The Interactive Fly

Zygotically transcribed genes

Calcium binding, calcium dependent enzymes and proteins, and calcium signaling

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
SMOC-1 interacts with both BMP and glypican to regulate BMP signaling in C. elegans
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
Voltage to Calcium Transformation Enhances Direction Selectivity in Drosophila T4 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>
Expanded polyQ aggregates interact with sarco-endoplasmic reticulum calcium ATPase and Drosophila inhibitor of apoptosis protein1 to regulate polyQ mediated neurodegeneration in Drosophila
Circadian pacemaker neurons display cophasic rhythms in basal calcium level and in fast calcium fluctuations
Proteolytic activation of Growth-blocking peptides triggers calcium responses through the GPCR Mthl10 during epithelial wound detection
Wolfram syndrome 1 regulates sleep in dopamine receptor neurons by modulating calcium homeostasis
PINK1 and Parkin regulate IP(3)R-mediated ER calcium release
Cholinergic neurons trigger epithelial Ca(2+) currents to heal the gut

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).

    SMOC-1 interacts with both BMP and glypican to regulate BMP signaling in C. elegans

    Secreted modular calcium-binding proteins (SMOCs) are conserved matricellular proteins found in organisms from Caenorhabditis elegans to humans. SMOC homologs characteristically contain 1 or 2 extracellular calcium (EC)-binding domain(s) and 1 or 2 thyroglobulin type-1 (TY) domain(s). SMOC proteins in Drosophila and Xenopus have been found to interact with cell surface heparan sulfate proteoglycans (HSPGs) to exert both positive and negative influences on the conserved bone morphogenetic protein (BMP) signaling pathway. This study used a combination of biochemical, structural modeling, and molecular genetic approaches to dissect the functions of the sole SMOC protein in C. elegans. CeSMOC-1 binds to the heparin sulfate proteoglycan GPC3 homolog LON-2/glypican, as well as the mature domain of the BMP2/4 homolog DBL-1. Moreover, CeSMOC-1 can simultaneously bind LON-2/glypican and DBL-1/BMP. The interaction between CeSMOC-1 and LON-2/glypican is mediated specifically by the EC domain of CeSMOC-1, while the full interaction between CeSMOC-1 and DBL-1/BMP requires full-length CeSMOC-1. Both in vitro biochemical and in vivo functional evidence id provided demonstrating that CeSMOC-1 functions both negatively in a LON-2/glypican-dependent manner and positively in a DBL-1/BMP-dependent manner to regulate BMP signaling. It was further shown that in silico, Drosophila and vertebrate SMOC proteins can also bind to mature BMP dimers. This work provides a mechanistic basis for how the evolutionarily conserved SMOC proteins regulate BMP signaling (DeGroot, 2023).

    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).

    Voltage to Calcium Transformation Enhances Direction Selectivity in Drosophila T4 Neurons

    An important step in neural information processing is the transformation of membrane voltage into calcium signals leading to transmitter release. However, the effect of voltage to calcium transformation on neural responses to different sensory stimuli is not well understood. This study used in vivo two-photon imaging of genetically encoded voltage and calcium indicators, ArcLight and GCaMP6f, respectively, to measure responses in direction-selective T4 neurons of female Drosophila Comparison between ArcLight and GCaMP6f signals reveals calcium signals to have a significantly higher direction selectivity compared with voltage signals. Using these recordings, a model was built which transforms T4 voltage responses into calcium responses. Using a cascade of thresholding, temporal filtering and a stationary nonlinearity, the model reproduces experimentally measured calcium responses across different visual stimuli. These findings provide a mechanistic underpinning of the voltage to calcium transformation and show how this processing step, in addition to synaptic mechanisms on the dendrites of T4 cells, enhances direction selectivity in the output signal of T4 neurons. Measuring the directional tuning of postsynaptic vertical system (VS)-cells with inputs from other cells blocked, this study found that, indeed, it matches the one of the calcium signal in presynaptic T4 cells (Mishra, 2023).

    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).

    Expanded polyQ aggregates interact with sarco-endoplasmic reticulum calcium ATPase and Drosophila inhibitor of apoptosis protein1 to regulate polyQ mediated neurodegeneration in Drosophila

    Polyglutamine (polyQ) induced neurodegeneration is one of the leading causes of progressive neurodegenerative disorders characterized clinically by deteriorating movement defects, psychiatric disability, and dementia. Calcium [Ca(2+)] homeostasis, which is essential for the functioning of neuronal cells, is disrupted under these pathological conditions. In this paper, we simulated Huntington's disease phenotype in the neuronal cells of the Drosophila eye, and [Ca(2+)] pump, sarco-endoplasmic reticulum calcium ATPase (SERCA), was identified as one of the genetic modifiers of the neurodegenerative phenotype. This paper shows genetic and molecular interaction between polyglutamine (polyQ) aggregates, SERCA and DIAP1. Evidence is presented that polyQ aggregates interact with SERCA and alter its dynamics, resulting in a decrease in cytosolic [Ca(2+)] and an increase in ER [Ca(2+)], and thus toxicity. Downregulating SERCA lowers the enhanced calcium levels in the ER and rescues, morphological and functional defects caused due to expanded polyQ repeats. Cell proliferation markers such as Yorkie (Yki), Scalloped (Sd), and phosphatidylinositol 3 kinases/protein kinase B (PI3K/Akt), also respond to varying levels of calcium due to genetic manipulations, adding to the amelioration of degeneration. These results imply that neurodegeneration due to expanded polyQ repeats is sensitive to SERCA activity, and its manipulation can be an important step toward its therapeutic measures (Maurya, 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).

    Proteolytic activation of Growth-blocking peptides triggers calcium responses through the GPCR Mthl10 during epithelial wound detection

    The presence of a wound triggers surrounding cells to initiate repair mechanisms, but it is not clear how cells initially detect wounds. In epithelial cells, the earliest known wound response, occurring within seconds, is a dramatic increase in cytosolic calcium. This study shows that wounds in the Drosophila notum trigger cytoplasmic calcium increase by activating extracellular cytokines, Growth-blocking peptides (Gbps; see Gbp1), which initiate signaling in surrounding epithelial cells through the G-protein-coupled receptor Methuselah-like 10 (Mthl10). Latent Gbps are present in unwounded tissue and are activated by proteolytic cleavage. Using wing discs, this study showed that multiple protease families can activate Gbps, suggesting that they act as a generalized protease-detector system. Experimental and computational evidence is presented that proteases released during wound-induced cell damage and lysis serve as the instructive signal: these proteases liberate Gbp ligands, which bind to Mthl10 receptors on surrounding epithelial cells, and activate downstream release of calcium (O'Connor, 2021).

    When a tissue is wounded, the cells surrounding the wound rapidly respond to repair the damage. Despite the non-specific nature of cellular damage, there is remarkable specificity to the earliest cellular response: cells around the wound increase cytosolic calcium, and this damage response is conserved across the animal kingdom. The calcium response is not limited to cells at the wound margin but extends even to distal cells. Multiple molecular mechanisms have been identified that regulate wound-induced gene expression or cell behavior downstream of calcium, but the upstream signals remain unclear. How exactly do cells detect wounds? Thia study investigate the molecular mechanisms by which a wound initiates cytosolic calcium signals (O'Connor, 2021).

    The immediate increase in cellular calcium in turn initiates repair or defense responses. Calcium has been well established as a versatile and universal intracellular signal that plays a role in the modulation of numerous intracellular processes. Several calcium-regulated processes are required for proper wound repair, including actomyosin dynamics, JNK pathway activation and plasma membrane repair. Unsurprisingly, an increase in cytosolic calcium is necessary for wound repair. Nonetheless, there is less clarity on the mechanisms that trigger increased cytosolic calcium in cells near to and distant from the wound. In some cases, wound-induced cytoplasmic calcium enters from the extracellular environment, either directly through plasma membrane damage. In others, calcium is released from the endoplasmic reticulum (ER) through the IP3 Receptor and initiated by an unknown G-protein-coupled receptor (GPCR) or receptor tyrosine kinase (RTK). Further, calcium responses can be initiated by mechanical stimuli alone. Elucidating the mechanisms by which calcium signaling is triggered in vivo is critical to understanding how wound information is transmitted through a tissue in order to change cellular behavior and properly repair the wound (O'Connor, 2021).

    By live imaging laser wounds in Drosophila pupae, previously work showed that damaged cells around wounds become flooded within milliseconds by extracellular calcium entering through microtears in the plasma membrane. Although this calcium influx expands one or two cell diameters through gap junctions, it does not extend to more distal cells. Strikingly, after a delay of 45-75 s, a second independent calcium response expands outward to reach a larger number of distal cells. This study identified the relevant signal transduction pathway and receptor, the GPCR Mthl10. Downstream, signals are relayed through Gαq and PLCβ to release calcium from the ER. Upstream, Mthl10 is activated around wounds by the cytokine ligands Growth-blocking peptides (Gbps). Further, experimental and computational evidence is provided that the initiating event for the distal calcium response in vivo is a wound-induced release of proteases that activate the latent Gbp cytokines, cleaving them from inactive/pro-forms into active signaling molecules (O'Connor, 2021).

    It was already known that Gbps are synthesized in an inactive pro-form, requiring proteolytic cleavage for activation, and that they are secreted by the fat body. Although Gbps are present in unwounded tissues, they activate Mthl10 only in the presence of a wound. Interestingly, Gbps have cleavage consensus sequences for multiple protease families. Further, the addition of cell lysate or the addition of the unrelated proteases trypsin or clostripain to unwounded tissue is sufficient to generate a calcium signal in wing discs through Mthl10/Gbp signaling. These results lead to a model in which the lysis of cells inherent in wounding releases non-specific cellular proteases into the extracellular environment. These proteases cleave and activate extracellular Gbps, which in turn activate the Mthl10 GPCR on cells around the wound, initiating wound-induced calcium signaling. Such cell lysis and protease release should be a general feature of cell destruction, whether caused by trauma, pathogen-induced lysis, or a lytic form of cell death such as pyroptosis or necroptosis (immunologically silent apoptosis may well be an exception). A variety of epithelial damage mechanisms may thus converge through the Gbps to signal via the GPCR Mthl10 and alert surrounding cells to the presence of a nearby wound. This molecular mechanism is supported by a computational model that accurately describes the pattern and timing of wound-induced calcium, predicted its dependence on wound size and initial levels of Gbps, and led to the observation that cell lysis is not immediate but rather takes place over tens of seconds. Thus, this study offers a model for how surrounding cells detect the damage of cell lysis, utilizing a Gbp-based protease-detector system (O'Connor, 2021).

    Two superimposed mechanisms increase cytoplasmic calcium levels around wounds Laser wounds generate complex yet reproducible patterns of increased cytoplasmic calcium, and the complexity of this pattern has undoubtedly made it difficult to unravel its underlying mechanisms. Within the first ~90 s after wounding, two mechanisms drive the increase of calcium, and the complexity is generated by the temporal and spatial superimposition of these two mechanisms. Previously, it was reported that a different type of cellular damage initiates a different mechanism for increasing cytoplasmic calcium. In that report, wound-induced microtears were identified in the plasma membranes of surviving cells, and these microtears provided an entry for extracellular calcium to flood into the cytoplasm and then flow out to neighboring undamaged cells via gap junctions. This direct entry of calcium through damaged plasma membranes is evident within milliseconds after wounding. In this report we describe a second mechanism that extends to more distal cells, initiated by cell lysis at wounding. The dynamics of protease release from lysed cells, along with the gradual accumulation of active Gbp and its rapid diffusion, all contribute to the appearance of this distal calcium response 45-75 s after wounding. The earliest and closest cells to be activated by Mthl10/Gbp signaling cannot be identified visually because the initial flood of calcium through microtears takes time to subside (O'Connor, 2021).

    Three tools allowed deciphering od these superimposed mechanisms. The first tool was the laser itself, which generates a highly stereotyped pattern of damage within a circular wound bed. Although cell lysis and plasma membrane damage are features of nearly every wound, their reproducible pattern in a laser wound allowed distinguishing the signaling mechanisms each type of damage potentiated. The second tool was a spatiotemporal analytical framework to measure radius over time, which clearly identified two peaks, the first induced by microtears and the second induced by cell lysis. The third tool was experimental, using RNAi-knockdown of genes in a limited region and comparing it with an internal control. The ability to identify asymmetry between the control and experimental sides of wounds allowed bypassing of concerns about variable wound sizes, which otherwise would have made it difficult to recognize patterns and interpret data. Complex overlapping patterns may have obscured the mechanisms upstream of wound-induced calcium in other systems as well as the current one (O'Connor, 2021).

    Previous studies identified other molecules and phenomena upstream of wound-induced calcium. Studies in cell culture found that wound-generated cell lysis releases ATP, which diffuses extracellularly to bind to purinergic receptors and activate calcium release from intracellular stores. Although reproducible in many types of cultured cells, there has been little evidence to support ATP signaling from lytic cells in vivo, likely because extracellular ATP is rapidly hydrolyzed by nucleotidases in vivo. Interestingly, ATP does appear to signal damage and promote motility in response to injuries associated with cell swelling in zebrafish, animals that inhabit a hypotonic aqueous environment; however, even in this wounding paradigm, ATP does not signal from lytic cells at an appreciable level. No evidence was found for ATP signaling upstream of calcium in the wounding experiments, as knockdown of the only fly adenosine receptor did not alter the calcium pattern around wounds (O'Connor, 2021).

    Some in vivo studies have implicated a TrpM ion channel upstream of calcium release. This role of TrpM was first identified in laser-wounding studies of the C. elegans hypodermis, a giant syncytial cell where great overlap in the spatial extent of microtear-initiated calcium, which would diffuse quickly throughout a syncytium, and receptor-mediated calcium released from the ER would be expected. In the hypodermis, loss of TrpM reduced by half the intensity of wound-induced calcium signaling, but without spatial and temporal analysis, the exact contribution of TrpM is not known. In the Drosophila notum, a previous study identified TrpM as a regulator of wound-induced actin remodeling, and a slight reduction in wound-induced calcium intensity over time was noted in TrpM knockdowns. In contrast, this study did not observe any change in the spatial or temporal aspects of the calcium response in TrpM knockdown cells compared with the internal control, and given wound-to-wound variability, it would have been hard to identify a small effect without an internal control. A study in the fly embryo determined that wound-induced calcium originates from both the external environment and internal stores, suggesting to that two superimposed calcium response mechanisms may have been at play in these experiments. That study found when TrpM was knocked down, calcium intensity was reduced by half, but again without spatial and temporal analysis or an internal control, it is difficult to know what pathway TrpM regulates. Tissue mechanics are upstream of increases in cytoplasmic calcium in a non-wounding context. Several labs have reported calcium flashes and waves in unwounded wing discs, dissected from larvae and cultured ex vivo. Cell and organ culture requires serum to support metabolism outside the organism, and in fly culture, this 'serum' is generated by grinding whole adult flies and collecting the supernatant. Because such serum would undoubtedly contain secreted signals from wounded cells, calcium signaling in wing discs ex vivo is probably a wound response; indeed, it was founs to be transduced by the same mechanism as wound signals, requiring protease, Gbps, and Mthl10. One aspect of calcium signaling in wing discs that we have not tested in our wounding model, however, is the role of mechanical tension. In carefully controlled mechanical experiments, fly serum was found to induce calcium flashes in wing discs specifically on the release of mechanical compression, indicating that tension is a requirement for calcium signaling in these wing discs. It is interesting to consider the TrpM results in light of these mechanical studies, as some TrpM channels can be mechanosensitive. Together, these data suggest that there may be a role for mechanical tension in wound-induced calcium responses (O'Connor, 2021).

    Two independent mechanisms were founs that increase cytoplasmic calcium, and in the cells at the wound margin these mechanisms would appear to act redundantly. Such redundancy indicates that the role of calcium in these cells is very important for wound healing. One biological pathway that may be downstream of calcium in these cells is recruitment of actin and myosin to the wound margin to form an actomyosin purse string that cinches the wound closed. What about calcium in the distal cells, regulated by Gbp/Mthl10? There are many possible functions, but currently, all of them are speculative. One possibility is that the cytosolic calcium response initiates distal epithelial cells to modify their cellular behavior from a stationary/non-proliferative state to a migratory and/or proliferative state necessary to repair the wound. Alternatively, an increase in cytosolic calcium may act to modulate an inflammatory response through DUOX leading to the formation of hydrogen peroxide to recruit inflammatory cells to the wound or through the calcium-dependent activation of cytoplasmic Phospholipase A2 leading to the rapid recruitment of immune cells to tissue damage. This possibility is intriguing because Gbp is known to activate an immune response leading to the upregulation of antimicrobial peptides and to increased activity of phagocytic plasmatocytes in a calcium-dependent manner. Interestingly, loss of Methuselah-like (Mthl) GPCRs results in increased lifespans, and Gbps are nutrition-sensitive peptides whose expression is reduced under starvation conditions. Increased lifespan, caloric restriction and decreased inflammation have all been linked, and Gbp/Mthl10 activation at wounds may be part of this link (O'Connor, 2021).

    Although the cytokine and GPCR families are widely conserved, Gbp and Mthl10 do not have direct orthologs in chordates. Nonetheless, similarities exist between the Gbp/Mthl10 mechanism and wound responses in other organisms. Damage- or pathogen-induced activation of proteins by proteolytic cleavage has been well documented in the cases of Spatzle in the Toll pathway, thrombin and fibrin in the blood coagulation pathway, and IL-1β and IL-18 in the pyroptosis pathway. Additionally, wound-defense signaling in plants relies on an immunomodulatory plant elicitor peptide that is cleaved into its active form by cysteine proteases upon damage-induced cytosolic calcium, and the plant defense hormone systemin is cleaved into its active form by phytaspases in response to damage or predation. Because the basic circuitry is similar across kingdoms, the current study suggests an ancient strategy for wound detection based on proprotein cleavage, activated by proteases released via cell lysis. As these examples make clear, proteases are already known to play critical roles in blood clotting and immune signaling, and this study finds that proteases are also instructive signals in epithelial wound detection (O'Connor, 2021).

    As noted above, the Gbp ligands and Mthl10 receptor are not present in mammals, so the extent of mechanistic conservation is unclear. Further, this study did not experimentally tested this wound-detection mechanism in other developmental stages of Drosophila. For the computational model, several simplifications were made: the use of one variable for all proteases and one variable for all Gbps, rather than having separate Gbp1 and Gbp2; the use of simplified receptor/ligand dynamics that do not include uptake or recycling; and the use of a ligand-receptor-binding threshold rather than inclusion of the signal transduction cascade between receptor-binding and calcium release. Finally, this study does not describe or address the mechanism behind the calcium flares that continue for at least one hour after wounding (O'Connor, 2021).

    Wolfram syndrome 1 regulates sleep in dopamine receptor neurons by modulating calcium homeostasis

    Sleep disruptions are quite common in psychological disorders, but the underlying mechanism remains obscure. Wolfram syndrome 1 (WS1) is an autosomal recessive disease mainly characterized by diabetes insipidus/mellitus, neurodegeneration and psychological disorders. It is caused by loss-of function mutations of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes an endoplasmic reticulum (ER)-resident transmembrane protein. Heterozygous mutation carriers do not develop WS1 but exhibit 26-fold higher risk of having psychological disorders. Since WS1 patients display sleep abnormalities, this study aimed to explore the role of WFS1 in sleep regulation so as to help elucidate the cause of sleep disruptions in psychological disorders. It was found in Drosophila that knocking down wfs1 in all neurons and wfs1 mutation lead to reduced sleep and dampened circadian rhythm. These phenotypes are mainly caused by lack of wfs1 in dopamine 2-like receptor (Dop2R) neurons which act to promote wake. Consistently, the influence of wfs1 on sleep is blocked or partially rescued by inhibiting or knocking down the rate-limiting enzyme of dopamine synthesis, suggesting that wfs1 modulates sleep via dopaminergic signaling. Knocking down wfs1 alters the excitability of Dop2R neurons, while genetic interactions reveal that lack of wfs1 reduces sleep via perturbation of ER-mediated calcium homeostasis. Taken together, a role is proposed for wfs1 in modulating the activities of Dop2R neurons by impinging on intracellular calcium homeostasis, and this in turn influences sleep. These findings provide a potential mechanistic insight for pathogenesis of diseases associated with WFS1 mutations (Hao, 2023).

    Sleep disruptions are common in individuals with psychiatric disorders, and sleep disturbances are risk factors for future onset of depression. However, the mechanism underlying sleep disruptions in psychiatric disorders are largely unclear. Wolfram Syndrome 1 (WS1) is an autosomal recessive neurodegenerative disease characterized by diabetes insipidus, diabetes mellitus, optic atrophy, deafness and psychiatric abnormalities such as severe depression, psychosis and aggression. It is caused by homozygous (and compound heterozygous) mutation of the WOLFRAM SYNDROME 1 (WFS1) gene, which encodes wolframin, an endoplasmic reticulum (ER) resident protein highly expressed in the heart, brain, and pancreas. On the other hand, heterozygous mutation of WFS1 does not lead to WS1 but increase the risk of depression by 26 fold. A study in mice further confirmed that WFS1 mutation is causative for depression. Consistent with the comorbidity of psychiatric conditions and sleep abnormalities, WS1 patients also experience increased sleep problems compared to individuals with type I diabetes and healthy controls. It has been proposed that sleep symptoms can be used as a biomarker of the disease, especially during relatively early stages, but the mechanisms underlying these sleep disturbances are unclear. Considering that heterozygous WFS1 mutation is present in up to 1% of the population and may be a significant cause of psychiatric disorder in the general population, it was decided to investigate the role of wolframin in sleep regulation so as to probe the mechanism underlying sleep disruptions in psychiatric disorders (Hao, 2023).

    Although the wolframin protein does not possess distinct functional domains, a number of ex vivo studies in cultured cells demonstrated a role for it in regulating cellular responses to ER stress and calcium homeostasis, as well as ER-mitochondria cross-talk. Mice that lack Wfs1 in pancreatic β cells develop glucose intolerance and insulin deficiency due to enhanced ER stress and apoptosis. Knocking out Wfs1 in layer 2/3 pyramidal neurons of the medial prefrontal cortex in mice results in increased depression-like behaviors in response to acute restraint stress. This is accompanied by hyperactivation of the hypothalamic-pituitary-adrenal axis and altered accumulation of growth and neurotrophic factors, possibly due to defective ER function. A more recent study in Drosophila found that knocking down wfs1 in the nervous system does not increase ER stress, but enhances the susceptibility to oxidative stress-, endotoxicity- and tauopathy-induced behavioral deficits and neurodegeneration (Sakakibara, 2018). Overall, the physiological function of wolframin in vivo, especially in the brain, remains elusive for the most part. This study identified a role for wolframin in regulating sleep and circadian rhythm in flies. Wfs1 deficiency in the dopamine 2-like receptor (Dop2R) neurons leads to reduced sleep, while inhibiting dopamine synthesis blocks the effect of wfs1 on sleep, implying that wfs1 influences sleep via dopaminergic signaling. It was further found that these Dop2R neurons function to promote wakefulness. Depletion of wfs1 alters neural activity, which leads to increased wakefulness and reduced sleep. Consistent with this, it was found that knocking down the ER calcium channel Ryanodine receptor (RyR) or 1,4,5-trisphosphate receptor (Itpr) rescues while knocking down the sarco(endoplasmic)reticulum ATPase SERCA synergistically enhances the short-sleep phenotype caused by wfs1 deficiency, indicating that wolframin regulates sleep by modulating calcium homeostasis. Taken together, these findings provide a potential mechanism for the sleep disruptions associated with WFS1 mutation, and deepen understanding of the functional role of wolframin in the brain (Hao, 2023).

    Sleep problems have been reported in WS1 patients. Their scores on Pediatric Sleep Questionnaire are more than 3 times higher than healthy controls and doubled compared to individuals with type I diabetes, indicating that the sleep issues are not merely due to metabolic disruptions. Indeed, this study suggests that the sleep problems in human patients are of neural origin, specifically in the wake-promoting Dop2R neurons. Given that the rebound sleep is not significantly altered in wfs1 depleted flies, it is believed that lack of wfs1 does not shorten sleep duration by impairing the sleep homeostasis system. Instead, wfs1 deficiency leads to excessive wakefulness which in turn results in decreased sleep. Considering that heterozygous WFS1 mutation is present in up to 1% of the population, it would be interesting to examine whether these heterozygous mutations contribute to sleep disruptions in the general population (Hao, 2023).

    In mouse, chick, quail and turtle, Wfs1 has been shown to be expressed in brain regions where dopamine receptor Drd1 is expressed. D1-like dopamine receptor binding is increased while striatal dopamine release is decreased in Wfs1-/- mice. The current results also implicate a role for wolframin in dopamine receptor neurons and that lack of wfs1 impacts dopaminergic signaling, as the effects of wfs1 deficiency on both sleep and mushroom body (MB) calcium concentration is blocked by the tyrosine hydroxylase inhibitor AMPT. Both Dop2RGAL4 and GoαGAL4 exhibit prominent expression in the MB, and to be more specific, in the α and β lobes of MB. Previous studies have shown that dopaminergic neurons innervate wake promoting MB neurons, and this study found Dop2R and Goα+ cells to be wake-promoting as well. Therefore, it is suspected that wolframin functions in MB Dop2R/Goα+ neurons to influence sleep. Taken together, these findings suggest an evolutionarily conserved role of wolframin within the dopaminergic system. As this system is also important for sleep/wake regulation in mammals, it is reasonable to suspect that wolframin functions in mammals to modulate sleep by influencing the dopaminergic tone as well (Hao, 2023).

    MB neural activity appears to be enhanced in wfs1 deficient flies based on the results obtained using CaLexA and spH reporters. This elevated activity is consistent with behavioral data, as activation of Dop2R/Goα+ cells reduces sleep, similar to the effects of wfs1 deficiency. Moreover, silencing Dop2R neurons rescues the short-sleep phenotype of wfs1 mutants, while over-expressing wfs1 restores the decreased sleep induced by activation of Dop2R neurons. These findings suggest that wolframin functions to suppress the excitability of MB Dop2R neurons, which in turn reduces wakefulness and promotes sleep. Comparable cellular changes have been observed in SERCA mutant flies. Electric stimulation leads to an initial increase followed by prolonged decrease of calcium concentration in mutant motor nerve terminal compared to the control, while action potential firing is increased in the mutants. This series of results underpin the importance of ER calcium homeostasis in determining membrane excitability and thus neural function (Hao, 2023).

    GCaMP6 monitoring reveals that wfs1 deficiency selectively reduces fluorescence signal in the MB both under baseline condition and after dopamine treatment, which should reflect a reduction of cytosolic calcium level that is usually associated with decreased excitability. Previous studies have shown that lack of wolframin leads to increased basal calcium level in neural progenitor cells derived from induced pluripotent stem (iPS) cells of WS1 patients and primary rat cortical neurons, but after stimulation the rise of calcium concentration is smaller in Wfs1 deficient neurons, resulting in reduced calcium level compared to controls. Similarly, evoked calcium increase is also diminished in fibroblasts of WS1 patients and MIN6 insulinoma cells with WFS1 knocked down. Notably, wolframin has been shown to bind to calmodulin (CaM) in rat brain, and is capable of binding with calcium/CaM complex in vitro and in transfected cells. This may undermine the validity of using GCaMP to monitor calcium level in wfs1 deficient animals and cells, and could potentially account for the contradictory data acquired using CaLexA vs GCaMP (Hao, 2023).

    It is intriguing that in this study wfs1 deficiency appears to selectively impair the function of Dop2R/Goα+ neurons. It has been shown that in the rodent brain Wfs1 is enriched in layer II/III of the cerebral cortex, CA1 field of the hippocampus, central extended amygdala, striatum, and various sensory and motor nuclei in the brainstem. Wfs1 expression starts to appear during late embryonic development in dorsal striatum and amygdala, and the expression quickly expands to other regions of the brain at birth. It is suspect that in flies wfs1 may be enriched in Dop2R/Goα+ cells during a critical developmental period, and that sufficient level of wolframin is required for their maturation and normal functioning in adults. Another possibility is that these cells are particularly susceptible to calcium dyshomeostasis induced by loss of wfs1. Indeed, this is believed to be an important cause of selective dopaminergic neuron loss in Parkinson's Disease, as dopaminergic neurons are unique in their autonomic excitability and selective dependence on calcium channel rather than sodium channel for action potential generation. It is reasoned that Dop2R/Goα+ neurons may also be more sensitive to abnormal intracellular calcium concentration, making them particularly vulnerable to wfs1 deficiency. The pathogenic mechanism underlying the neurodegeneration of WS1 is quite complex, possibly involving brain-wide neurodegenerative processes and neurodevelopmental dis-regulations. The findings of this study provide some evidence supporting a role for altered dopaminergic system during development. Obviously, much more needs to be done to test these hypotheses (Hao, 2023).

    The precise role of wolframin in ER calcium handling is not yet clear. It has been shown in human embryonic kidney (HEK) 293 cells that knocking down WFS1 reduces while over-expressing WFS1 increases ER calcium level. The authors concluded that wolframin upregulates ER calcium concentration by increasing the rate of calcium uptake. Consistently, this study found by genetic interaction that knocking down RyR or Itpr (which act to reduce ER calcium level and thus knocking down either one will increase ER calcium level) rescues the short-sleep phenotype caused by wfs1 mutation, while knocking down SERCA (which acts to increase ER calcium level and thus knocking down this gene will reduce ER calcium level) synergistically enhances the short-sleep phenotype. Based on the results of these genetic interactions, it is proposed that lack of wfs1 increases cytosolic calcium while decreasing ER calcium, leading to hyperexcitability of Dop2R neurons and thus reduced sleep. Knocking down RyR or Itpr decreases cytosolic calcium and increases ER calcium, counteracting the influences of wfs1 deficiency and thus rescuing the short-sleep phenotype. On the other hand, knocking down SERCA further increases cytosolic calcium and decreases ER calcium, rendering an enhancement of the short-sleep phenotype. In line with this, study conducted in neural progenitor cells derived from iPS cells of WS1 patients demonstrated that pharmacological inhibition of RyR can prevent cell death caused by WFS1 mutation. In addition, inhibiting the function of IP3R may mitigate ER stress in wolframin deficient cells. One caveat is that SERCA protein level is increased in primary islets isolated from Wfs1 conditional knock-out mice, as well as in MIN6 cells and neuroblastoma cell line with WFS1 knocked down. It is reasoned that this may be a compensatory increase to make up for the reduced ER calcium level due to wolframin deficiency. It is acknowledged that the hypothesis proposed in in the papert is not supported by GCaMP data, which indicates decreased cytosolic calcium level in Dop2R neurons of wfs1 deficient flies. It is suspected that since the sleep phenotype associated with lack of wfs1 is of developmental origin, it is possible there is an initial increase of cytosolic calcium during critical developmental period in wfs1 deficient flies and this influences the function of Dop2R neurons in adults. Clearly, further characterizations need to be done to fully elucidate this issue, and preferably another calcium indicator independent of the GCaMP system should be employed (Hao, 2023).

    In conclusion, this study identified a role for wolframin in the wake-promoting Dop2R neurons. wfs1 depletion in these cells lead to impaired calcium homeostasis and altered neural activity, which in turn leads to enhanced wakefulness and reduced sleep. This study may provide some insights for the mechanisms underlying the sleep disruptions in individuals with WFS1 mutation, as well as for the pathogenesis of WS1 (Hao, 2023).

    PINK1 and Parkin regulate IP(3)R-mediated ER calcium release

    Although defects in intracellular calcium homeostasis are known to play a role in the pathogenesis of Parkinson's disease (PD), the underlying molecular mechanisms remain unclear. This study shows that loss of PTEN-induced kinase 1 (PINK1) and Parkin leads to dysregulation of inositol 1,4,5-trisphosphate receptor (IP(3)R) activity, robustly increasing ER calcium release. In addition, CDGSH iron sulfur domain 1 (CISD1, also known as mitoNEET) functions were identifed downstream of Parkin to directly control IP(3)R. Both genetic and pharmacologic suppression of CISD1 and its Drosophila homolog CISD (also known as Dosmit) restore the increased ER calcium release in PINK1 and Parkin null mammalian cells and flies, respectively, demonstrating the evolutionarily conserved regulatory mechanism of intracellular calcium homeostasis by the PINK1-Parkin pathway. More importantly, suppression of CISD in PINK1 and Parkin null flies rescues PD-related phenotypes including defective locomotor activity and dopaminergic neuronal degeneration. Based on these data, it is proposed that the regulation of ER calcium release by PINK1 and Parkin through CISD1 and IP(3)R is a feasible target for treating PD pathogenesis (Ham, 2023).

    This study provides a new insight into the mechanistic connection between the dysregulation of intracellular calcium homeostasis and PD pathogenesis induced by PINK1 or Parkin deficiency. PINK1 or Parkin KO mammalian cells exhibit increased IP3R activity, leading to increased ER calcium release and cytosolic calcium levels. CISD1, a substrate of Parkin, directly controls IP3R activity and ER calcium release, indicating that PINK1, Parkin, CISD1, and IP3R all function in the same essential pathway that regulates ER and cytosolic calcium homeostasis. Loss of CISD or treatment with the CISD inhibitor pioglitazone restores the elevated ER calcium release in PINK1 and Parkin null flies and fully rescues their PD-related phenotypes. Taken together, the increased IP3R activity and ER calcium release caused by PINK1 and Parkin deficiency are key to PD pathogenesis, all of which can be rescued by suppression of CISD1 activity (Ham, 2023).

    In humans, there are three isoforms of CISD, CISD1, CISD2 (also known as ERIS, Miner1, NAF-1, WFS2, and ZCD2), and CISD3 (also called as MiNT). Among the three isoforms, CISD1 and CISD2 contain a single CDGSH domain and a transmembrane domain that facilitates their anchoring to the outer membrane of mitochondria and the ER, respectively. These two isoforms form homodimers within their respective organelles. CISD3 functions as a monomer and contains two CDGSH domains. CISD3 localizes specifically to the mitochondrial matrix. However, no isoforms exist in Drosophila CISD and this single protein shows sequence similarity with both human CISD1 and CISD2. Isoform CISD1 was selected for the experiment, as CISD1 is a much better substrate for Parkin E3 ligase compared to CISD2. Furthermore, it is well known that Parkin localizes to the mitochondria upon its activation and subsequently, ubiquitinates mitochondrial protein substrates. When subcellular localization of human CISD1/2 and Drosophila CISD proteins were observed, human CISD1 and Drosophila CISD were localized in the mitochondria; however, human CISD2 was localized in the ER. Considering these points, human CISD1 was selected as the mammalian counterpart of Drosophila CISD and the experiments were performed accordingly. Interestingly, it has been demonstrated previously that CISD2 is required for BCL2 to suppress IP3R activity. Thus, this study on the regulatory mechanism of IP3R activity through CISD1 is distinct from the earlier study on the regulation of IP3R activity by CISD2. Despite the structural and functional similarities between CISD1 and CISD2, the two proteins have distinct subcellular localizations and are involved in roles independent of one another, due to their interactions with different proteins. Overall, the results of the prior and current studies suggest that both CISD1 and CISD2 are modulators of IP3R activity, but they do so via their unique mechanisms that are distinctive of each other. (Ham, 2023).

    Previous studies reported that CISD1 is involved in iron homeostasis and the downregulation of CISD1 causes iron accumulation and ROS production in mitochondria. In light of these effects, ROS levels were measured in PINK1 and Parkin WT or KO mammalian cells and Drosophila, and an increase was confirmed in ROS levels in PINK1 and Parkin KO MEF cells and Drosophila. An increase was also observed in ROS levels in CISD KD and KO flies, compared to control flies. Interestingly, CISD1/CISD KD or KO in PINK1 and Parkin KO cells and Drosophila resulted in similar ROS levels compared to PINK1 and Parkin KO cells and Drosophila. Furthermore, the increased ROS levels in PINK1 or Parkin KO cells and Drosophila were not restored when CISD1/CISD was knocked down or deleted. These results implicated that the rise in ROS levels induced by loss of CISD1/CISD is not directly involved in the rescue of PD phenotypes, which was observed in CISD1/CISD loss-of-function experiments (Ham, 2023).

    The Fe-S binding capability of CISD1 may play a role in its interactions with IP3R. CISD1 has been reported to interact with several proteins, including CISD2, VDAC1, and transferrin receptor (TfR). CISD249, VDAC115,50, and TfR51 proteins have been shown to interact with each other, and this interaction has been implicated in the regulation of iron homeostasis, redox signaling, and Fe-S cluster synthesis in the mitochondria. However, whether the Fe-S binding motif of CISD1 plays an essential role in protein-protein interactions is unclear. Whether the functions of CISD1 related to Fe-S binding are important to regulate IP3R activity were tested, and it was identified that the cysteine 74 residue in the Fe-S binding motif (in the CDGSH domain) of CISD1 is critical for the interaction with IP3R1. However, it was also confirmed that the Fe-S binding motif of CISD1 binds with IP3R despite C72A substitution and CDGSH pentapeptide deletion mutations. Thus, it was postulate that the structural change in the Fe-S binding motif of CISD1 does not affect the binding between CISD1 and IP3R and that pioglitazone reduces the binding of CISD1 with IP3R regardless of the stability of Fe-S binding. Altogether, the Fe-S binding ability of CISD1 is not directly related to regulating IP3R activity (Ham, 2023).

    Flies with either CISD RNAi or CISD KO exhibited lower ER calcium release and cytosolic calcium levels compared to mef2-GAL4 control flies. Upon crossing with CISD RNAi or CISD KO flies, PINK1 or Parkin null flies displayed a greater reduction in ER calcium release and cytosolic calcium levels than mef2-GAL4 control flies. This observation can be explained by the varying amounts of endogenous CISD in the flies. Notably, CISD RNAi flies exhibited significantly lower endogenous CISD amounts compared to the control flies, while PINK1 or Parkin KO flies presented higher levels. The elevated amount of endogenous CISD protein in PINK1 or Parkin KO flies contributes to the increased ER calcium release observed in these flies, while the reduced endogenous CISD protein in CISD RNAi or CISD KO flies results in a more significant decrease in ER calcium release compared to the control flies. Moreover, flies resulting from the crossing of PINK1/Parkin KO with CISD RNAi/CISD KO demonstrated lower endogenous CISD levels compared to the control flies, leading to a larger reduction in ER calcium release or cytosolic calcium levels. Collectively, these findings proposed that ER calcium release and cytosolic calcium levels are modulated proportionally to the amount of endogenous CISD protein present (Ham, 2023).

    Defects in ER calcium homeostasis can also have profound effects on other organelles through physical contact sites, including the ER-mitochondria interconnections known as Mitochondria-associated membranes (MAMs). MAMs are enriched with the MCU complex in the inner mitochondrial membrane and IP3R on the ER membrane. MCU and IP3R are coupled via the glucose-regulated protein 75 (Grp75), which links IP3R to the VDAC1 on the outer mitochondrial membrane, establishing connections that allow calcium exchange between the ER and mitochondria. Interestingly, previous studies show that inhibition of MCU or VDAC1 partially rescues the PD phenotypes of PINK1- and Parkin-deficient flies, suggesting that the disruption of MAMs may alleviate PD pathogenesis. Previous studies also report that the level of MAM contacts was increased in cultured human fibroblasts from PD patients carrying PINK1 or Parkin pathogenic mutations and PINK1 and Parkin null mutant flies60. In addition, our present results demonstrate that CISD1 directly binds to and regulates IP3R activity, and CISD1 is localized at MAMs and the mitochondrial outer membrane12. These data therefore suggest that CISD1 and the PINK1-Parkin pathway are crucial for the formation and maintenance of MAM structure and ER-mitochondrial calcium transduction, which in turn are critical for mitochondria-related physiology and pathologic phenotypes including calcium-dependent metabolic changes, ROS production, mitophagy, mitochondrial permeability transition, and apoptosis (Ham, 2023).

    Through extensive studies, it is understood that loss of PINK1 or Parkin impairs mitophagy and that defective mitophagy is one of the potential contributing factors to the onset of PD. Furthermore, a recent study has shown increased mitophagy in thoraces and neurons of CISD KO or KD Drosophila, and the reduced mitophagy in PINK1 or Parkin KO flies was alleviated by crossing them with CISD KO or KD flies. In the current study, it was observed that loss of CISD1/CISD reduced the elevated cytosolic calcium levels observed in PINK1 or Parkin KO cells and Drosophila. Intracellular calcium signaling is an important factor in mitophagy regulation. Nix, also known as BCL2 interacting protein 3 like (BNIP3L), exhibits biological activity at both the mitochondria and the ER. At the mitochondria, Nix functions as a selective autophagy receptor, facilitating the recruitment of LC3B71. In muscle, during a mitophagy response, Nix promotes ER-dependent calcium signaling to activate the mitochondrial fission regulator dynamin-related protein 1 (DRP1), indicating the contribution of Nix to mitophagy. During hypoxia, mitochondrial Lon protein promotes FUNDC1-ULK1-mediated mitophagy at the MAMs, which depends on its binding with mitochondrial Na+/Ca2+ exchanger (NCLX). This interaction stabilizes the FUNDC1-ULK1 at the MAMs and initiates the mitophagy by regulating calcium levels between the mitochondria and cytosol. This process occurs independently of PINK1 and Parkin. Furthermore, other calcium-sensitive proteins and pathways may also contribute to PINK1-Parkin-independent mitophagy. For example, CaMKII-AMP-activated protein kinase (AMPK) pathway has been implicated in the regulation of mitophagy. Activation of AMPK by CaMKII can promote mitophagy by phosphorylating and activating proteins involved in autophagy initiation. This suggests the possibility that mitophagy could be activated by the decreased cytosolic calcium levels in CISD1/CISD KO or KD cells and Drosophila. Collectively, the regulation of mitophagy by CISD1/CISD holds the potential to alleviate PD pathogenesis caused by loss of PINK1 or Parkin. However, further investigation is required to unravel the molecular mechanism underlying mitophagy regulation by CISD1 and its interplay with intracellular calcium signaling (Ham, 2023).

    Although degeneration of DA neurons is known to occur in PD, how such selective neurodegeneration occurs remains unknown. The current results show that DA neuronal loss and locomotor impairments in PINK1 and Parkin KO flies can be rescued by adjusting ER calcium release, suggesting that ER and mitochondrial calcium dysregulation may cause selective DA neuronal death. Intracellular calcium signaling in DA neurons is extremely fine-tuned as it controls many cellular processes including gene transcription, membrane excitability, dopamine neurotransmitter secretion, and synaptic plasticity. Furthermore, energy production in neurons is tightly regulated by ER and mitochondrial calcium. DA neurons promote mitochondria calcium influx from the ER to stimulate OXPHOS and the production of ATP. This bioenergetic control system is costly, as enhancing OXPHOS in the absence of strong ATP demand leads to mitochondrial hyperpolarization, retrograde electron flux through the electron transport chain, and increased production of ROS. Therefore, continuous dysregulation of calcium homeostasis in DA neurons along with exposure to risk factors (i.e., aging, mitochondrial toxins, mutations) may selectively induce metabolic stress and mitochondrial damages, leaving DA neurons more vulnerable than other neuronal populations to death (Ham, 2023).

    While the importance of calcium regulation in PD pathogenesis has been recognized, previous trials of calcium-related drugs had failed to improve symptoms in PD patients. This study proposes that pioglitazone, a thiazolidinedione (TZD) and antidiabetic drug, can alleviate PD pathogenesis. Though previous clinical studies have reported mixed results on the effectiveness of pioglitazone against PD, the results clearly demonstrate that feeding pioglitazone to flies rescues PD-related phenotypes induced by PINK1 or Parkin deficiency. In addition, pioglitazone treatment reverses the increased ER calcium release and cytosolic calcium levels in PINK1 and Parkin KO MEF cells. This study thus established that pioglitazone can specifically protect PD pathogenesis caused by dysregulation of intracellular calcium homeostasis, calling for future clinical studies of pioglitazone and its analogs to be conducted specifically on PD patients that harbor PINK1 or Parkin mutations (Ham, 2023).

    Cholinergic neurons trigger epithelial Ca(2+) currents to heal the gut

    A fundamental and unresolved question in regenerative biology is how tissues return to homeostasis after injury. Answering this question is essential for understanding the aetiology of chronic disorders such as inflammatory bowel diseases and cancer. This study used the Drosophila midgut to investigate this and discovered that during regeneration a subpopulation of cholinergic neurons triggers Ca(2+) currents among intestinal epithelial cells, the enterocytes, to promote return to homeostasis. It was found that downregulation of the conserved cholinergic enzyme Acetylcholine esterase in the gut epithelium enables acetylcholine from specific Eiger (TNF in mammals)-sensing cholinergic neurons to activate nicotinic receptors in innervated enterocytes. This activation triggers high Ca(2+), which spreads in the epithelium through Innexin2-Innexin7 gap junctions, promoting enterocyte maturation followed by reduction of proliferation and inflammation. Disrupting this process causes chronic injury consisting of ion imbalance, Yki (YAP in humans) activation, cell death and increase of inflammatory cytokines reminiscent of inflammatory bowel disease. Altogether, the conserved cholinergic pathway facilitates epithelial Ca(2+) currents that heal the intestinal epithelium. These findings demonstrate nerve- and bioelectric-dependent intestinal regeneration and advance current understanding of how a tissue returns to homeostasis after injury (Petsakou, 2023).


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    Zygotically transcribed genes

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