Limits...
Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.

Smith RM, Baibakov B, Ikebuchi Y, White BH, Lambert NA, Kaczmarek LK, Vogel SS - J. Cell Biol. (2000)

Bottom Line: We tested whether channel distribution can account for the localization of retrieval at exocytotic sites.We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis.Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Medical College of Georgia, Augusta, Georgia 30912-2630, USA.

ABSTRACT
Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169375&req=5

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.

Mentions: Immunolocalization of ω-agatoxin was used to determine if toxin does bind to channels on the surface of the unfertilized egg and whether there is an increase in the number of ω-agatoxin binding sites after fertilization, as expected if the channels are present on cortical granules. We used a polyclonal antibody that recognizes both free ω-agatoxin and the toxin when it is bound to the P-type channel (Calbiochem). Confocal microscopy was used to image the surface fluorescence of unfertilized and fertilized eggs treated with ω-agatoxin, the anti-agatoxin antibody, and a fluorescent secondary antibody. We found that there was a dramatic increase in the surface labeling of the egg following egg activation (Fig. 6 B). Immunogold electron microscopy with this antibody confirmed that there was no specific labeling in the egg before activation (Fig. 7A and Fig. C), but agatoxin binding sites were observed on the membranes of subcortical vesicular structures (SVM) and on the membranes of translucent vesicles (TVM) of fertilized eggs (Fig. 7B and Fig. D). Specific binding was not observed on microvilli or the flat portions of the plasma membrane. We did observe nonspecific binding of gold particles to the core contents of cortical granules and yolk granules (data not shown). Because the density of gold particles on these structures was the same when probed with anti-agatoxin IgG, nonimmune IgG, and when IgG was omitted completely, we conclude that this binding is not specific for agatoxin. Low levels (<2 gold particles/μm2) of nonspecific labeling were also occasionally observed on mitochondria and cytoplasm (data not shown). Our findings are consistent with the hypothesis that new ω-agatoxin binding sites are inserted into the surface by cortical granule exocytosis after egg activation.


Exocytotic insertion of calcium channels constrains compensatory endocytosis to sites of exocytosis.

Smith RM, Baibakov B, Ikebuchi Y, White BH, Lambert NA, Kaczmarek LK, Vogel SS - J. Cell Biol. (2000)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169375&req=5

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.
Mentions: Immunolocalization of ω-agatoxin was used to determine if toxin does bind to channels on the surface of the unfertilized egg and whether there is an increase in the number of ω-agatoxin binding sites after fertilization, as expected if the channels are present on cortical granules. We used a polyclonal antibody that recognizes both free ω-agatoxin and the toxin when it is bound to the P-type channel (Calbiochem). Confocal microscopy was used to image the surface fluorescence of unfertilized and fertilized eggs treated with ω-agatoxin, the anti-agatoxin antibody, and a fluorescent secondary antibody. We found that there was a dramatic increase in the surface labeling of the egg following egg activation (Fig. 6 B). Immunogold electron microscopy with this antibody confirmed that there was no specific labeling in the egg before activation (Fig. 7A and Fig. C), but agatoxin binding sites were observed on the membranes of subcortical vesicular structures (SVM) and on the membranes of translucent vesicles (TVM) of fertilized eggs (Fig. 7B and Fig. D). Specific binding was not observed on microvilli or the flat portions of the plasma membrane. We did observe nonspecific binding of gold particles to the core contents of cortical granules and yolk granules (data not shown). Because the density of gold particles on these structures was the same when probed with anti-agatoxin IgG, nonimmune IgG, and when IgG was omitted completely, we conclude that this binding is not specific for agatoxin. Low levels (<2 gold particles/μm2) of nonspecific labeling were also occasionally observed on mitochondria and cytoplasm (data not shown). Our findings are consistent with the hypothesis that new ω-agatoxin binding sites are inserted into the surface by cortical granule exocytosis after egg activation.

Bottom Line: We tested whether channel distribution can account for the localization of retrieval at exocytotic sites.We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis.Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Medical College of Georgia, Augusta, Georgia 30912-2630, USA.

ABSTRACT
Proteins inserted into the cell surface by exocytosis are thought to be retrieved by compensatory endocytosis, suggesting that retrieval requires granule proteins. In sea urchin eggs, calcium influx through P-type calcium channels is required for retrieval, and the large size of sea urchin secretory granules permits the direct observation of retrieval. Here we demonstrate that retrieval is limited to sites of prior exocytosis. We tested whether channel distribution can account for the localization of retrieval at exocytotic sites. We find that P-channels reside on secretory granules before fertilization, and are translocated to the egg surface by exocytosis. Our study provides strong evidence that the transitory insertion of P-type calcium channels in the surface membrane plays an obligatory role in the mechanism coupling exocytosis and compensatory endocytosis.

Show MeSH
Related in: MedlinePlus