Morgan AJ , Davis LC , Wagner SK , Lewis AM , Parrington J , Churchill GC , Galione A .

Wellcome Trust , BB/G008523/1 Biotechnology and Biological Sciences Research Council

	Figure 1. Characterization of pHL changes in response to photolysis of caged NAADP. Sea urchin eggs were microinjected with caged NAADP (∼0.5 µM cytosolic concentration) and photolysis effected with a UV laser as indicated. Images are pseudocolored ratios of one channel (pHL) or two channels (Ca2+), and time after photolysis indicated in seconds in the corner. The inset cartoons indicate the regions of interest from which the traces are derived. (A) In ratiometric pHL recordings, global photolysis (70% UV) evoked a larger response in the periphery (red) than the center (green). n ≥ 14 eggs. (B) Ratiometric Ca2+ recordings. The traces correspond to the bottom cell exposed globally to 70% UV laser. n ≥ 6 eggs. (C) Quantification of the initial rapid pHL responses in the periphery (red) and center (green) as a function of UV intensity. Data are mean ± SEM of 3–19 eggs. (D) Same data as C normalized to the maximal pHL response recorded in each region. (E) Magnitude of the central response as a percentage of the corresponding peripheral change. (F) Focal uncaging of NAADP elicits a pHL response (50% UV, irradiated at box indicated). (G) pHL changes at the UV site (solid line) and antipode (dotted line) in the periphery and center. (H) Summary of changes at the UV site (UV) and antipode (AP); mean ± SEM of n = 22 eggs (***, P < 0.001; ###, P < 0.001 compared with Peri UV). Bars, 50 µm.
	Figure 2. Inhibition of the NAADP receptor inhibits the fertilization-induced pHLash. (A–C) Ned-19 was preincubated with eggs for 30 min (at 80 µM) or 60–90 min (at 160 µM) or with DMSO vehicle (0.16% vol/vol) as a control. Eggs were loaded with Acridine orange plus LysoTracker red for the final 15–20 min of the preincubation period. Peripheral (red) and central (green) pHL responses to sperm in single eggs treated with DMSO (A) or 160 µM Ned-19 (B). At the end, 10 mM NH4Cl was applied. Inset brightfield images show unfertilized (minus sperm, −Sp) and fertilized eggs (plus sperm, +Sp), with the yellow arrows highlighting the boundary of the fertilization envelope. Bars, 50 µm. (C) Summary of the effect of Ned-19 on the rapid pHLash (red) and central slow central acidification (green) expressed as the percentage of DMSO-treated eggs; n = 45–83 eggs. The bar chart (gray boxes) quantifies Ned-19 loading into single eggs measured as its intrinsic fluorescence at 351 nm (n = 77–130 eggs). (D) Effect of Ned-19 (160 µM, 30 min) upon the pHL response to microinjection of free NAADP (50 µM pipette). Eggs loaded with Acridine orange were injected with Alexa Fluor 647 dextran alone (Mock), or Alexa Fluor 647 dextran plus NAADP. Bar chart summarizes the pHLash in 8–28 eggs, the underlying traces normalized to their initial fluorescence. (E) Summary of the effect of nifedipine and PPADS on pHL responses to sperm, normalized to the respective responses in untreated eggs. 100 µM nifedipine (or DMSO) was included during the dye-loading period (20 min), n = 28–102 eggs; PPADS (10 mM pipette) was microinjected into dye-loaded eggs, n = 39–45. (F) Effect of diltiazem upon sperm-induced pHL changes. (Fi) Diltiazem was included during the dye-loading period (20 min), and responses normalized to the peripheral pHLash or central acidification responses in control eggs (Ctrl) in the absence of inhibitor (n = 64–426 eggs). (Fii) Peripheral pHL records in the absence or presence of 50 µM diltiazem (±Dzm). NH4Cl = 10 mM. Traces are the mean of 19 (−Dzm) or 36 (+Dzm) eggs, respectively. (G) Preincubation with 50 µM diltiazem inhibited the response to NAADP injection (50 µM pipette), both in terms of the peripheral pHLash (bar chart) and fertilization envelope lifting (inset micrographs); n = 11–12. Bar, 50 µm.
	Figure 3. Ca2+ increases pHL and NAADP levels. (A–E) Eggs loaded with Acridine orange and LysoTracker red were exposed to sperm, 1 µM ionomycin, or 10 mM NH4Cl. Representative traces of the peripheral (red) and central (green) pHL changes with sperm (A) or ionomycin (B). Exocytosis results in movement out of the peripheral ROI (Morgan and Galione, 2007a), hence the break in the red traces. (C) Summary of the rapid peripheral pHLash (red) and the slow central acidification (green) responses with sperm and ionomycin, n = 48–49 eggs. (D and E) Spatial nature of the single-cell peripheral pHL (n = 48–49 eggs) and Ca2+ (n = 8–21 eggs) responses to sperm (top) and ionomycin (bottom). For clarity, traces are normalized to the initial (F0) Acridine orange or rhod-dextran fluorescence and derived from the three regions of interest illustrated in the inset egg schematic. (F) NAADP levels measured in populations of eggs stimulated by sperm (gray) or 1–2 µM ionomycin (black). Data are from a single preparation, typical of four. (G–I) Effect of NAADP antagonists on 1–2 µM ionomycin-induced pHL changes. Ned-19 (n = 40–60 eggs) and diltiazem (n = 91–200 eggs) were preincubated according to the protocols in Fig. 2; SKF96365 (or 0.1–0.2% DMSO vehicle) was coincubated with the pHL dyes in the presence of 0.05% Pluronic F127 to promote inhibitor loading (n = 72–428 eggs). NAADP antagonists had a significantly greater effect upon the pHLash than the acidification (P < 0.001) at all concentrations except 30 µM SKF96365 (P > 0.05). *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. 0 µM control.
	Figure 5. SERCA inhibition and caged Ca2+ couple weakly to the pHLash. (A–C) Effect of the SERCA inhibitor, cyclopiazonic acid (CPA, 150 µM) upon Ca2+. (A) Two types of single-cell CPA responses were observed: a predominant small, monotonic increase (black trace) or a large, biphasic increase (gray trace). 2 µM ionomycin (iono) was then added. (B) Bar chart summarizing the peak responses from all cells (n = 14). (C) Summary of CPA responses sorted into the two categories with the percentage of eggs exhibiting them indicated above the bars. (D–F) CPA is a poor pHLash stimulus. (D) Most eggs failed to give a pHL response to 150 µM CPA (nonresponders, black trace), but a minority showed a peripheral pHLash after a delay. (E) Summary of peak responses in all eggs (n = 132). (F) Data were categorized as CPA responders (n = 35) or CPA nonresponders (n = 97) and the CPA and subsequent ionomycin responses plotted accordingly (the percentage of eggs is indicated). (G–I) Comparison of caged Ca2+ and fertilization upon Ca2+ signals in eggs. (G) Eggs were injected with caged Ca2+ (NP-EGTA, ∼250 µM cytosolic) and focal photolysis initiated in a cortical region of the egg by UV laser. The trace represents the Ca2+ response within the photolysis region; the inset shows the Ca2+ peak in a single egg as an F/F0 image, with the photolysis region of interest labeled as “UV”. (H) Whole-cell Ca2+ signal in response to sperm. (I) Summary of peak Ca2+ amplitudes (n = 24, caged Ca2+ ; n = 7, sperm). (J and K) pHLash response to photolysis of caged Ca2+ (J) or sperm (K). Most cells (18/20) did not respond to uncaging, only 2/20 cells gave a pHLash (J, dotted line). (L) Summary of the peak pHL responses in all cells (n = 20, caged Ca2+; n = 64, sperm). ***, P < 0.001 compared with uncaging.
	Figure 6. Pharmacology of pHL responses and fertilization envelope lifting. (A) Schematic indicating the possible routes that IP3 and cADPR could affect pHL. (B) Representative traces of the pHL responses to messenger injection (pipette concentrations, 50 µM NAADP, 20–30 µM cADPR, and 1 mM IP3). Left-hand column shows control (Ctrl) responses in the absence of inhibitors (but in the presence of vehicle). Other columns depict egg treatment: injected with EGTA or BAPTA (pipette concentrations of 250 mM); preincubated for 60 min with 160 µM Ned-19; preincubated with 50 µM diltiazem (Dzm); or 200 µM SKF96365 (plus 0.05% Pluronic F127) during dye loading for 15–20 min. (C) Summary of the messenger-induced pHLash expressed as a percentage of the NAADP control (Ctrl); n = 9–124 eggs. (D) Quantification of fertilization envelope lifting in the same eggs as pHL was recorded. Data are expressed as the number of eggs that showed a partial/full lifting as a percentage of the total number of eggs injected with each messenger. The effect of BAPTA on cADPR and IP3 was not determined (nd).
	Figure 8. Effect of inhibiting ER Ca2+ signaling upon the fertilization-induced pHL changes. (A–D) Effect of cytosolic EGTA upon Ca2+ and pHL. Micropipettes containing 1 mM rhod-dextran ± 250 mM EGTA were used to inject eggs loaded with 1 µM Acridine orange. (A) Fertilization-induced peripheral Ca2+ responses in the absence (dark blue) or presence (light blue) of EGTA. (B) Peripheral pHL changes in single eggs were recorded in the absence (dark orange) or presence (light orange) of EGTA. Dotted parts of the traces indicate artifacts due to changes in shape. The inset in A is a summary of 11–15 eggs. (C) Localized pHL responses in a single EGTA-injected egg (n = 3). Images are pseudocolored F/F0 ratios of the Acridine orange fluorescence (time in seconds) in response to sperm added at 78 s. Bottom brightfield images depict the sperm entry point at the region contained within the dotted box. The local pHL change is overlayed. −Sp, minus sperm; +Sp, plus sperm. Bars: (main panels) 50 µm; (magnified) 10 µm. (D) Fluorescence changes at the color-matched ROIs depicted on the first image in C. The arrow in both C and D indicates the point of a second local response. (E–H) Effect of ER Ca2+ channel blockade upon fertilization-induced pHL responses. Eggs were injected with or without heparin plus 8-NH2-cADPR (500 mg/ml and 500 µM pipette concentration, respectively). (E) Pseudocolored Acridine orange F/F0 images with time in seconds (sperm were added at 53 s). (F) Fluorescence traces from the ROIs drawn in E. Summary of the peripheral pHL responses at the sperm entry point and antipode plotting the amplitude (G) and kinetics (H). Data represent the mean ± SEM of 11–13 eggs.
	Figure 9. Localization of acidic vesicles and ER. Acidic vesicles were labeled with LysoTracker green and the ER with microinjected DiI and consecutive 1-µm slices were collected in an egg quarter. An equatorial slice is shown, with an arc of exocytotic cortical granules (CG) docked at the plasma membrane. Deeper vesicles show close apposition with the ER (inset). Images are representative of at least 10 eggs.

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