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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.

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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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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.

Mentions: To confirm that the subcortical structures labeled by BC-α1A and by the agatoxin-specific sera represent a transient intermediate structure of the exocytosis-endocytosis pathway, we measured the number of cortical granules and subcortical vesicles observed in electron micrographs of unfertilized eggs, and in fertilized eggs at 5 and 15 min after fertilization. In both control and agatoxin-treated eggs the cortical granules should disappear upon fertilization. Furthermore, the number of subcortical vesicles which appear upon fertilization in agatoxin-treated eggs (at 5 and 15 min post fertilization) should be approximately equivalent to the number of cortical granules in unfertilized eggs because membrane retrieval is inhibited by agatoxin. In contrast, the number of subcortical vesicles observed at 5 and 15 min after fertilization in control eggs should decrease as these structures are internalized by compensatory endocytosis and should be transported into the cytoplasm. These dynamic predictions were observed in our micrographs (Fig. 9). In agatoxin-treated eggs the number of cortical granules (37.2 ± 3.8 structures/micrograph, mean ± SD, n = 40) observed in unfertilized eggs and the number of subcortical vesicles at 5 (34.5 ± 5.1 structures/micrograph) and 15 min after fertilization (30.6 ± 4.7 structures/micrograph) were similar. In control eggs the number of subcortical vesicles observed 5 min after fertilization (12.9 ± 2.5 structures/micrograph) was less than the number of cortical granules in unfertilized eggs (36.4 ± 4.1 structures/micrograph), and the number of subcortical vesicles declined with time (compare 5 and 15 min after fertilization in Fig. 9 A).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

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.
Mentions: To confirm that the subcortical structures labeled by BC-α1A and by the agatoxin-specific sera represent a transient intermediate structure of the exocytosis-endocytosis pathway, we measured the number of cortical granules and subcortical vesicles observed in electron micrographs of unfertilized eggs, and in fertilized eggs at 5 and 15 min after fertilization. In both control and agatoxin-treated eggs the cortical granules should disappear upon fertilization. Furthermore, the number of subcortical vesicles which appear upon fertilization in agatoxin-treated eggs (at 5 and 15 min post fertilization) should be approximately equivalent to the number of cortical granules in unfertilized eggs because membrane retrieval is inhibited by agatoxin. In contrast, the number of subcortical vesicles observed at 5 and 15 min after fertilization in control eggs should decrease as these structures are internalized by compensatory endocytosis and should be transported into the cytoplasm. These dynamic predictions were observed in our micrographs (Fig. 9). In agatoxin-treated eggs the number of cortical granules (37.2 ± 3.8 structures/micrograph, mean ± SD, n = 40) observed in unfertilized eggs and the number of subcortical vesicles at 5 (34.5 ± 5.1 structures/micrograph) and 15 min after fertilization (30.6 ± 4.7 structures/micrograph) were similar. In control eggs the number of subcortical vesicles observed 5 min after fertilization (12.9 ± 2.5 structures/micrograph) was less than the number of cortical granules in unfertilized eggs (36.4 ± 4.1 structures/micrograph), and the number of subcortical vesicles declined with time (compare 5 and 15 min after fertilization in Fig. 9 A).

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