Davis LC , Morgan AJ , Ruas M , Wong JL , Graeff RM , Poustka AJ , Lee HC , Wessel GM , Parrington J , Galione A .

Medical Research Council , Wellcome Trust , T32 GM007601 NIGMS NIH HHS

	Figure 1. Distribution of ARCα, ARCβ, and ARCγ in Sea Urchin Eggs(A) Specificity of ARC antibodies confirmed in immunoblots with recombinant GST-ARCs.(B–M) Eggs stained with Lysotracker Red DND-99 (red) were fixed, permeabilized (unless otherwise indicated), and labeled (green) with antibodies against ARCα (B–E), ARCβ (F–I), and ARCγ (J–M). The following are shown: cortical staining with ARC antibodies (B, F, and J); staining blocked with competing antigenic peptides (C, G, and K); nonpermeabilized eggs (D, H, and L); and 3D reconstruction of sequential z sections (E, I, and M).(N–V) Stratified eggs studies. (N) Schematic representation of the stratification of intracellular organelles. Staining for the cortical granule marker protein, hyalin, or ARCs in the absence (O–R) or presence (S–V) of urethane is shown. Scale bars represent 2 μm (B) and 10 μm (C).
	Figure 2. ARCβ and ARCγ Localize to the Cortical Granule LumenSchematic representation (A) of cortical lawn preparation by the shearing of eggs adhering to coverslips (B–G). Cortical lawns stained (green) for ARCα (B and C), ARCβ (D and E), ARCγ (F and G) in fixed, permeabilized (B, D, and F), or live, unpermeabilized samples (C, E, and G). Fixed samples were costained for the cortical granule protein hyalin (red). Fluorescence images are shown (left) and are overlayed with DIC images (right).(H–K) Immunogold localization and quantification of ARCβ (H and I) and ARCγ (J and K). For clarity, micrographs represent magnified cortical regions that do not include the entire region used for quantification. (I and K) Quantification of labeling in cytosol (cyt), endoplasmic reticulum (ER), cortical granules (CG), and yolk platelets (yolk); vertical bars display standard deviation for tallies from six different eggs.The images in (B)–(E) are depicted on a common scale; those in (F) and (G) share a different scale. Scale bars represents 10 μm (B) and 1 μm (F, H, and J).
	Figure 3. ARC Distribution and Activity in Egg Subcellular Fractions(A) Immunoblotting of egg fractions for ARCα, ARCβ, and ARCγ. ARC isoforms are present as tetramers or monomers, as seen with other ARCs [38, 39].(B–D) ARC activity assessed by [32P]cADPR and [32P]ADPR production. (B) CSCs and CGs were resuspended in either isotonic (Intact) or hypotonic buffers (Lysis). (C) CGs were lysed in hypotonic buffer at pH 7 or pH 5 and separated into soluble (S) and particulate (P) fractions. (D) pH dependency of recombinant ARCα or ARCβ is shown. Inset shows western blots before (−) and after (+) induction in yeast by methanol. ∗∗, p < 0.01; ∗∗∗, p < 0.001 (n = 3).Data are presented as the mean ± SEM of n populations.
	Figure 4. Nucleotide Transport in Cortical Granules Is Required for Fertilization Ca2+ Responses(A–D) Effect of dipyridamole (DPM) upon fertilization-induced Ca2+ signals. Ca2+ waves in control (plus DMSO) (A) and DPM-treated eggs (B) are shown; image numbers represent the time (s) after wave initiation. The intracellular [DPM] is ∼10% of the extracellular concentration (Figure S5). The effect of DPM upon the global main Ca2+ spike (C) and wave kinetics (D) is shown (n = 4–32 eggs).(E and F) Effect of transport inhibitors with or without heparin (250 mg/ml pipette concentration) upon fertilization-evoked Ca2+ signals. (E) Traces represent global Ca2+ signals upon fertilization in control (Ctrl) and heparin-injected (hep) eggs incubated with either DMSO or 30 μM DPM. (F) A summary of the effect of DPM, indoprofen (indo), and nitrobenzylthioinosine (NBTI) upon the main Ca2+ spike amplitude in eggs injected with or without heparin is shown (n = 12–32). Heparin increased the lag from 13 ± 1 s to 38 ± 2 s (n = 29–33, p < 0.001). Responses are normalized to the ΔF/F0 in control eggs (ΔF/F0 = 2.38 ± 0.19, n = 32). Eggs were treated with 30 μM DPM for 3 min, 500 μM indoprofen for 5 min, or 100 μM NBTI for 45–90 min (∗∗, p < 0.01 and ∗∗∗, p < 0.001, comparing inhibitors with or without heparin; ##, p < 0.01 and ###, p < 0.001 versus heparin plus DMSO).(G–I) Effect of DPM upon signals in response to nucleotide injection: 30 μM DPM inhibits Ca2+ responses to microinjection of β-NAD+ (5–10 mM pipette concentration [G and I]) but not to cADPR (30 μM pipette concentration [H and I]); n = 44–51 eggs. (G and H) Control ΔF/F0: β-NAD+, 2.71 ± 0.31 (n = 44); cADPR, 2.19 ± 0.19 (n = 50); without nucleotides, 0.43 ± 0.10 (n = 13). I: ΔF/F0 expressed as percentage of control.(J) Kinetics of the accumulation of 32P in CSCs incubated with [32P]β-NAD+ in the presence of DMSO vehicle (blue) or 10 μM DPM (red). Digitonin (300 μM) and 100 μM unlabeled (“cold”) β-NAD+ were added where indicated (n = 3). ∗, p < 0.05 and ∗∗, p < 0.01 versus control (radioactivity at time zero = 1947 ± 116 disintegrations per minute [d.p.m.]).(K) Net luminal 32P accumulation (Intact minus digitonin) after a 5 min incubation of CSC with [32P]β-NAD+ in the presence of 0.5% DMSO, 10 μM DPM, 10 μM NBTI, or 100 μM indoprofen (n = 5–7, ∗p < 0.05 versus DMSO). Raw radioactivity of DMSO control, 3251 ± 658 d.p.m.(L) Identification of luminal 32P-labeled nucleotides by TLC analysis, after an 18 min incubation of CSC with [32P]β-NAD+ in the presence of 0.8% DMSO or 16 μM DPM (n = 4, ∗∗p < 0.01 versus DMSO). Spot intensities (arbitrary units) with DMSO are as follows: β-NAD+, 1696 ± 274; cADPR, 354 ± 94. Note that in these experiments, [32P]ADPR levels were not consistently above the level of detection.Data are presented as the mean ± SEM of n populations.

Bacher, Channelling of substrate promiscuity of the skeletal-muscle ADP-ribosyl cyclase isoform. 2004, Pubmed

Bacher, Channelling of substrate promiscuity of the skeletal-muscle ADP-ribosyl cyclase isoform. 2004, Pubmed
Billington, A transport mechanism for NAADP in a rat basophilic cell line. 2006, Pubmed
Billington, Characterization of NAD uptake in mammalian cells. 2008, Pubmed
Bruzzone, Dimeric and tetrameric forms of catalytically active transmembrane CD38 in transfected HeLa cells. 1998, Pubmed
Ceni, Evidence for an intracellular ADP-ribosyl cyclase/NAD+-glycohydrolase in brain from CD38-deficient mice. 2003, Pubmed
Churamani, Molecular characterization of a novel intracellular ADP-ribosyl cyclase. 2007, Pubmed , Echinobase
Churchill, Spatial control of Ca2+ signaling by nicotinic acid adenine dinucleotide phosphate diffusion and gradients. 2000, Pubmed , Echinobase
Clapper, Pyridine nucleotide metabolites stimulate calcium release from sea urchin egg microsomes desensitized to inositol trisphosphate. 1987, Pubmed , Echinobase
De Flora, Autocrine and paracrine calcium signaling by the CD38/NAD+/cyclic ADP-ribose system. 2004, Pubmed
Galione, Redundant mechanisms of calcium-induced calcium release underlying calcium waves during fertilization of sea urchin eggs. 1993, Pubmed , Echinobase
Graeff, Cyclic GMP-dependent and -independent effects on the synthesis of the calcium messengers cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate. 1998, Pubmed , Echinobase
Haley, Regulated proteolysis by cortical granule serine protease 1 at fertilization. 2004, Pubmed , Echinobase
Hirata, ADP ribosyl cyclase activity of a novel bone marrow stromal cell surface molecule, BST-1. 1994, Pubmed
Hylander, The effect of local anesthetics and ammonia on cortical granule-plasma membrane attachment in the sea urchin egg. 1981, Pubmed , Echinobase
Jedlitschky, The nucleotide transporter MRP4 (ABCC4) is highly expressed in human platelets and present in dense granules, indicating a role in mediator storage. 2004, Pubmed
Leckie, The NO pathway acts late during the fertilization response in sea urchin eggs. 2003, Pubmed , Echinobase
Lee, Calcium mobilization by dual receptors during fertilization of sea urchin eggs. 1993, Pubmed , Echinobase
Lee, Cyclic ADP-ribose and its metabolic enzymes. 1995, Pubmed
Lee, Structure and enzymatic functions of human CD38. 2006, Pubmed
Malavasi, Evolution and function of the ADP ribosyl cyclase/CD38 gene family in physiology and pathology. 2008, Pubmed
McNiven, Vesicle formation at the plasma membrane and trans-Golgi network: the same but different. 2006, Pubmed
Miyata, Development of calcium releasing activity induced by inositol trisphosphate and cyclic ADP-ribose during in vitro maturation of sea urchin oocytes. 2006, Pubmed , Echinobase
Morgan, Fertilization and nicotinic acid adenine dinucleotide phosphate induce pH changes in acidic Ca(2+) stores in sea urchin eggs. 2007, Pubmed , Echinobase
Morita, Cyclic ADP-ribose mediates formyl methionyl leucyl phenylalanine (fMLP)-induced intracellular Ca(2+) rise and migration of human neutrophils. 2008, Pubmed
Munshi, The homo-dimeric form of ADP-ribosyl cyclase in solution. 1998, Pubmed
Palade, The hunt for an alternate way to generate NAADP. Focus on "NAADP as a second messenger: neither CD38 nor base-exchange reaction are necessary for in vivo generation of NAADP in myometrial cells". 2007, Pubmed
Poustka, Generation, annotation, evolutionary analysis, and database integration of 20,000 unique sea urchin EST clusters. 2003, Pubmed , Echinobase
Reid, The human multidrug resistance protein MRP4 functions as a prostaglandin efflux transporter and is inhibited by nonsteroidal antiinflammatory drugs. 2003, Pubmed
Sawada, Identification of a vesicular nucleotide transporter. 2008, Pubmed
Sethi, Nicotinamide inhibits cyclic ADP-ribose-mediated calcium signalling in sea urchin eggs. 1996, Pubmed , Echinobase
Soares, NAADP as a second messenger: neither CD38 nor base-exchange reaction are necessary for in vivo generation of NAADP in myometrial cells. 2007, Pubmed
Sodergren, The genome of the sea urchin Strongylocentrotus purpuratus. 2006, Pubmed , Echinobase
Todisco, Identification of the mitochondrial NAD+ transporter in Saccharomyces cerevisiae. 2006, Pubmed
Trubiani, Insights into nuclear localization and dynamic association of CD38 in Raji and K562 cells. 2008, Pubmed
Vacquier, The isolation of intact cortical granules from sea urchin eggs: calcium lons trigger granule discharge. 1975, Pubmed , Echinobase
Whalley, Direct membrane retrieval into large vesicles after exocytosis in sea urchin eggs. 1995, Pubmed , Echinobase
Wilson, Differential regulation of nicotinic acid-adenine dinucleotide phosphate and cADP-ribose production by cAMP and cGMP. 1998, Pubmed , Echinobase
Zhang, Studies of nucleoside transporters using novel autofluorescent nucleoside probes. 2006, Pubmed
Zocchi, Self-aggregation of purified and membrane-bound erythrocyte CD38 induces extensive decrease of its ADP-ribosyl cyclase activity. 1995, Pubmed