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Intracellular Ca Signaling and Store-Operated Ca Entry Are Required in Drosophila Neurons for Flight

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Intracellular Ca Signaling and Store-Operated Ca Entry Are Required in Drosophila Neurons for Flight Intracellular Ca2؉ signaling and store-operated Ca2؉ entry are required in Drosophila neurons for flight Gayatri Venkiteswaran and Gaiti Hasan1 National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bellary Road, Bangalore 560065, India Edited by Michael Rosbash, Brandeis University, Waltham, MA, and approved April 22, 2009 (received for review March 23, 2009) -Neuronal Ca2؉ signals can affect excitability and neural circuit for mation. Ca2؉ signals are modified by Ca2؉ flux from intracellular stores as well as the extracellular milieu. However, the contribution of intracellular Ca2؉ stores and their release to neuronal processes is poorly understood. Here, we show by neuron-specific siRNA deple- tion that activity of the recently identified store-operated channel encoded by dOrai and the endoplasmic reticulum Ca2؉ store sensor encoded by dSTIM are necessary for normal flight and associated patterns of rhythmic firing of the flight motoneurons of Drosophila melanogaster. Also, dOrai overexpression in flightless mutants for the Drosophila inositol 1,4,5-trisphosphate receptor (InsP3R) can par- tially compensate for their loss of flight. Ca2؉ measurements show that Orai gain-of-function contributes to the quanta of Ca2؉-release 2؉ through mutant InsP3Rs and elevates store-operated Ca entry in Drosophila neurons. Our data show that replenishment of intracel- .lular store Ca2؉ in neurons is required for Drosophila flight calcium homeostasis ͉ flight patterns ͉ inositol 1,4,5-trisphosphate receptor ͉ sarco-endoplasmic reticulum-associated Ca2ϩ ATPase ͉ STIM everal aspects of neuronal function are regulated by ionic Scalcium (Ca2ϩ). Specific attributes of a Ca2ϩ‘‘signature’’ such as amplitude, duration, and frequency of the signal can determine the morphology of a neural circuit by affecting the outcome of cell migration, the direction taken by a growth-cone, dendritic devel- opment, and synaptogenesis (1). Ca2ϩ signals also determine the nature and strength of neural connections in a circuit by specifying neurotransmitters and receptors (2). Most of these Ca2ϩ signals have been attributed to the entry of extracellular Ca2ϩ through voltage-operated channels or ionotropic receptors. However, other Fig. 1. Intracellular Ca2ϩ homeostasis in larval neurons is altered on expres- 2ϩ 2ϩ 2ϩ components of the ‘‘Ca tool-kit’’ coupled to Ca release from sion of dsdOrai and dsdSTIM. (A) pseudocolor images represent [Ca ]ER and 2ϩ intracellular Ca stores are also present in neurons. These mole- SOCE in primary cultures of neurons loaded with Fluo-4 from WT larvae and ϩ cules include the store-operated Ca2 (SOC) channel, encoded by those expressing dsdOrai or dsdSTIM. (Scale bar, 10 ␮m.) (B) Single cell traces the Orai gene, identified recently in siRNA screens for molecules of SOCE by Ca2ϩ add-back after store depletion. Two cells of each genotype that reduce or abolish Ca2ϩ influx from the extracellular milieu are shown. Arrow heads represent peak values of response, which have been after intracellular Ca2ϩ store depletion (3–5). Several reports have plotted as a box chart in C.(C) Box plots of ⌬F/F values of SOCE. The bigger confirmed its identity as the pore forming subunit of the Ca2ϩ- boxes represent the data spread, smaller squares represent mean, and the 2ϩ diamonds on either side represent outlier values. (D) Box plot comparison of release activated Ca (CRAC) channel (6–8). Activation of this 2ϩ [Ca ]ER between neurons of indicated genotypes. (E) Kolmogorov–Smirnov CRAC channel is mediated by the endoplasmic reticulum (ER) 2ϩ (K-S) plot analyzing the distribution of [Ca ]i in neurons loaded with Indo-1. resident protein STIM (stromal interaction molecule), also identi- The frequency distribution is significantly shifted to the left for cells with fied in an RNAi screen for molecules that regulate SOC influx (9, dSTIM RNAi (dsdSTIM), indicating a higher frequency of cells with reduced 2ϩ 2ϩ 2ϩ 10). STIM senses the drop in ER Ca levels, and interacts with [Ca ]i (PK-SϽ0.05). (F) Box plot representation of [Ca ]i in neurons with Ն Ͻ Ͻ Orai by a mechanism which is only just being understood (11). Orai dsdOrai or dsdSTIM.(n 150 cells; *, PANOVA 0.05; **, PANOVA 0.01). and STIM function in conjunction with the sarco-endoplasmic 2ϩ 2ϩ reticular Ca -ATPase pump (SERCA) to maintain ER store Ca 2ϩ and basal Ca2ϩ. The importance of intracellular Ca2ϩ homeostasis gene (12). The InsP3R is a ligand gated Ca -channel present on the membranes of intracellular Ca2ϩ stores. It is thought to be critical and SOC entry (E) in neural circuit formation and in neuronal for various aspects of neuronal function (1, 13). Mutants in the gene function and physiology remains to be elucidated. Here, we report how Orai and STIM mediated Ca2ϩ influx and 2ϩ Ca homeostasis in Drosophila neurons contribute to cellular and Author contributions: G.V. and G.H. designed research; G.V. performed research; G.V. and systemic phenotypes. Reduced SOCE measured in primary neu- G.H. contributed new reagents/analytic tools; G.V. analyzed data; and G.V. and G.H. wrote ronal cultures is accompanied by a range of defects in adults, the paper. including altered wing posture, increased spontaneous firing, and The authors declare no conflict of interest. loss of rhythmic flight patterns. These phenotypes mirror the This article is a PNAS Direct Submission. spontaneous hyperexcitability of flight neuro-muscular junctions 1To whom correspondence should be addressed. E-mail: [email protected]. and loss of rhythmic flight patterns observed in Drosophila mutants This article contains supporting information online at www.pnas.org/cgi/content/full/ of the inositol 1,4,5-trisphosphate receptor (InsP3R, itpr, CG1063) 0902982106/DCSupplemental. 10326–10331 ͉ PNAS ͉ June 23, 2009 ͉ vol. 106 ͉ no. 25 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0902982106 Downloaded by guest on September 28, 2021 Fig. 2. RNAi knockdown of dOrai or dSTIM in subsets of neurons gives rise to flight motor defects. Pan- neuronal knockdown of dOrai and dSTIM induces (A) change in wing posture, (B) flight defects (n Ն 100 flies), (C) higher levels of spontaneous firing (n Ն 15 flies), and (D) defects in air-puff-induced flight pat- terns. (E) Representative traces of spontaneous firing activity from the DLMs of the indicated genotypes. Histograms represent mean Ϯ SE; (*, P Ͻ 0.05; Stu- dent’s t test). Both GAL4 control strains were tested and found to be similar to WT. Data shown are for glutamatergic GAL4 flies. 2ϩ coding for the mouse InsP3R1 are ataxic (14). Cerebellar slices from when levels of a plasma membrane localized ligand-gated Ca - InsP3R1 knockout mice show reduced long-term depression, indi- channel are reduced. Pan-neural expression of dsdStim followed by 2ϩ 2ϩ cating that altered synaptic plasticity of the cognate neural circuits Ca imaging revealed significant reduction of SOCE, [Ca ]ER 2ϩ 2ϩ NEUROSCIENCE could underlie the observed ataxia (15). (Fig. 1 B–D), and resting cytosolic Ca ([Ca ]i;Fig.1E and F). 2ϩ To understand the temporal and spatial nature of intracellular A significantly higher frequency of cells with lower [Ca ]i were Ca2ϩ signals required during flight circuit development and func- present among the neuronal population with dsdSTIM. However, 2ϩ tion, dOrai (CG11430) and dSERCA (encoded by CaP-60A gene, dsdOrai expression had no effect on [Ca ]i. The efficacy of the CG3725) function was modulated by genetic means in itpr mutants. dsRNA strains used was ascertained by semiquantitative RT-PCR, This modulation can restore flight to flightless adults, by altering which showed a consistent reduction in the levels of the appropriate several parameters of intracellular Ca2ϩ homeostasis including transcripts (Fig. S1 A–D). As expected, reduction in the level of SOCE. Our results suggest that components of the central pattern dNR1 transcripts did not affect store Ca2ϩ or SOCE (Fig. S1 E and generator (CPG) required for maintenance of normal rhythmic F). These results demonstrate that Ca2ϩ influx, leading to replen- flight in adults have a stringent requirement for SOCE after InsP3R ishment of ER stores through the STIM–Orai pathway, is con- stimulation. served in Drosophila neurons. Also, the single STIM-encoding gene 2ϩ 2ϩ in Drosophila appears to regulate both [Ca ]ER and [Ca ]i.In Results mammalian systems, these cellular properties are regulated inde- SOC Entry in Neurons of Drosophila Requires Orai and STIM. Genes pendently by STIM1 and STIM2, respectively (17). encoding the SOC channel (Orai1) and the store Ca2ϩ sensor (Stim1) are known to maintain intracellular Ca2ϩ store levels Reduced SOCE in Drosophila Neurons Causes Flight Defects. To deter- 2ϩ 2ϩ ([Ca ]ER) in stimulated T cells. The replenishment of [Ca ]ER in mine whether reduced SOCE in Drosophila neurons affects neu- T cells is required for their prolonged activation (16). Homologs of ronal function, motor coordination defects were measured in the mammalian Orai and Stim exist in Drosophila as single genes, and appropriate genotypes. No obvious changes were visible in larvae perform similar cellular functions in S2 cells, where their depletion expressing dsRNA for either dOrai or dSTIM. The larvae were by gene specific double-stranded (ds)RNA leads to abrogation of viable and pupated normally. However, adult flies with pan-neural SOCE (3–5). To investigate SOC channel activity in Drosophila expression of dsdOrai and dsdSTIM had defective wing posture with neurons, we reduced levels of dOrai transcripts using dsRNA in significant loss of flight as seen in the ‘‘cylinder drop’’ test assay (Fig. primary neuronal cultures derived from larval brains. SOCE was 2 A and B) (18). Whereas Ͼ50% flies with dOrai knockdown were monitored by Ca2ϩ imaging of cultured neurons in Ca2ϩ add-back flightless, dSTIM knockdown resulted in a complete loss of flight experiments, after depletion of ER stores with thapsigargin in very (Fig.
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