Smith RM , Baibakov B , Ikebuchi Y , White BH , Lambert NA , Kaczmarek LK , Vogel SS .

NS36455 NINDS NIH HHS , R01 NS036455 NINDS NIH HHS , R29 NS036455 NINDS NIH HHS

	Figure 1. Compensatory endocytosis excludes retrieval of plasma membrane proteins but not lipids. (a) The vitelline membrane of eggs in suspension was removed, surface proteins were labeled with Alexa 488 maleimide, and then imaged by confocal microscopy (see A). Next, eggs were activated with 25 μM A23187 and 30 μM tetramethylrhodamine dextran was added as a fluid phase marker of endocytosis. After 15 min the eggs were washed three times in ASW and green (Alexa 488) and red (tetramethylrhodamine) fluorescence was imaged (B). In control experiments the eggs were treated as in the first experiment except Alexa 488 maleimide was not removed before egg activation (see bottom time line). Eggs were imaged 15 min after activation with calcium ionophore (C). All pictures in a are representative micrographs, n = 8, from five different egg preparations. (b) The vitelline membrane was removed and surface proteins were labeled with a green fluorescent conjugate of concanavalin A and Oregon green 488 (2 μg/ml). Next, eggs were activated with A23187 and perfused with a red fluorescent conjugate of concanavalin A and Texas red to label any new exposed concanavalin A binding sites. After a 15-min incubation, the activated eggs were washed three times with ASW and imaged by confocal microscopy (D). A Z-axis series of 15 images spaced 1 μm apart was used in conjunction with a look-through algorithm to generate a three-dimensional rendition of the egg viewed from directly above (0°) or after being rotated by 50°. Pictures in b are representative micrographs, n = 9, from nine different egg preparations. (c) Eggs were labeled with the lipidic fluorescent dye octadeclyrhodamine and imaged by confocal microscopy (see E). Eggs were activated with a 1:1,000 dilution of sperm and the same egg was imaged again 15 min later (see F). Note the formation of fluorescent intracellular inclusions and a few elongated microvilli extending out from the surface. All pictures in c are representative micrographs, n = 11, from five different egg preparations. Bars: (A) 5 μm; (D) 10 μm; (E and F) 1 μm.
	Figure 2. Compensatory endocytosis selectively retrieves cortical granule membrane proteins. The vitelline membranes of eggs in suspension were removed. Next, eggs were washed three times in ASW and incubated with ASW containing Alexa 488 maleimide to label-free sulfhydryl groups on the egg surface. Eggs were then washed three times with ASW to remove dye that had not covalently attached. Eggs were activated with 25 μM A23187 and 3 min later placed into calcium-free ASW to trap cortical granule membranes in the cell surface. Newly introduced sulfhydryl groups on the egg surface were now labeled with Alexa 594 maleimide (4 μM in ASW). After three washes with ASW to remove nonincorporated Alexa 594, the Alexa 488 (green) and Alexa 594 (red) fluorescence was imaged by confocal microscopy (A). The two indicators were primarily distributed on the surface of the egg. Membrane depolarization and the subsequent compensatory endocytosis was triggered by the addition of 9.3 mM calcium chloride and 50 mM potassium chloride as indicated on the protocol time line. Subsequent imaging revealed a dramatic segregation of the two fluorescent indicators (B). All pictures are representative micrographs, n = 5, from five different egg preparations. Bar, (A) 5 μm.
	Figure 3. Compensatory endocytosis occurs at sites where exocytosis has occurred. Sea urchin eggs were attached to a polylysine-treated coverslip as previously described (Vogel et al. 1999). Eggs were incubated in artificial sea water (ASW) containing 100 μM tetramethylrhodamine dextran (3,000 mol wt; Molecular Probes) and activated by focal application of the calcium ionophore A23187 (50 μM) with a micropipette. After partial elevation of the fertilization envelope was observed, the eggs were washed in sea water to remove exogenous tetramethylrhodamine dextran. Bar, 20 μm.
	Figure 4. Depolarization triggers compensatory retrieval only when granule components are present on the egg surface. Eggs were activated with a 1:1,000 dilution of sperm and washed with either calcium-free artificial sea water (see Retrieval Arrested time line) or normal ASW (see Normal Retrieval time line) 3 min after addition of sperm. The normal extracellular calcium concentration (9.3 mM) was restored 10 min after sperm addition for the Retrieval Arrested eggs. Note: Extracellular calcium addition at t = 10 min does not trigger retrieval because the egg membrane potential at that time is hyperpolarized thus keeping P-type calcium channels closed (Vogel et al. 1999). 15 min after fertilization eggs were either treated with 50 mM potassium chloride to depolarize the egg membrane potential or mock treated. The fluid phase uptake marker (100 μM tetramethylrhodamine dextran) was also added at 15 min to monitor endocytotic retrieval (black bar in time line). At t = 30 min eggs were washed and percent retrieval was measured. The depolarization-induced membrane retrieval was measured (+ Potassium; grey bars) and compared with eggs that were not depolarized (− Potassium; white bars) for eggs placed in calcium-free ASW (Retrieval Arrested) and eggs that had been allowed to undergo their normal retrieval reactions in normal ASW (Normal Retrieval). All points are mean ± SD, n = 6.
	Figure 5. P-type channels are inaccessible to toxin before fertilization. Eggs were fertilized in artificial seawater and 100 μM tetramethylrhodamine dextran was added at 5 min after fertilization and net tetramethylrhodamine dextran uptake was determined after a 15-min incubation. All points are normalized to a positive control in the absence of any inhibitor (white bars). Before fertilization some eggs were preincubated with either 500 μM cadmium or 5 μM conotoxin MVIIC for 5 min and then washed to remove free inhibitor (green bars). These eggs were then fertilized and net membrane retrieval was determined. Other eggs in parallel were fertilized in the presence of either cadmium or conotoxin and after three min the eggs were washed to remove free inhibitor (yellow bars). Note the complete inhibition with the irreversible inhibitor, conotoxin. Finally, some eggs were fertilized in the presence of either cadmium or conotoxin with preincubation (red bars) and without preincubation (blue bars), and net membrane retrieval was determined after 15 min. 5 μM conotoxin MVIIC had no effect on sperm-induced fertilization envelope elevation (Vogel et al. 1999). All points are mean ± SD, n = 6 normalized relative to the control.
	Figure 6. Egg activation increases the number of agatoxin binding sites on the egg surface. (A) Unfertilized eggs were incubated with 100 nM ω-agatoxin–IVA for 7 min, washed, and then fixed. (B) Eggs were activated in ASW containing 50 μM A23187 and 100 nM agatoxin–IVA for 7 min before fixation. (C) Eggs were treated as those in B but agatoxin was absent. After fixation all samples were washed, blocked, and incubated in a 1:500 dilution of a rabbit anti-agatoxin antibody. Next, samples were incubated with a 1:2,000 dilution of a goat anti–rabbit IgG conjugated to tetramethylrhodamine, washed, and visualized by confocal microscopy. Note that there was virtually no fluorescence observed in unfertilized eggs or in fertilized eggs that were not treated with agatoxin. All pictures are representative micrographs, n = 4, from four different egg preparations, excluding eggs damaged by excessive DTT and/or ionophore treatment. Bar, 20 μm.
	Figure 7. Immunogold electron microscopy of ω-agatoxin binding sites. (A and B) Representative micrographs of sea urchin egg thin sections of unfertilized (A), and 15 min post-fertilized eggs (B) that had been pretreated with ω-agatoxin TK, washed, fixed, and incubated successively with a rabbit anti-agatoxin antibody, and then with a goat anti–rabbit IgG coupled to 15-nm gold particles. The black arrows indicate examples of the membrane structures analyzed: MV, microvilli; CGM, cortical granule membrane; PM, plasma membrane; TVM, translucent vesicle membrane; SVM, subcortical vesicle membrane; and YGM, yolk granule membrane. Note that a typical gold particle can be observed at the tip of the arrow marked SVM in B. Bar, 0.5 μm. (C and D) Gold particle density per micron of membrane in unfertilized (C) and 15 min post-fertilization eggs (D) for the different sea urchin egg membrane structures when treated with nonimmune IgG (white bars), no IgG (grey bars), or anti-agatoxin IgG (black bars). All points are mean ± SD, n = 4 experiments with 40 micrographs analyzed in each experiment.
	Figure 8. Immunolocaliza-tion of sea urchin egg P-type calcium channels. (A) Membranes were prepared from isolated sea urchin cortical granules and separated on a 7.5% SDS-PAGE. Proteins were electroblotted onto nitrocellulose, blocked and incubated with 1 μg/ml of: Aplysia P-type–specific affinity-purified rabbit antisera, BC-α1A; L-type–specific antisera, BC-α1D; BC-α1A preincubated with a twofold excess of immune peptide, Pre-Ab; and the preimmune sera of the rabbit which produced BC-α1A, Pre-Im. Immunoreactivity was detected with a goat anti–rabbit antisera conjugated to alkaline phospha–tase. Arrows on left show major components unique to the P-type–specific antisera. Arrows on right are size markers. (B and C) Gold particle density per micron of membrane in unfertilized (B) and 15 min post-fertilization eggs (C) for the different sea urchin egg membrane structures (MV, microvilli; CGM, cortical granule membrane; PM, plasma membrane; TVM, translucent vesicle membrane; SVM, subcortical vesicle membrane; and YGM, yolk granule membrane) when treated with preimmune IgG (white bars), preabsorbed BC-α1A (grey bars), or BC-α1A (black bars). All points are mean ± SD, n = 4 experiments with 40 micrographs analyzed in each experiment. (D–F) Representative micrographs of unfertilized sea urchin egg thin sections treated with BC-α1A (D), preimmune sera (E), or BC-α1A preabsorbed with immune peptide (F), and then treated with a goat anti–rabbit IgG coupled to 15-nm gold particles. The black arrows indicate gold particles on the cortical granule surface. Note: The nonspecific binding of gold particles over yolk and cortical granule contents in all three micrographs. Bar, 0.5 μm.
	Figure 9. Subcortical vesicles are a transient compartment of the exocytosis–endocytosis cycle. The average number of cortical granules (grey bars) and subcortical vesicles (black bars) per micrograph was determined in unfertilized eggs, and at 5 and 15 min after fertilization in the absence (A) or presence (B) of ω-agatoxin. All points are mean ± SD, from 40 micrographs.

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