Immunofluorescence as a Method to Study Golgi Organization in Larval Salivary Glands of Drosophila melanogaster.

Immunofluorescence is an important research tool in cell biology that reveals structural organization of subcellular organelles by detecting their associated constituents. Here, we describe an antibody staining method to detect Golgi-associated proteins in Drosophila larval salivary glands, using the cis-Golgi protein Lava lamp and the clathrin adaptor AP-1 as a suitable example. Golgi bodies immunostained using this protocol can be visualized using confocal or structured illumination microscopy.

Mutant IP3 receptors attenuate store-operated Ca2+ entry by destabilizing STIM-Orai interactions in Drosophila neurons.

Store-operated Ca2+ entry (SOCE) occurs when loss of Ca2+ from the endoplasmic reticulum (ER) stimulates the Ca2+ sensor, STIM, to cluster and activate the plasma membrane Ca2+ channel Orai (encoded by Olf186-F in flies). Inositol 1,4,5-trisphosphate receptors (IP3Rs, which are encoded by a single gene in flies) are assumed to regulate SOCE solely by mediating ER Ca2+ release. We show that in Drosophila neurons, mutant IP3R attenuates SOCE evoked by depleting Ca2+ stores with thapsigargin. In normal neurons, store depletion caused STIM and the IP3R to accumulate near the plasma membrane, association of STIM with Orai, clustering of STIM and Orai at ER-plasma-membrane junctions and activation of SOCE. These responses were attenuated in neurons with mutant IP3Rs and were rescued by overexpression of STIM with Orai. We conclude that, after depletion of Ca2+ stores in Drosophila, translocation of the IP3R to ER-plasma-membrane junctions facilitates the coupling of STIM to Orai that leads to activation of SOCE.

The genetics of calcium signaling in Drosophila melanogaster.

BACKGROUND: Genetic screens for behavioral and physiological defects in Drosophila melanogaster, helped identify several components of calcium signaling of which some, like the Trps, were novel. For genes initially identified in vertebrates, reverse genetic methods have allowed functional studies at the cellular and systemic levels. SCOPE OF REVIEW: The aim of this review is to explain how various genetic methods available in Drosophila have been used to place different arms of Ca2+ signaling in the context of organismal development, physiology and behavior. MAJOR CONCLUSION: Mutants generated in genes encoding a range of Ca2+ transport systems, binding proteins and enzymes affect multiple aspects of neuronal and muscle physiology. Some also affect the maintenance of ionic balance and excretion from malpighian tubules and innate immune responses in macrophages. Aspects of neuronal physiology affected include synaptic growth and plasticity, sensory transduction, flight circuit development and function. Genetic interaction screens have shown that mechanisms of maintaining Ca2+ homeostasis in Drosophila are cell specific and require a synergistic interplay between different intracellular and plasma membrane Ca2+ signaling molecules. GENERAL SIGNIFICANCE: Insights gained through genetic studies of conserved Ca2+ signaling pathways have helped understand multiple aspects of fly physiology. The similarities between mutant phenotypes of Ca2+ signaling genes in Drosophila with certain human disease conditions, especially where homologous genes are causative factors, are likely to aid in the discovery of underlying disease mechanisms and help develop novel therapeutic strategies. This article is part of a Special Issue entitled Biochemical, biophysical and genetic approaches to intracellular calcium signalling.