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Recent studies by our group and others demonstrated a required and conserved role of Stim in store-operated Ca 2+ influx and Ca 2+ release-activated Ca 2+ (CRAC) channel activity. By using an unbiased genome-wide RNA interference screen in Drosophila S2 cells, we now identify 75 hits that strongly inhibited Ca 2+ influx upon store emptying by thapsigargin. Among these hits are 11 predicted transmembrane proteins, including Stim , and one, olf186-F , that upon RNA interference-mediated knockdown exhibited a profound reduction of thapsigargin-evoked Ca 2+ entry and CRAC current, and upon overexpression a 3-fold augmentation of CRAC current. CRAC currents were further increased to 8-fold higher than control and developed more rapidly when olf186-F was cotransfected with Stim . olf186-F is a member of a highly conserved family of four-transmembrane spanning proteins with homologs from Caenorhabditis elegans to human. The endoplasmic reticulum (ER) Ca 2+ pump sarco-/ER calcium ATPase (SERCA) and the single transmembrane-soluble N -ethylmaleimide-sensitive (NSF) attachment receptor (SNARE) protein Syntaxin5 also were required for CRAC channel activity, consistent with a signaling pathway in which Stim senses Ca 2+ depletion within the ER, translocates to the plasma membrane, and interacts with olf186-F to trigger CRAC channel activity.

Dap160/Intersectin is a multidomain adaptor protein that colocalizes with endocytic machinery in the periactive zone at the Drosophila NMJ. We have generated severe loss-of-function mutations that eliminate Dap160 protein from the NMJ. dap160 mutant synapses have decreased levels of essential endocytic proteins, including dynamin, endophilin, synaptojanin, and AP180, while other markers of the active zone and periactive zone are generally unaltered. Functional analyses demonstrate that dap160 mutant synapses are unable to sustain high-frequency transmitter release, show impaired FM4-64 loading, and show a dramatic increase in presynaptic quantal size consistent with defects in synaptic vesicle recycling. The dap160 mutant synapse is grossly malformed with abundant, highly ramified, small synaptic boutons. We present a model in which Dap160 scaffolds both endocytic machinery and essential synaptic signaling systems to the periactive zone to coordinately control structural and functional synapse development.

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.

In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the heterotetrameric adaptor protein AP3 and a small molecular weight GTPase of the ADP ribosylation factor (ARF) family. We have now discovered a second pathway that sorts the synaptic vesicle-associated membrane protein (VAMP) into similarly sized vesicles. For this pathway the plasma membrane is the precursor rather than endosomes. Both pathways require cytosol and ATP and are inhibited by GTPγS. The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive. The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not. The VAMP-containing, plasma membrane–derived vesicles can be readily separated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled plasma membrane preparations at 37°C. Dynamin- interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes. Thus, VAMP is sorted into small vesicles by AP3 and ARF1 at endosomes and by AP2 and clathrin at the plasma membrane.

For efficient development of an immune response, T lymphocytes require long-lasting calcium influx through calcium release-activated calcium (CRAC) channels and the formation of a stable immunological synapse (IS) with the antigen-presenting cell (APC). Recent RNAi screens have identified Stim and Orai inDrosophilacells, and their corresponding mammalian homologs STIM1 and Orai1 in T cells, as essential for CRAC channel activation. Here, we show that STIM1 and Orai1 are recruited to the immunological synapse between primary human T cells and autologous dendritic cells. Both STIM1 and Orai1 accumulated in the area of contact between either resting or super-antigen (SEB)-pretreated T cells and SEB-pulsed dendritic cells, where they were colocalized with T cell receptor (TCR) and costimulatory molecules. In addition, imaging of intracellular calcium signaling in T cells loaded with EGTA revealed significantly higher Ca2+concentration near the interface, indicating Ca2+influx localized at the T cell/dendritic cell contact area. Expression of a dominant-negative Orai1 mutant blocked T cell Ca2+signaling but did not interfere with the initial accumulation of STIM1, Orai1, and CD3 in the contact zone. In activated T cell blasts, mRNA expression for endogenous STIM1 and all three human homologs of Orai was up-regulated, accompanied by a marked increase in Ca2+influx through CRAC channels. These results imply a positive feedback loop in which an initial TCR signal favors up-regulation of STIM1 and Orai proteins that would augment Ca2+signaling during subsequent antigen encounter.

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knock-down of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca 2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels. © The Rockefeller University Press.

Here we report the description of the Drosophila gene futsch, which encodes a protein recognized by the monoclonal antibody 22C10 that has been widely used to visualize neuronal morphology and axonal projections. The Futsch protein is 5327 amino acids in length. It localizes to the microtubule compartment of the cell and associates with microtubules in vitro. The N- and C-terminal domains of Futsch are homologous to the vertebrate MAP1B microtubule-associated protein. The central domain of the Futsch protein is highly repetitive and shows sequence similarity to neurofilament proteins of which no Drosophila homologs have been reported. Loss-of-function analyses demonstrate that during embryogenesis Futsch is necessary for dendritic and axonal growth. Gain-of-function analyses demonstrate a functional interaction of Futsch with other MAPs. In addition, we show that during development, futsch expression is negatively regulated in nonneuronal tissues.

We present evidence that Futsch, a novel protein with MAP1B homology, controls synaptic growth at the Drosophila neuromuscular junction through the regulation of the synaptic microtubule cytoskeleton. Futsch colocalizes with microtubules and identifies cytoskeletal loops that traverse the lateral margin of select synaptic boutons. An apparent rearrangement of microtubule loop architecture occurs during bouton division, and a genetic analysis indicates that Futsch is necessary for this process. futsch mutations disrupt synaptic microtubule organization, reduce bouton number, and increase bouton size. These deficits can be partially rescued by neuronal overexpression of a futsch MAP1B homology domain. Finally, genetic manipulations that increase nerve-terminal branching correlate with increased synaptic microtubule loop formation, and both processes require normal Futsch function. These data suggest a common microtubule-based growth mechanism at the synapse and growth cone.