent Ca2+ signaling in the three important properties of the circadian clock, i.e., cell-autonomous oscillation, temperature compensation, and entrainment. The circadian Ca2+ oscillation is observed in mice lacking Bmal1 or Cry1/Cry2, implicating that the Ca2+ oscillator is an upstream regulator of the TTFL in mammals (Kon, 2021).

    The effects of NCX2 and NCX3 deficiencies on the regulation of mouse behavioral rhythms (Fig. 7, A to C) suggest involvement of Na+/Ca2+ exchanging activity in the Ca2+ dynamics of the SCN. Previous studies showed that L-type Ca2+ channel (LTCC) and voltage-gated Na+ channel (VGSC) are required for high-amplitude Ca2+ rhythms in the SCN. Because NCX activities are regulated by local concentrations of Na+/Ca2+ and the membrane potential, cooperative actions of LTCC, VGSC, and NCX seem to play important roles in generation mechanism of the robust Ca2+ oscillations in the SCN (Kon, 2021).

    It should be emphasized that the role of Ca2+/calmodulin-dependent protein kinases is conserved among clockworks in insects, fungi, and plants, suggesting that the Ca2+ oscillator might be a core timekeeping mechanism in their common ancestor (see Involvement of ancient Ca2+ signaling for temperature-compensated circadian rhythms). After divergence of each lineage, a subset of clock genes should have independently evolved in association with the Ca2+ oscillator. It is noteworthy that NCX is also required for temperature compensation of PTO-based cyanobacterial clock. Because intracellular Ca2+ in cyanobacteria is elevated in response to temperature decrease, YrbG-mediated Ca2+ signaling may regulate the PTO in vivo. Conservation of NCX among eukaryotes, eubacteria, and archaea suggests that NCX-dependent temperature signaling is essential for adaptation of a wide variety of organisms to environment. Further studies on NCX-regulated Ca2+ flux will provide evolutionary insights into the origin of the circadian clocks (Kon, 2021).

    From spikes to intercellular waves: Tuning intercellular calcium signaling dynamics modulates organ size control

    Information flow within and between cells depends significantly on calcium (Ca2+) signaling dynamics. However, the biophysical mechanisms that govern emergent patterns of Ca2+ signaling dynamics at the organ level remain elusive. Recent experimental studies in developing Drosophila wing imaginal discs demonstrate the emergence of four distinct patterns of Ca2+ activity: Ca2+ spikes, intercellular Ca2+ transients, tissue-level Ca2+ waves, and a global "fluttering" state. This study used a combination of computational modeling and experimental approaches to identify two different populations of cells within tissues that are connected by gap junction proteins. These two subpopulations were termed "initiator cells," defined by elevated levels of Phospholipase C (PLC) activity, and "standby cells," which exhibit baseline activity. The type and strength of hormonal stimulation and extent of gap junctional communication were found to jointly determine the predominate class of Ca2+ signaling activity. Further, single-cell Ca2+ spikes are stimulated by insulin, while intercellular Ca2+ waves depend on Gαq activity. A computational model successfully reproduces how the dynamics of Ca2+ transients varies during organ growth. Phenotypic analysis of perturbations to Gαq and insulin signaling support an integrated model of cytoplasmic Ca2+ as a dynamic reporter of overall tissue growth. Further, perturbations to Ca2+ signaling tuned the final size of organs. This work provides a platform to further study how organ size regulation emerges from the crosstalk between biochemical growth signals and heterogeneous cell signaling states (Soundarrajan, 2021).

    A mathematical model of calcium signals around laser-induced epithelial wounds

    Cells around epithelial wounds must first become aware of the wound's presence in order to initiate the wound healing process. An initial response to an epithelial wound is an increase in cytosolic calcium followed by complex calcium signaling events. While these calcium signals are driven by both physical and chemical wound responses, cells around the wound will all be equipped with the same cellular components to produce and interact with the calcium signals. This study developed a mathematical model in the context of laser-ablation of the Drosophila pupal notum that integrates tissue-level damage models with a cellular calcium signaling toolkit. The model replicates experiments in the contexts of control wounds as well as knockdowns of specific cellular components, but it also provides new insights that are not easily accessible experimentally. The model suggests that cell-cell variability is necessary to produce calcium signaling events observed in experiments, it quantifies calcium concentrations during wound-induced signaling events, and it shows that intercellular transfer of the molecule IP(3) is required to coordinate calcium signals across distal cells around the wound. The mathematical model developed in this study serves as a framework for quantitative studies in both wound signaling and calcium signaling in the Drosophila system (Stevens, 2022).

    A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo

    Calcium induced calcium release signaling (CICR) plays a critical role in many biological processes. Every cellular activity from cell proliferation and apoptosis, development and ageing, to neuronal synaptic plasticity and regeneration have been associated with Ryanodine receptors (RyRs). Despite the importance of calcium signaling, the exact mechanism of its function in early development is unclear. As an organism with a short gestational period, the embryos of Drosophila melanogaster are prime study subjects for investigating the distribution and localization of CICR associated proteins and their regulators during development. However, because of their lipid-rich embryos and chitin-rich chorion, their utility is limited by the difficulty of mounting embryos on glass surfaces. This work introduceS a practical protocol that significantly enhances the attachment of Drosophila embryo onto slides and detail methods for successful histochemistry, immunohistochemistry, and in-situ hybridization. The chrome alum gelatin slide-coating method and embryo pre-embedding method dramatically increases the yield in studying Drosophila embryo protein and RNA expression. To demonstrate this approach, DmFKBP12/Calstabin, a well-known regulator of RyR during early embryonic development of Drosophila melanogaster, was studied. DmFKBP12 was identified in as early as the syncytial blastoderm stage, and the dynamic expression pattern of DmFKBP12 during development: initially as an evenly distributed protein in the syncytial blastoderm, then preliminarily localizing to the basement layer of the cortex during cellular blastoderm, before distributing in the primitive neuronal and digestion architecture during the three-gem layer phase in early gastrulation. This distribution may explain the critical role RyR plays in the vital organ systems that originate in from these layers: the suboesophageal and supraesophageal ganglion, ventral nervous system, and musculoskeletal system (Zhang, 2022).

    Independently paced calcium oscillations in progenitor and differentiated cells in an ex vivo epithelial organ

    Cytosolic calcium is a highly dynamic, tightly regulated, and broadly conserved cellular signal. Calcium dynamics have been studied widely in cellular monocultures, yet organs in vivo comprise heterogeneous populations of stem and differentiated cells. This study examined calcium dynamics in the adult Drosophila intestine, a self-renewing epithelial organ in which stem cells continuously produce daughters that differentiate into either enteroendocrine cells or enterocytes. Live imaging of whole organs ex vivo reveals that stem cell daughters adopt strikingly distinct patterns of calcium oscillations after differentiation: Enteroendocrine cells exhibit single-cell calcium oscillations, while enterocytes exhibit rhythmic, long-range calcium waves. These multicellular waves do not propagate through immature progenitors (stem cells and enteroblasts), whose oscillation frequency is approximately half that of enteroendocrine cells. Organ-scale inhibition of gap junctions eliminates calcium oscillations in all cell types--even, intriguingly, in progenitor and enteroendocrine cells that are surrounded only by enterocytes. These findings establish that cells adopt fate-specific modes of calcium dynamics as they terminally differentiate and reveal that the oscillatory dynamics of different cell types in a single, coherent epithelium are paced independently (Kim, 2022).

    Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure

    Changes in cell morphology require the dynamic remodeling of the actin cytoskeleton. Calcium fluxes have been suggested as an important signal to rapidly relay information to the actin cytoskeleton, but the underlying mechanisms remain poorly understood. This study identified the EF-hand domain containing protein EFhD2/Swip-1 as a conserved lamellipodial protein strongly upregulated in Drosophila macrophages at the onset of metamorphosis when macrophage behavior shifts from quiescent to migratory state. Loss- and gain-of-function analysis confirm a critical function of EFhD2/Swip-1 in lamellipodial cell migration in fly and mouse melanoma cells. Contrary to previous assumptions, TIRF-analyses unambiguously demonstrate that EFhD2/Swip-1 proteins efficiently cross-link actin filaments in a calcium-dependent manner. Using a single-cell wounding model, this study showed that EFhD2/Swip-1 promotes wound closure in a calcium-dependent manner. Mechanistically, these data suggest that transient calcium bursts reduce EFhD2/Swip-1 cross-linking activity and thereby promote rapid reorganization of existing actin networks to drive epithelial wound closure (Lehne, 2022).

    Insect nephrocyte function is regulated by a store operated calcium entry mechanism controlling endocytosis and Amnionless turnover

    Insect nephrocytes are ultrafiltration cells that remove circulating proteins and exogenous toxins from the haemolymph. Experimental disruption of nephrocyte development or function leads to systemic impairment of insect physiology as evidenced by cardiomyopathy, chronic activation of immune signalling and shortening of lifespan. The genetic and structural basis of the nephrocyte's ultrafiltration mechanism is conserved between arthropods and mammals, making them an attractive model for studying human renal function and systemic clearance mechanisms in general. Although dynamic changes to intracellular calcium are fundamental to the function of many cell types, there are currently no studies of intracellular calcium signalling in nephrocytes. This work aimed to characterise calcium signalling in the pericardial nephrocytes of Drosophila melanogaster. To achieve this, a genetically encoded calcium reporter (GCaMP6) was expressed in nephrocytes to monitor intracellular calcium both in vivo within larvae and in vitro within dissected adults. Larval nephrocytes exhibited stochastically timed calcium waves. A calcium signal could be initiated in preparations of adult nephrocytes and abolished by EGTA, or the store operated calcium entry (SOCE) blocker 2-APB, as well as RNAi mediated knockdown of the SOCE genes Stim and Orai. Neither the presence of calcium-free buffer nor EGTA affected the binding of the endocytic cargo albumin to nephrocytes but they did impair the subsequent accumulation of albumin within nephrocytes. Pre-treatment with EGTA, calcium-free buffer or 2-APB led to significantly reduced albumin binding. Knock-down of Stim and Orai was non-lethal, caused an increase to nephrocyte size and reduced albumin binding, reduced the abundance of the endocytic cargo receptor Amnionless and disrupted the localisation of Dumbfounded at the filtration slit diaphragm. These data indicate that pericardial nephrocytes exhibit stochastically timed calcium waves in vivo and that SOCE mediates the localisation of the endocytic co-receptor Amnionless. Identifying the signals both up and downstream of SOCE may highlight mechanisms relevant to the renal and excretory functions of a broad range of species, including humans (Sivakumar, 2022).

    Visceral organ morphogenesis via calcium-patterned muscle constrictions

    Organ architecture is often composed of multiple laminar tissues arranged in concentric layers. During morphogenesis, the initial geometry of visceral organs undergoes a sequence of folding, adopting a complex shape that is vital for function. Genetic signals are known to impact form, yet the dynamic and mechanical interplay of tissue layers giving rise to organs' complex shapes remains elusive. This study traced the dynamics and mechanical interactions of a developing visceral organ across tissue layers, from sub-cellular to organ scale in vivo. Combining deep tissue light-sheet microscopy for in toto live visualization with a novel computational framework for multilayer analysis of evolving complex shapes, this study found a dynamic mechanism for organ folding using the embryonic midgut of Drosophila as a model visceral organ. Hox genes, known regulators of organ shape, control the emergence of high-frequency calcium pulses. Spatiotemporally patterned calcium pulses trigger muscle contractions via myosin light chain kinase. Muscle contractions, in turn, induce cell shape change in the adjacent tissue layer. This cell shape change collectively drives a convergent extension pattern. Through tissue incompressibility and initial organ geometry, this in-plane shape change is linked to out-of-plane organ folding. This analysis follows tissue dynamics during organ shape change in vivo, tracing organ-scale folding to a high-frequency molecular mechanism. These findings offer a mechanical route for gene expression to induce organ shape change: genetic patterning in one layer triggers a physical process in the adjacent layer - revealing post-translational mechanisms that govern shape change (Mitchell, 2022).

    CBP-Mediated Acetylation of Importin α Mediates Calcium-Dependent Nucleocytoplasmic Transport of Selective Proteins in Drosophila Neurons

    For proper function of proteins, their subcellular localization needs to be monitored and regulated in response to the changes in cellular demands. In this regard, dysregulation in the nucleocytoplasmic transport (NCT) of proteins is closely associated with the pathogenesis of various neurodegenerative diseases. However, it remains unclear whether there exists an intrinsic regulatory pathway(s) that controls NCT of proteins either in a commonly shared manner or in a target-selectively different manner. To dissect between these possibilities, this study investigated the molecular mechanism regulating NCT of truncated ataxin-3 (ATXN3) proteins of which genetic mutation leads to a type of polyglutamine (polyQ) diseases, in comparison with that of TDP-43. In Drosophila dendritic arborization (da) neurons, dynamic changes were observed in the subcellular localization of truncated ATXN3 proteins between the nucleus and the cytosol during development. Moreover, ectopic neuronal toxicity was induced by truncated ATXN3 proteins upon their nuclear accumulation. Consistent with a previous study showing intracellular calcium-dependent NCT of TDP-43, NCT of ATXN3 was also regulated by intracellular calcium level and involves Importin α3 (Imp α3). Interestingly, NCT of ATXN3, but not TDP-43, was primarily mediated by CBP. It was further shown that acetyltransferase activity of CBP is important for NCT of ATXN3, which may acetylate Imp α3 to regulate NCT of ATXN3. These findings demonstrate that CBP-dependent acetylation of Imp α3 is crucial for intracellular calcium-dependent NCT of ATXN3 proteins, different from that of TDP-43, in Drosophila neurons (Cho, 2022).

    Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling

    Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H(+), Na(+), K(+) and anions have been discovered or engineered, Ca(2+)-selective ChRs have not been reported to date. This study analysed ChRs and mutant derivatives with regard to their Ca(2+) permeability and improve their Ca(2+) affinity by targeted mutagenesis at the central selectivity filter. The engineered channels, termed CapChR1 and CapChR2 for calcium-permeable channelrhodopsins, exhibit reduced sodium and proton conductance in connection with strongly improved Ca(2+) permeation at negative voltage and low extracellular Ca(2+) concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca(2+) concentrations. Moreover, CapChR2 can robustly trigger Ca(2+) signalling in hippocampal neurons. When expressed together with genetically encoded Ca(2+) indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca(2+) entry in brain explants (Lahore, 2022).

    The multimodal action of G alpha q in coordinating growth and homeostasis in the Drosophila wing imaginal disc

    G proteins mediate cell responses to various ligands and play key roles in organ development. This study employed the Gal4/UAS binary system to inhibit or overexpress Gαq in the wing disc, followed by phenotypic analysis. This study characterized how the G protein subunit Gαq tunes the size and shape of the wing in the larval and adult stages of development. Downregulation of Gαq in the wing disc reduced wing growth and delayed larval development. Gαq overexpression is sufficient to promote global Ca (2+) waves in the wing disc with a concomitant reduction in the Drosophila final wing size and a delay in pupariation. The reduced wing size phenotype is further enhanced when downregulating downstream components of the core Ca (2+) signaling toolkit, suggesting that downstream Ca (2+) signaling partially ameliorates the reduction in wing size. In contrast, Gαq -mediated pupariation delay is rescued by inhibition of IP (3) R, a key regulator of Ca (2+) signaling. This suggests that Gαq regulates developmental phenotypes through both Ca (2+) -dependent and Ca (2+) -independent mechanisms. RNA seq analysis shows that disruption of Gαq homeostasis affects nuclear hormone receptors, JAK/STAT pathway, and immune response genes. Notably, disruption of Gαq homeostasis increases expression levels of Dilp8, a key regulator of growth and pupariation timing. It is concluded that Gαq activity contributes to cell size regulation and wing metamorphosis. Disruption to Gαq homeostasis in the peripheral wing disc organ delays larval development through ecdysone signaling inhibition. Overall, Gαq signaling mediates key modules of organ size regulation and epithelial homeostasis through the dual action of Ca (2+) -dependent and independent mechanisms (Velagala, 2023).

    Circadian pacemaker neurons display cophasic rhythms in basal calcium level and in fast calcium fluctuations

    Circadian pacemaker neurons in the Drosophila brain display daily rhythms in the levels of intracellular calcium. These calcium rhythms are driven by molecular clocks and are required for normal circadian behavior. To study their biological basis, this study employed genetic manipulations in conjunction with improved methods of in vivo light-sheet microscopy to measure calcium dynamics in individual pacemaker neurons over complete 24-h durations at sampling frequencies as high as 5 Hz. This technological advance unexpectedly revealed cophasic daily rhythms in basal calcium levels and in high-frequency calcium fluctuations. Further, the rhythms of basal calcium levels and of fast calcium fluctuations were found to reflect the activities of two proteins that mediate distinct forms of calcium fluxes. One is the inositol trisphosphate receptor (ITPR), a channel that mediates calcium fluxes from internal endoplasmic reticulum calcium stores, and the other is a T-type voltage-gated calcium channel, which mediates extracellular calcium influx. These results suggest that Drosophila molecular clocks regulate ITPR and T-type channels to generate two distinct but coupled rhythms in basal calcium and in fast calcium fluctuations. It is proposed that both internal and external calcium fluxes are essential for circadian pacemaker neurons to provide rhythmic outputs and thereby, regulate the activities of downstream brain centers (Liang, 2022).

    Circadian rhythms in multiple aspects of cellular physiology help organisms across taxa, from unicellular cyanobacteria to multicellular animals, adapt to environmental day-night changes. In mammals, neurons in the hypothalamic suprachiasmatic nucleus (SCN) show circadian rhythms in gene expression, intracellular calcium, neural activity, and other cellular properties. Circadian rhythms in SCN neuronal outputs coordinate circadian rhythms in other cells throughout the body and generate behavioral rhythms. The rhythms of SCN neuronal outputs can be generated cell intrinsically by the negative transcription/translation feedback loop of core clock genes as a molecular clock, which then generates 24-h oscillations in a series of genes. These gene oscillations then regulate different aspects of membrane physiology, such as the expression levels of channels for potassium, sodium, and calcium. The mechanisms by which the molecular clockworks coordinate complex membrane physiology to generate neural activity rhythms within individual circadian pacemakers remain to be defined (Liang, 2022).

    Calcium signaling regulates many cellular processes, such as neural excitability, neurotransmitter release, and gene expression. Cytoplasmic calcium can be regulated from extracellular calcium influx as well as from intracellular calcium stored in the endoplasmic reticulum (ER) and mitochondria. Studies on SCN neurons in vitro and recently, in vivo measured circadian calcium rhythms (CCRs) in SCN neurons. Some studies suggested that calcium rhythms were driven by neuronal firing and voltage-gated calcium channels, while others suggested they were driven by intracellular stores via the ER channel ryanodine receptor (RyR). These alternative hypotheses may derive from the technical differences in the various studies, including the details of in vitro preparations, but also, due to a lack of single-cell resolution in the calcium measurements (Liang, 2022).

    In Drosophila, circadian pacemaker neurons also show clock-driven CCRs. The dynamics can be resolved across all five major pacemaker groups (the small ventral lateral neurons [s-LNv], the large ventral lateral neurons [l-LNv], the dorsal lateral neurons [LNd], the group #1 dorsal neurons [DN1], and the group #3 Dorsal Neurons [DN3]), and each group exhibits distinct and sequential daily peak phases. Within such groups, the rhythms can be measured in single identified cells. The multihour phase diversity exhibited by this network requires a series of delays effected by environmental light and by noncell-autonomous modulation mediated by different neuropeptides. Precisely how neuropeptide signaling regulates calcium activity in pacemaker neurons over long (many-hour) durations is unknown. To begin to understand these critical mechanisms of pacemaker modulation, this study began by addressing the cellular and molecular basis of pacemaker calcium rhythms with physiological, genetic, and behavioral measures (Liang, 2022).

    In this study, in vivo calcium imaging began at single-cell resolution, using a high-speed light-sheet microscope termed OCPI-2; the acronym OCPI stands for objective-coupled planar illumination. OCPI-2 represents a fundamental technical advance because it permits sampling frequencies to capture stacks of large tissue volumes, without compromising photon efficiency or spatial resolution. Whereas OCPI-1 methods permitted sampling a whole-brain volume once every 10 min across the 24-h day, OCPI-2 methods permit sampling volumetrically at rates as high as 5 Hz. Thus, both basal calcium levels and fast calcium fluctuations were simultaneously measured at single-cell resolution over entire 24-h durations. Circadian rhythmicity was found in both measures. The fast fluctuations are considered to represent events closely coupled to neuronal firing, as have previous studies conducted in much more restricted temporal durations (i.e., not circadian). In all the Drosophila pacemaker neurons studied, these two layers of calcium rhythms shared the same daily temporal pattern (i.e., they were cophasic). To gain insights into the mechanism of these patterns, the fact was exploited that in Drosophila, many calcium channels are encoded by single genes, and genetics was used to study the roles of individual channels in generating daily pacemaker calcium rhythms. This study presents results of experiments in which RNAs encoding different calcium channels were knocked down selectively in all or a subset of pacemakers. The impact of individual channels in setting both slow daily changes in basal calcium levels and in fast fluctuations was evaluated. Finally, PERIOD (PER) protein staining levels and behavior were measure to determine which channels provide feedback to the molecular clock and which are required for normal circadian output from the pacemaker network (Liang, 2022).

    This study used in vivo 24-h high-frequency calcium imaging and genetic screening to study the cellular biology of daily calcium rhythms in circadian pacemaker neurons of Drosophila. It was found that the calcium rhythm is in fact a composite; it reflects daily fluctuations in both a slow component (basal levels) and a fast one (high-frequency fluctuations). Fast calcium fluctuations were interpreted as representations of calcium dynamics that occur as neurons fire single action potentials or bursts of them. While it is not sufficient to resolve single action potentials, GCaMP6-induced fluorescence is a good index of neuronal electrical activity. For individual identified pacemakers, these two calcium rhythms share the same daily pattern, yet distinct calcium sources appear to contribute differentially to these two rhythms. An extracellular calcium influx, through plasma membrane calcium channels that include the α1T subunit, is critical for the fast calcium fluctuations. In contrast, calcium fluxes from the ER via the channel ITPR are required for both the slow rhythms in the basal calcium levels and the fast ones. Importantly, both channels are essential for normal circadian behavior. Thus, the molecular clocks may drive circadian rhythms in pacemaker neuron output by regulating different calcium sources to generate coordinate but distinct rhythms in its calcium activities (Liang, 2022).

    CCRs are widespread across taxa. Calcium rhythms are required for circadian pacemaker functions in both rodents and Drosophila. Studies on mammalian circadian pacemakers in the SCN are controversial regarding the temporal relationship between the CCR and rhythms in electrical activity, such as in spontaneous firing rate (SFR) and in resting membrane potential (RMP). Recordings from SCN slice cultures showed that the phases of CCR in individual pacemakers are diverse and could be different from the populational phase of SFR rhythms (16, 33). However, the populational SFR phase is composed of many diverse phases of SFR rhythms on the individual cell level; it is unclear whether SFR phases align with the phases of CCR. Imaging with both voltage sensor and calcium sensors in SCN slices, another study concluded that RMP rhythms and CCR were in phase, yet even a third study concluded that RMP rhythms and CCR were in phase in the ventral SCN but that in dorsal SCN, the CCR phase led the RMP rhythms by ~2 h. Because the voltage sensor signal measured from dorsal SCN may derive from the neural processes of ventral SCN neurons, the cellular interpretation of these results is unclear. Another source for the inconsistency might be the culture conditions; in fact, when SCN neurons were recorded in vivo by photometry, the rhythms in fast calcium activity were in phase with slow calcium rhythms. In general, comparisons of population rhythms and rhythms in single cells are not easily reconciled. Recordings that tracked pacemaker neurons from different identified groups in vivo for 24 h showed that at the single cell level, slow calcium rhythms (CCRs) were unambiguously in phase with rhythms in fast calcium fluctuations; the latter likely reflects rhythms in SFR (Liang, 2022).

    To measure the fast calcium fluctuations, a high-frequency light-sheet scanning microscope termed OCPI-2 was used. OCPI-2 resolved limitations present in previous microscope designs that created a bottleneck in terms of the rate at which tissue volumes can be repeatedly scanned. Importantly, it does so without compromising photon efficiency or spatial resolution. Its use was instrumental in allowing sampling of the whole-brain volume at frequencies as high as 5 Hz periodically throughout the day. cry01 flies were used to avoid the direct light responses of pacemaker neurons, and it was found that all pacemaker groups displayed slow calcium rhythms, with patterns that were comparable with those previously reported, except for that of l-LNv. That group displayed an additional calcium peak in the early evening and another just before lights on. Because l-LNv innervates the optic lobes and receives large-scale visual inputs, it is speculated that the repeated optical scanning might anomalously activate l-LNv in the evening via visual systems. In the later experiments, when the illumination duration per hour was reduced from 31.5 s to 2.4 s, l-LNv did not show the additional evening peaks; this was true even when measured in flies wild type for cry. Shorter durations of light exposure permit all pacemaker groups to display slow calcium rhythms with phases similar to those obtained with the slow-frequency (every 10-min) recording sessions. Therefore, the concern to avoid technical artifacts when imaging from light-sensitive pacemaker appears especially acute in the case of l-LNv. Nevertheless, by carefully tuning the illumination intensity for calcium imaging, it is proposed that it is possible to monitor normal slow calcium rhythms and fast calcium fluctuations from the same individual pacemaker neurons (Liang, 2022).

    The causal relationships between clock gene rhythms, calcium rhythms, and electrical activity rhythms in the SCN remain generally unresolved. Treating SCN slices with Tetrodotoxin (TTX, to block Na-dependent action potentials) diminished SFR rhythms, partially affected RMP rhythms and CCRs, and slowly affected clock gene rhythms over several days. Dispersed SCN cells in vitro showed a TTX-resistant CCR, suggesting that CCR is driven by clock gene rhythms. Thus, the variation in CCR sensitivity to TTX treatment might be caused by the degree to which clock gene rhythms in vitro become progressively dysfunctional. In Drosophila, the findings suggested that clock gene rhythms drive two components of the CCR-both basal calcium levels and fast calcium fluctuations-via circadian regulation of the ER channel ITPR and membrane voltage-gated calcium channel α1T. Both channels might then contribute to SFR and RMP rhythms. Similarly, in SCN pacemakers, pharmacologically blocking another ER channel RyR affected both CCR and SFR rhythms, suggesting that rhythms in basal calcium levels are regulated by calcium from ER and are required for fast electric activity rhythms. In addition, SCN pacemakers also showed a circadian rhythm in fast calcium activity mediated by L-type voltage-gated calcium channels. Pharmacologically blocking these membrane channels affected SFR rhythms and in some case, affected CCR. In these studies, manipulating a membrane voltage-gated calcium channel in all or a subset of pacemakers selectively affected rhythms in fast calcium fluctuations, which likely reflected SFR rhythms and thus, impaired circadian outputs; however, it did not significantly affect the slow rhythms in basal calcium levels. Manipulating the ER calcium channel IP3R in all pacemakers affected rhythms in both slow and fast calcium rhythms. Therefore, in parallel to mammalian SCN neurons, Drosophila circadian pacemakers generate calcium rhythms by regulating both ER and extracellular calcium sources. Since these results suggest little or no role for the RyR channel, the daily rhythmic regulation in fly pacemakers likely acts on a different set of ER and cytoplasmic membrane channels from those in mammalian pacemakers (Liang, 2022).

    This study also presented RNAi evidence implicating the Itpr and Ca-&alpha1T genes in pacemaker cell calcium fluxes. However, for each, only a single RNAi proved effective in reducing rhythmic power and increasing percentage of arrhythmicity. Corroboration for these findings is provided by a prior report, which found that the same RNAi line used decreases Itpr RNA levels. Likewise, corroboration was found in a prior report, that described a specific Ca-alpha1T insertion line as a protein null mutation that exhibited reduced percentage of rhythmicity and power in DD but without a change in circadian period or in the daily per rhythm. They concluded that Ca-alpha1T expression does not affect the central clock mechanism and surmised instead that it likely affects the output of the circadian system, specifically via a requirement for the channel to permit normal physiological activation of pacemaker neurons. That work provides independent genetic confirmation of the RNAi results, and its conclusions align precisely with the current work (Liang, 2022).

    The RNAi knockdown experiments indicate a role for the ER calcium channel SERCA in supporting slow calcium rhythms in Drosophila pacemakers and behavioral rhythms. However, SERCA knockdown also produced high rates of lethality and among survivors, strong effects on the PER molecular oscillation. It is concluded that SERCA, which maintains the ER cytoplasmic calcium gradient, is essential for the normal physiology of the cells but that these additional phenotypes precluded an assessment of its precise role in circadian rhythmicity. In contrast to the situation with SERCA, the results support a hypothesis that ITPR is a crucial actuator of the molecular clock. Previous transcriptomic analysis also supports that possibility; in circadian neurons, Itpr displays rhythmic expression, while SERCA does not. Knocking down Itpr in all circadian neurons (with tim-GAL4) generally caused stronger deficits in both behavioral rhythms and calcium rhythms than in just the PDF-positive neurons (with pdf-GAL4). One possibility might be that the RNAi expression level is lower when driven by pdf-GAL4 than when driven by tim-GAL4. However, in SI knocking down Itpr in all circadian neurons caused stronger deficits in the amplitude of calcium rhythms of non-PDF-positive neurons (LNd, DN1, and DN3) than in those of PDF-positive neurons (s-LNv and l-LNv). Hence, a difference in the vulnerability to IP3R disruption between PDF-negative and PDF-positive neurons might provide an additional or alternative explanation for why the pdf-GAL4-driven knockdown of Itpr affected neither calcium rhythms nor behavior. It is also noted that the literature provides examples, wherein a single RNAi line produces stronger behavioral effects when coupled with pdf-Gal4 than with tim-GAL4. Such findings suggest that the final result in such experiments will represent a mixture of GAL4 driver line strength and the differential contributions of the UAS:Responder gene product across different positions within an affected neural circuit (Liang, 2022).

    Finally, the RNAi knockdown of the plasma membrane calcium channel α1T indicated a role for voltage-gated T-type calcium channels in the final rhythmic output of the pacemakers. Consistent with a role in the presumed output pathway, impairing the rhythm of fast calcium activity strongly affected circadian behavior but did not affect the molecular clock or the slow calcium rhythms. T-type channels play a crucial role in other pacemakers, such as the Sino-Atrial node of the mammalian heart. Their conduction in the hyperpolarized state and closure at more depolarized potentials are central to their role in generating bursting dynamics with periods much longer than the membrane time constant. Given the power spectrum of the fluctuations observed, it seems possible that α1T channels play a similar role in the fast (~0.1-Hz) fluctuations of Drosophila circadian neurons. Remarkably, the expression of α1T also displays a circadian rhythm, with distinct phases in different groups of pacemaker neurons. Collectively, these results suggest that Itpr and α1T channel activity are together critical to produce clock regulation of rhythms in both slow and fast calcium activities of key pacemaker neurons (Liang, 2022).


    REFERENCES

    Asteriti, S., Liu, C. H. and Hardie, R. C. (2017). Calcium signalling in Drosophila photoreceptors measured with GCaMP6f. Cell Calcium 65: 40-51. PubMed ID: 28238353

    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

    Cho, J. H., Jo, M. G., Kim, E. S., Lee, N. Y., Kim, S. H., Chung, C. G., Park, J. H. and Lee, S. B. (2022). CBP-Mediated Acetylation of Importin α Mediates Calcium-Dependent Nucleocytoplasmic Transport of Selective Proteins in Drosophila Neurons. Mol Cells 45(11): 855-867. PubMed ID: 36172977

    Kim, A. A., Nguyen, A., Marchetti, M., Du, X., Montell, D. J., Pruitt, B. L. and O'Brien, L. E. (2022). Independently paced calcium oscillations in progenitor and differentiated cells in an ex vivo epithelial organ. J Cell Sci. PubMed ID: 35722729

    Kon, N., Wang, H. T., Kato, Y. S., Uemoto, K., Kawamoto, N., Kawasaki, K., Enoki, R., Kurosawa, G., Nakane, T., Sugiyama, Y., Tagashira, H., Endo, M., Iwasaki, H., Iwamoto, T., Kume, K. and Fukada, Y. (2021). Na+/Ca2+ exchanger mediates cold Ca2+ signaling conserved for temperature-compensated circadian rhythms. Sci Adv 7(18). PubMed ID: 33931447

    Lahore, R. G. F., Pampaloni, N. P., Peter, E., Heim, M. M., Tillert, L., Vierock, J., Oppermann, J., Walther, J., Schmitz, D., Owald, D., Plested, A. J. R., Rost, B. R. and Hegemann, P. (2022). Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling. Nat Commun 13(1): 7844. PubMed ID: 36543773

    Lehne, F., Pokrant, T., Parbin, S., Salinas, G., Grosshans, J., Rust, K., Faix, J. and Bogdan, S. (2022). Calcium bursts allow rapid reorganization of EFhD2/Swip-1 cross-linked actin networks in epithelial wound closure. Nat Commun 13(1): 2492. PubMed ID: 35524157

    Liang, X., Holy, T. E. and Taghert, P. H. (2022). Circadian pacemaker neurons display cophasic rhythms in basal calcium level and in fast calcium fluctuations. Proc Natl Acad Sci U S A 119(17): e2109969119. PubMed ID: 35446620

    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

    Mitchell, N. P., Cislo, D. J., Shankar, S., Lin, Y., Shraiman, B. I. and Streichan, S. J. (2022). Visceral organ morphogenesis via calcium-patterned muscle constrictions. Elife 11. PubMed ID: 35593701

    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

    Sivakumar, S., Miellet, S., Clarke, C. and Hartley, P. S. (2022). Insect nephrocyte function is regulated by a store operated calcium entry mechanism controlling endocytosis and Amnionless turnover. J Insect Physiol 143: 104453. PubMed ID: 36341969

    Soundarrajan, D. K., Huizar, F. J., Paravitorghabeh, R., Robinett, T. and Zartman, J. J. (2021). From spikes to intercellular waves: Tuning intercellular calcium signaling dynamics modulates organ size control. PLoS Comput Biol 17(11): e1009543. PubMed ID: 34723960

    Stevens, A. C., O'Connor, J. T., Pumford, A. D., Page-McCaw, A. and Hutson, M. S. (2022). A mathematical model of calcium signals around laser-induced epithelial wounds. Mol Biol Cell: mbcE22080361. PubMed ID: 36322412

    Velagala, V., Soundarrajan, D. K., Unger, M. F., Gazzo, D., Kumar, N., Li, J. and Zartman, J. (2023). The multimodal action of G alpha q in coordinating growth and homeostasis in the Drosophila wing imaginal disc. bioRxiv. PubMed ID: 36711848

    Zhang, W., Lei, X., Zhou, X., He, B., Xiao, L., Yue, H., Wang, S., Sun, Y., Wu, Y., Wang, L., Ghartey-Kwansah, G., Jones, O. D., Bryant, J. L., Xu, M., Ma, J. and Xu, X. (2022). A Protocol for Immunohistochemistry and RNA In-situ Distribution within Early Drosophila Embryo. J Vis Exp(183). PubMed ID: 35604165

    Zygotically transcribed genes

    Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

    The Interactive Fly resides on the
    Society for Developmental Biology's Web server.