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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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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.
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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.

Mentions: Exocytosis inserts granule proteins and lipids into the cell surface. Coupled endocytotic membrane retrieval removes membrane from the cell surface, but in sea urchins it is not known if the retrieved membrane is comprised of components whose origin is solely the cortical granule membrane, components from the plasma membrane, or a mixture of components from both sources. We devised an imaging strategy to directly test if plasma membrane components are specifically retrieved by compensatory retrieval. Egg membrane surface proteins were labeled with Alexa 488 maleimide before fertilization (Fig. 1 a). The plasma membrane components labeled by this reagent (membrane proteins with free sulfhydryl groups) were not internalized by endocytotic membrane retrieval (i.e., they did not colocalize with the fluid phase marker tetramethylrhodamine dextran; see Fig. 1 a, B) upon activation with calcium ionophore. Occasionally, we did observe a limited amount of fluorescent plasma membrane marker internalization. However, internalized Alexa 488 did not colocalize with the fluid phase marker. Presumably under certain circumstances Alexa 488 maleimide can be transported into the egg by a separate mechanism. In control experiments, we left Alexa 488 maleimide on after fertilization and confirmed that under these conditions some Alexa dye does colocalize in the same intracellular inclusions as the fluid phase marker (Fig. 1 a, C). Colocalization with the fluid phase marker suggests that Alexa 488 maleimide labels both plasma membrane proteins as well as components tightly associated with the membrane surface. Upon membrane retrieval, some of these labeled components extend into the lumen of these intracellular inclusions. A similar labeling pattern was observed with the fluorescent membrane probe FM1-43 in neuroendocrine cells (Angleson et al. 1999). Plasma membrane components prelabeled with a green fluorescent conjugate of concanavalin A were not retrieved by compensatory endocytosis, while a red conjugate applied after egg activation was retrieved (Fig. 1 b). It is known that concanavalin A binds to glucosans attached to the egg plasma membrane, as well as with components on the granule membrane (Veron and Shapiro 1977; Veron et al. 1977). Because the binding specificity of maleimide and concanavalin A are quite different, and because these reagents will bind to a broad spectrum of membrane proteins, we conclude that the majority of proteins retrieved by compensatory endocytosis did not originate in the plasma membrane.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: Exocytosis inserts granule proteins and lipids into the cell surface. Coupled endocytotic membrane retrieval removes membrane from the cell surface, but in sea urchins it is not known if the retrieved membrane is comprised of components whose origin is solely the cortical granule membrane, components from the plasma membrane, or a mixture of components from both sources. We devised an imaging strategy to directly test if plasma membrane components are specifically retrieved by compensatory retrieval. Egg membrane surface proteins were labeled with Alexa 488 maleimide before fertilization (Fig. 1 a). The plasma membrane components labeled by this reagent (membrane proteins with free sulfhydryl groups) were not internalized by endocytotic membrane retrieval (i.e., they did not colocalize with the fluid phase marker tetramethylrhodamine dextran; see Fig. 1 a, B) upon activation with calcium ionophore. Occasionally, we did observe a limited amount of fluorescent plasma membrane marker internalization. However, internalized Alexa 488 did not colocalize with the fluid phase marker. Presumably under certain circumstances Alexa 488 maleimide can be transported into the egg by a separate mechanism. In control experiments, we left Alexa 488 maleimide on after fertilization and confirmed that under these conditions some Alexa dye does colocalize in the same intracellular inclusions as the fluid phase marker (Fig. 1 a, C). Colocalization with the fluid phase marker suggests that Alexa 488 maleimide labels both plasma membrane proteins as well as components tightly associated with the membrane surface. Upon membrane retrieval, some of these labeled components extend into the lumen of these intracellular inclusions. A similar labeling pattern was observed with the fluorescent membrane probe FM1-43 in neuroendocrine cells (Angleson et al. 1999). Plasma membrane components prelabeled with a green fluorescent conjugate of concanavalin A were not retrieved by compensatory endocytosis, while a red conjugate applied after egg activation was retrieved (Fig. 1 b). It is known that concanavalin A binds to glucosans attached to the egg plasma membrane, as well as with components on the granule membrane (Veron and Shapiro 1977; Veron et al. 1977). Because the binding specificity of maleimide and concanavalin A are quite different, and because these reagents will bind to a broad spectrum of membrane proteins, we conclude that the majority of proteins retrieved by compensatory endocytosis did not originate in the plasma membrane.

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