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First published online 9 November 2004
doi: 10.1242/jcs.01518

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ß subunits of heterotrimeric G-proteins contribute to Ca²⁺ release at fertilization in the sea urchin

Department of Molecular and Cell Biology and Biochemistry, Brown University, 69 Brown Street, Providence, RI 02912, USA

View larger version (72K): [in a new window]   Fig. 2. (A-C) Inhibition of the signaling mediated by two G-proteins, Gq and Gs, interferes with fertilization envelope (FE) formation upon fertilization. Percentage of fertilized eggs forming normal FEs was determined for each treatment (A, Gs-inhibiting reagents; B, Gq-inhibiting reagents; C, Gi-inhibiting reagents). In each case, the fertilization event was detected by sperm incorporation into the egg by Hoechst staining. Plotted are the average values of two or three independent experiments, each involving 10-12 cells (error bars, 1±s.d.). Reagents used: U, untreated; P, inhibitory peptides; Ab, antibody (whole IgG); dAb, denatured antibody; pAb, preimmune (irrelevant) antibody; Fab, affinity-purified Fab fragments; I, inositol (1,4,5)-trisphosphate injected following Fab fragment injection (FE formed independent of fertilization); M, mastroparan (FE formed only upon fertilization); Pt, pertussis toxin; dPt, denatured pertussis toxin. The cytoplasmic concentration of introduced reagents was 100 µg/ml for all antibodies, 4.5 µg/ml for Fab fragments, 50 µM for inhibitory peptides, 0.2 µg/ml for pertussis toxin and 10 µM for mastoparan. (D,E) Example of normal FE formation. Note single sperm pronucleus (sn). (F,G) Example of aberrant FE formation upon microinjection of Gs antibody. Note multiple sperm pronuclei (sn). (D,F) DIC images; (E,G) epifluorescent images of zygotes stained with Hoechst. Arrows, female pronuclei; arrowheads, oil droplets resulting from microinjection; fe, fertilization envelope.

View larger version (75K): [in a new window]   Fig. 1. Gi, Gq and Gs proteins localize to the cortex of sea urchin eggs. (A-D) Mature sea urchin eggs labeled with hyalin (cortical granule marker; red) and anti-G antibodies (green). A single cell is outlined in B. (a-d) Fragments of images in A-D (outlined by dotted lines) showing red and green channels separately. Immunolocalizations in thick paraffin sections were performed as described in the Materials and Methods. Bar, 50 µm. (E) Cell-surface complex (csc; plasma membrane and cortical granules) and whole eggs lysate (e) protein samples were analyzed by immunoblotting (loading µg amounts indicated below lanes). Positions of molecular weight markers (in kDa) are shown at the left. The blots were probed with anti-MGB (1:1000) (Haley and Wessel, 2004), anti-YP30 (1:4000) (Wessel et al., 2000), anti-Gi (1:500), anti-Gq (4.5 µg/ml), anti-Gs (1:500) and anti-G12 (1:100) (Voronina and Wessel, 2004).

View larger version (30K): [in a new window]   Fig. 3. Quantification of cortical granule exocytosis (CGE) at fertilization: input of G-proteins. (A) Increase in FM1-43 fluorescence indicates CGE. (B) Gi inhibitor (competitor peptide; 50 µM final concentration in egg) changes exocytosis dynamics. (C) Gs inhibitor (inhibitory antibody; 100 µg/ml final concentration in egg) decreases exocytosis. (D) Gq inhibitor (competitor peptide; 50 µM final concentration in egg) decreases exocytosis. Each data point represents an average of 3-6 recordings (error bars, 1±s.d.).

View larger version (39K): [in a new window]   Fig. 6. (A) Injection of phosducin or ßARKct prevents normal Ca2+ release at fertilization. Each data point represents an average of 6 or 8 recordings (error bars, 1±s.d.). (B) Delay of Ca2+ release during fertilization of sea urchin eggs injected with 80 µg/ml phosducin. Fluorescence intensity over the individual eggs (injected with the indicated reagents) is shown over time, with the number of recordings in parentheses. Asterisks over traces indicate gamete fusion. Phosducin-injected eggs exhibited either a dramatically delayed and attenuated Ca2+ transient (top trace) or a mildly delayed and attenuated Ca2+ transient (bottom trace). The average delays for each group are summarized in below; *, values are significantly different from the denatured control by non-parametric Wilcoxin Rank Sum test, P<0.02. (C) Injection of inositol (1,4,5)trisphosphate [Ins(1,4,5)P3] overrides phosducin effects and causes cortical granule exocytosis (CGE). Two eggs were injected with 80 µg/ml phosducin with Rhodamin dextran (red fluorescence). After 1 hour incubation, the left egg was injected with 28 nM Ins(1,4,5)P3 with Alexa Fluor488 dextran (green fluorescence; orange when overlaid with red), which caused CGE and formation of fertilization envelope (FE). Arrowheads indicate oil droplets resulting from microinjection. (D) Response of phosducin-injected eggs to suboptimal amounts of Ins(1,4,5)P3 (10 nM final concentration) is indistinguishable from that of control eggs. Shown are the results of a representative experiment; 6-15 eggs were analyzed per group. One hour after microinjection of the control or test reagent, the eggs were challenged with 10 nM Ins(1,4,5)P3, and the time to initiation of CGE was monitored (white diamonds: the average time for the group; error bars, 1±s.d.). Black bars represent the percentage of eggs that were able to initiate CGE within 1 minute of Ins(1,4,5)P3 microinjection.

View larger version (37K): [in a new window]   Fig. 4. Inhibitors of Gs- and Gq-mediated signaling interfere with normal Ca2+ release at fertilization. (A) Ratiometric quantification of normal Ca2+ release at fertilization. (B) Anti-Gs (100 µg/ml final concentration in egg) and Ca2+ release at fertilization. (C) Gq peptide (50 µM final concentration in egg) and Ca2+ release at fertilization. Each data point represents an average of 3-8 recordings (error bars, 1±s.d.). (D) Ca2+ wave in a control egg (top left; pseudocolored); compare with the abnormal pattern in a Gq peptide-injected egg (bottom right, observed in 42.5% of eggs). The images were independently adjusted to better reveal the features (wave versus spikes); colors reflect normalized fluorescence ratios of individual pixels (see side bars).

View larger version (33K): [in a new window]   Fig. 5. (A) Sequestration of ß subunits in the egg achieved by using two alternative reagents (100 µg/ml phosducin or 200 µg/ml ßARKct, unless otherwise indicated) leads to abnormal fertilization envelope (FE) formation. Percentage of eggs forming normal FEs was determined for each treatment. Plotted are the average values of three or four independent experiments (10 eggs analyzed per each experiment; error bars, 1±s.d.). (B) ß subunits might mediate signaling initiated by disparate G subunit activation. (C) High concentrations of injected phosducin prevent FE formation altogether, whereas lower ones cause low or partial FE elevation (10-20 eggs analyzed per each injected concentration). (D) Sequestration of ß subunits leads to lower amount of membrane added to cell surface at fertilization. Each data point represents an average of 3 or 4 recordings (error bars, 1±s.d.).

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