Recombinant Drosophila melanogaster Stromal interaction molecule homolog (Stim), partial

What is the molecular nature of Drosophila Stromal Interaction Molecule (Stim)?

Drosophila STIM (dSTIM) is an ER-resident transmembrane protein that functions as a calcium sensor. When ER calcium stores are depleted, dSTIM undergoes dimerization and structural rearrangements that facilitate binding to Orai, a plasma membrane calcium channel. This interaction activates Store-operated Calcium entry (SOCE). The Drosophila genome contains a single STIM homolog that is structurally similar to both mammalian STIM1 and STIM2 . In contrast to mammals with multiple STIM proteins, Drosophila has one dSTIM that interacts with a single dOrai protein (olf186-F) to activate calcium entry through SOC channels .

How does dSTIM function in calcium homeostasis?

In resting cells, cytosolic Ca2+ concentration is maintained at low levels through active sequestration into organelles, primarily the ER. Upon activation, ligand-activated Ca2+ channels on the ER (such as IP3R) release ER-store Ca2+ into the cytosol. This loss of ER-Ca2+ causes dSTIM to dimerize and undergo structural rearrangements, facilitating binding to Orai on the plasma membrane . This interaction triggers an influx of extracellular Ca2+ into the cytosol, a process known as Store-operated Ca2+ entry (SOCE). This mechanism is crucial for maintaining calcium homeostasis and proper cellular signaling .

What developmental roles does dSTIM play in Drosophila?

dSTIM plays essential roles in multiple developmental processes:

• Larval development and survival: dSTIM is required for normal larval development, particularly under nutrient restriction conditions .

• Imaginal disc development: dSTIM regulates growth and patterning of imaginal discs, suggesting interactions with key developmental pathways such as Notch and Wingless signaling .

• Tissue-specific functions: dSTIM is involved in specification of mechanosensory bristles in the notum and determination of wing vein thickness .

• Neuropeptide regulation: dSTIM controls neuropeptides required for development under nutrient restriction, including Corazonin and short Neuropeptide F .

What expression patterns does dSTIM exhibit during development?

dSTIM is widely expressed throughout Drosophila development. Expression studies have demonstrated that dSTIM is present in both embryonic and larval tissues . In particular, dSTIM is found in the dorsolateral peptidergic (DLP) neurons, which co-express neuropeptides Corazonin and short Neuropeptide F . The broad expression pattern reflects the diverse roles of dSTIM in multiple tissues and developmental stages.

What phenotypes are associated with dSTIM dysfunction?

Partial loss of dSTIM function results in several phenotypes:

• Accumulation of neuropeptides: Reduced dSTIM causes peptide accumulation in DLP neurons and decreased systemic Corazonin signaling .

• Developmental defects: dSTIM dysfunction leads to defects in specification of mechanosensory bristles and abnormal wing vein thickness .

• Impaired nutrient response: Under nutrient restriction, larvae with reduced dSTIM fail to show the normal increase in peptide levels in DLP neurons, compromising their ability to complete development .

What methodological approaches can be used to study dSTIM function in neuropeptide regulation?

To investigate dSTIM's role in neuropeptide regulation, researchers can employ multiple approaches:

• UAS-GAL4 expression system: Generate transgenic Drosophila expressing full-length UAS-dSTIM cDNA or UAS-dSTIM RNAi constructs under the control of tissue-specific GAL4 drivers . This allows manipulation of dSTIM levels in specific cell types, including neuropeptide-producing neurons.

• Immunohistochemistry: Quantify neuropeptide levels (such as Corazonin and sNPF) in specific neuronal populations using antibody staining .

• Genetic rescue experiments: Test whether wild-type dSTIM can rescue phenotypes in dSTIM mutant backgrounds, particularly focusing on neuropeptide levels and developmental outcomes .

• Calcium imaging: Monitor calcium dynamics in neuropeptide-producing neurons using genetically encoded calcium indicators to assess how dSTIM affects calcium signaling patterns that may regulate peptide release .

• Behavioral assays: Examine feeding behavior, development timing, and stress responses in dSTIM mutants to correlate cellular phenotypes with organismal outcomes .

How can researchers generate and utilize recombinant dSTIM for functional studies?

For generating recombinant dSTIM:

• Construct design: Clone the full-length or partial dSTIM cDNA into appropriate expression vectors. Consider including epitope tags (e.g., His, FLAG) for purification and detection.

• Expression systems:

  • Bacterial expression (E. coli) for structural domains

  • Insect cell expression (Sf9, S2 cells) for full-length protein with proper folding and modifications

  • Cell-free translation systems for rapid protein production

• Functional testing: Assess protein activity through:

  • In vitro calcium binding assays

  • Pull-down experiments with dOrai

  • Liposome reconstitution systems to study membrane interactions

• Structure-function analysis: Generate truncation and point mutations to map functional domains and critical residues for calcium sensing, dimerization, and Orai binding.

What experimental strategies can reveal dSTIM's interactions with developmental signaling pathways?

To investigate dSTIM's interactions with Notch, Wingless, and other signaling pathways:

• Genetic modifier screens: Test whether mutations in signaling pathway components enhance or suppress dSTIM-related phenotypes . This approach has been powerful in identifying functional interactions in Drosophila, as demonstrated by studies on Notch pathway components .

• Transcriptome analysis: Perform RNA-seq on tissues with altered dSTIM expression to identify changes in signaling pathway target genes.

• Double mutant analysis: Generate flies carrying both dSTIM mutations and mutations in candidate pathway components to assess epistatic relationships.

• Reporter assays: Use pathway-specific transcriptional reporters (e.g., Notch response elements driving GFP) to visualize signaling activities in dSTIM mutant backgrounds.

• Protein interaction studies: Conduct co-immunoprecipitation or proximity ligation assays to detect physical interactions between dSTIM and signaling pathway components.

How does dSTIM function in nutrient-dependent developmental regulation?

The role of dSTIM in nutrient-dependent development can be investigated through:

• Nutrient restriction protocols: Implement standardized protocols to subject larvae to defined nutrient restriction conditions and monitor developmental outcomes with varying dSTIM levels .

• Metabolic measurements: Assess changes in energy metabolism (e.g., ATP levels, lipid storage) in dSTIM mutants under normal and restricted nutrient conditions.

• Neuropeptide signaling analysis:

  • Quantify Corazonin and sNPF levels in DLP neurons under different nutritional states

  • Assess downstream signaling of these neuropeptides using receptor activity assays or phospho-specific antibodies

  • Determine if dSTIM primarily affects peptide synthesis, transport, or release

• Genetic epistasis experiments: Test whether overexpression of Corazonin or sNPF can rescue developmental defects in dSTIM mutants under nutrient restriction.

What methods can be used to study dSTIM's role in hypoxia tolerance?

Given the potential connection between calcium signaling and hypoxia response:

• Hypoxia exposure systems: Develop controlled O2 environments to test survival of dSTIM mutants under varying hypoxic conditions, similar to the approach used to select hypoxia-tolerant Drosophila strains .

• Gene expression analysis: Compare transcriptional responses to hypoxia between wild-type and dSTIM mutant flies, focusing on known hypoxia-responsive genes.

• Genetic interaction studies: Test interactions between dSTIM and hypoxia-tolerance genes identified through selection experiments (e.g., Best1, broad, CG7102, dunce, lin19-like, sec6) .

• Calcium imaging during hypoxia: Monitor cellular calcium dynamics during hypoxic challenge in wild-type versus dSTIM mutant tissues to correlate calcium signaling with hypoxic adaptations.

• Transgenic rescue experiments: Determine if tissue-specific expression of dSTIM can restore hypoxia tolerance in mutant backgrounds.

What techniques are available for analyzing calcium dynamics in dSTIM mutants?

Advanced calcium imaging approaches include:

• Genetically encoded calcium indicators (GECIs): Express GCaMP or other fluorescent calcium sensors in specific tissues of interest in dSTIM mutant backgrounds.

• Ex vivo tissue preparations: Isolate tissues (e.g., neurons, imaginal discs) from dSTIM mutants for calcium imaging under controlled stimulation conditions.

• Optogenetic stimulation: Combine calcium imaging with optogenetic tools to precisely trigger calcium release from ER stores and monitor SOCE in real-time.

• Patch-clamp electrophysiology: Record CRAC (Calcium Release-Activated Calcium) currents in dSTIM mutant cells to directly measure SOCE function.

• Pharmacological manipulations: Use compounds that deplete ER calcium (thapsigargin, ionomycin) or block specific calcium channels to dissect the role of dSTIM in different calcium signaling pathways.

How can researchers distinguish between dSTIM's roles in neuropeptide synthesis versus release?

To differentiate between effects on synthesis and release:

• Quantitative microscopy: Compare the ratio of cell body to axon terminal neuropeptide staining intensity as a measure of peptide transport and release defects .

• Pulse-chase experiments: Use conditional expression systems to pulse-express tagged neuropeptide precursors and track their processing and release over time.

• Secretion assays: Collect hemolymph from larvae and quantify secreted neuropeptides using ELISA or mass spectrometry.

• Transcriptional analysis: Measure mRNA levels of neuropeptide genes (Corazonin, sNPF) to determine if transcriptional regulation is affected by dSTIM.

• Vesicle dynamics: Image neuropeptide vesicle trafficking and fusion events using tagged vesicle proteins in combination with dSTIM manipulations.

The available evidence suggests that dSTIM primarily compromises neuroendocrine function by interfering with neuropeptide release, though under chronic stimulation, it also appears to regulate neuropeptide synthesis .

What genetic tools are most effective for tissue-specific manipulation of dSTIM?

For precise manipulation of dSTIM expression:

• GAL4-UAS system: Utilize the extensive collection of GAL4 driver lines to express UAS-dSTIM or UAS-dSTIM-RNAi in specific tissues or cell types . This approach has been successfully used to study dSTIM functions in various contexts.

• Split-GAL4 approach: For even more precise expression patterns, use split-GAL4 lines that restrict expression to the intersection of two expression domains.

• Temperature-sensitive GAL80: Incorporate GAL80ts to achieve temporal control over dSTIM manipulation, allowing stage-specific studies.

• CRISPR-Cas9 genome editing: Generate precise mutations or tagged versions of endogenous dSTIM for functional studies at physiological expression levels.

• FLP-FRT system: Create mosaic animals with clones of dSTIM mutant cells in otherwise wild-type tissues to study cell-autonomous functions.

Rescue experiments in which dSTIM expression is restored specifically in THD' marked neurons have demonstrated the effectiveness of the GAL4-UAS system for dissecting dSTIM function in particular neuronal populations .

Key Molecular Interactions of dSTIM