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Curr Biol
2008 Oct 28;1820:1612-8. doi: 10.1016/j.cub.2008.09.024.
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Ca(2+) signaling occurs via second messenger release from intraorganelle synthesis sites.
Davis LC
,
Morgan AJ
,
Ruas M
,
Wong JL
,
Graeff RM
,
Poustka AJ
,
Lee HC
,
Wessel GM
,
Parrington J
,
Galione A
.
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Cyclic ADP-ribose is an important Ca(2+)-mobilizing cytosolic messenger synthesized from beta-NAD(+) by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCbeta and ARCgamma are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a beta-NAD(+) transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca(2+) wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol.
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.
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