Search

Key points This work confirms previous reports that CM4620, a small molecule inhibitor of Ca2+ entry via store operated Ca2+ entry (SOCE) channels formed by stromal interaction molecule 1 (STIM1)/Orai complexes, attenuates acinar cell pathology and acute pancreatitis in mouse experimental models. Here we report that intravenous administration of CM4620 reduces the severity of acute pancreatitis in the rat, a hitherto untested species. Using CM4620, we probe further the mechanisms whereby SOCE via STIM1/Orai complexes contributes to the disease in pancreatic acinar cells, supporting a role for endoplasmic reticulum stress/cell death pathways in these cells. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes neutrophil oxidative burst and inflammatory gene expression during acute pancreatitis, including in immune cells which may be either circulating or invading the pancreas. Using CM4620, we show that SOCE via STIM1/Orai complexes promotes activation and fibroinflammatory gene expression within pancreatic stellate cells. Abstract Key features of acute pancreatitis include excess cellular Ca2+ entry driven by Ca2+ depletion from the endoplasmic reticulum (ER) and subsequent activation of store-operated Ca2+ entry (SOCE) channels in the plasma membrane. In several cell types, including pancreatic acinar, stellate cells (PaSCs) and immune cells, SOCE is mediated via channels composed primarily of Orai1 and stromal interaction molecule 1 (STIM1). CM4620, a selective Orai1 inhibitor, prevents Ca2+ entry in acinar cells. This study investigates the effects of CM4620 in preventing or reducing acute pancreatitis features and severity. We tested the effects of CM4620 on SOCE, trypsinogen activation, acinar cell death, activation of NFAT and NF-κB, and inflammatory responses in ex vivo and in vivo rodent models of acute pancreatitis and human pancreatic acini. We also examined whether CM4620 inhibited cytokine release in immune cells, fibro-inflammatory responses in PaSCs, and oxidative burst in neutrophils, all cell types participating in pancreatitis. CM4620 administration to rats by i.v. infusion starting 30 min after induction of pancreatitis significantly diminished pancreatitis features including pancreatic oedema, acinar cell vacuolization, intrapancreatic trypsin activity, cell death signalling and acinar cell death. CM4620 also decreased myeloperoxidase activity and inflammatory cytokine expression in pancreas and lung tissues, fMLF peptide-induced oxidative burst in human neutrophils, and cytokine production in human peripheral blood mononuclear cells (PBMCs) and rodent PaSCs, indicating that Orai1/STIM1 channels participate in the inflammatory responses of these cell types during acute pancreatitis. These findings support pathological Ca2+ entry-mediated cell death and proinflammatory signalling as central mechanisms in acute pancreatitis pathobiology.

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.

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca2+-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca2+-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20–50 pA at −110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing ∼300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of ∼50 nM, and was also blocked by 20 μM SKF 96365 and by 20 μM 2-APB. At concentrations between 5 and 14 μM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.

Two new polyprenyl products in addition to dehydrodolichol and dolichol were detected by two-plate silica gel thin layer chromatography of nonpolar products formed from [1-14C]isopentenyl diphosphate and farnesyl diphosphate in the reaction with a crude 1,000 x g supernatant of yeast homogenates in the presence of NADPH. The new products were indistinguishable from authentic dehydrodolichal and dolichal. Analyses of the time-dependent and pH-dependent formation of the four products including dehydrodolichal and dolichal suggested that the biosynthetic pathway from dehydrodolichol leading to dolichal is different from that to dolichol. In double-labeled experiments with a combination of -l-14C-isopentenyl diphosphate and a [4B-3H]NADPH-generating system, the ratio of 3H- and 14C-derived radioactivities found in dolichal was six times higher than that in dolichol. A small amount of 3H-labeled dehydrodolichol was also detected. Considering the fact that dolichol is synthesized from dehydrodolichol (Sagami, H., Kurisaki, A., and Ogura, K. (1993) J. Biol. Chem. 268, 10109-10113), we propose that dehydrodolichol is a common branch point intermediate in the biosynthetic pathways leading to dolichal and dolichol and that dehydrodolichal is an intermediate in the pathway from dehydrodolichol to dolichal.

Sodium vanadate is an effective drug for the enrichment of yeast mutants defective in glycosylation reactions that are carried out in the Golgi complex. We have isolated vanadate-resistant, hygromycin B-sensitive mutants that act at very early steps of N-linked glycosylation, occurring in the endoplasmic reticulum. Here we describe the phenotypic characterization of ost4, a vanadate-resistant mutant that is defective in oligosaccharyltransferase (OTase) activity both in vivo and in vitro. The OST4 open reading frame is unusual in that it predicts a protein of only 36 amino acids. We demonstrate that the OST4 gene product is, in fact, an unusually small protein of approximately 3.6 kDa, predicted to lie almost entirely in the hydrophobic environment of the membrane. Strains carrying a disruption of the OST4 gene are viable but grow poorly at 25 degrees C. The null mutant is inviable at 37 degrees C, demonstrating that the OST4 gene product is essential for growth at high temperatures. Deletion of the OST4 gene greatly diminishes OTase activity but does not abolish it. These results suggest that the OST4 gene encodes a subunit or accessory component of OTase that is essential at high temperature.

In budding yeast, one of three G1 cyclins is required for progression though START, when cells commit to a further round of cell division. We have identified mutations in ALG1 (ERC14), a gene required for N-glycosylation, which are inviable in a cln1 cln2 background but are rescued by over-expression of CLNs. CLN1 and CLN2 are much more efficient than CLN3 in rescuing the erc14-1 allele. The erc14-1 allele results in a significant N-glycosylation defect, and no rescue of this defect by CLN1 over-expression was detected. These data suggest that CLN over-expression could be allowing cells to live with lower levels of N-glycosylation, possibly by overcoming a checkpoint sensitive to N-glycosylation capacity. A plasmidsuppressor of alg1, PSA1, encodes a 361 amino-acid protein with homology to NDP-hexose pyrophosphorylases, the enzymes that catalyze the formation of activated sugar nucleotides. PSA1 is an essential gene, and PSA1 transcription is nearly co-ordinately regulated with CLN2 transcription, peaking near START. Co-ordinate regulation of glycosylation, sugar nucleotide metabolism, and cell-cycle progression through G1 may be a feature that ensures adequate cell-wall precursors are present before bud emergence.

In budding yeast, one of three G1 cyclins is required for progression though START, when cells commit to a further round of cell division. We have identified mutations inALG1 (ERC14), a gene required for N-glycosylation, which are inviable in acln1 cln2 background but are rescued by over-expression ofCLNs.CLN1 andCLN2 are much more efficient thanCLN3 in rescuing theerc14-1 allele. Theerc14-1 allele results in a significant N-glycosylation defect, and no rescue of this defect byCLN1 over-expression was detected. These data suggest thatCLN over-expression could be allowing cells to live with lower levels of N-glycosylation, possibly by overcoming a checkpoint sensitive to N-glycosylation capacity. A plasmid suppressor ofalg1, PSA1, encodes a 361 amino-acid protein with homology to NDP-hexose pyrophosphorylases, the enzymes that catalyze the formation of activated sugar nucleotides.PSA1 is an essential gene, andPSA1 transcription is nearly co-ordinately regulated withCLN2 transcription, peaking near START. Co-ordinate regulation of glycosylation, sugar nucleotide metabolism, and cell-cycle progression through G1 may be a feature that ensures adequate cell-wall precursors are present before bud emergence.

Dolichol in the form of dolichyl phosphate participates in the synthesis of N- and O-linked glycoproteins and phosphatidylinositol-linked proteins in the yeast Saccharomyces cerevisiae. In this organism, as well as in higher eukaryotes, a number of the enzymes in the polyisoprenoid and glycoprotein biosynthetic pathways have not been identified. In this study, we have developed a convenient, highly sensitive assay that uses one of the end products of the dolichylphosphate synthetic pathway, oligosaccharide-diphosphodolichol, and a 125I-labeled peptide substrate for N-linked glycosylation to screen a collection of temperature-sensitive yeast mutants for defects in protein glycosylation. By using a combination of biochemical and genetic procedures, the defective mutants were grouped into three categories: those containing defects in dolichyl-phosphate synthesis (class 1), lipid-linked oligosaccharide assembly (class 2), or oligosaccharide transferase activity (class 3). Among the mutants identified by this screen were sec59 (which encodes dolichol kinase) and a mutant that affects the activity of the ALG1-encoded mannosyltransferase that forms dolichol-PP-(GlcNAc)2Man1. Of particular interest was a mutant that exhibits a temperature-sensitive defect in oligosaccharide transferase activity. This mutant, meg1 (microsomal protein essential for glycosylation 1) assembles a complete oligosaccharide chain and, therefore, is likely to be a class 3 mutant. We report the cloning of MEG1, the gene that rescues the oligosaccharide transferase activity defect in this mutant. A number of criteria distinguish this gene from previously described genes in this pathway.

A morphological study suggests a new view of synapse formation by demonstrating that presynaptic elements are assembled into ‘prototerminals’ before being transported down axons by fast, saltatory movement. These structures stop moving when the axon contacts a dendrite.

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.