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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 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.
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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.

Mentions: By definition, compensatory endocytosis is preceded by exocytosis, and in the sea urchin egg exocytosis is the physiologically relevant trigger for membrane retrieval. Nonetheless, the mechanism of compensatory endocytosis may not inherently require prior exocytosis. For example, retrieval might be triggered by a plasma-membrane flacidity sensor, or by the binding of secreted ligands to cell surface receptors. Activation of the hypothetical cell surface sensors would then activate a signaling pathway to initiate membrane retrieval. If such a coupling mechanism was operant in the sea urchin egg, one could imagine that activation of the appropriate signaling pathway might trigger retrieval even in the absence of exocytotic activity. Clearly, it is important to test if exocytosis is required for retrieval. Specific retrieval of granule components suggests that prior exocytosis is required for compensatory endocytosis. If prior cortical granule exocytosis is an absolute requirement for compensatory endocytosis, (a) membrane retrieval should occur only at sites where exocytosis has occurred, and (b) depolarization-triggered gating of P-type calcium channels should only trigger retrieval when granule components are present on the cell surface. We exploited the ability to trigger exocytosis focally (Lawson et al. 1978; Chambers and Hinkley 1979) to test the first prediction. We used focal application of a calcium ionophore to trigger exocytosis focally (rather than globally). Eggs were placed in sea water containing the fluid phase marker tetramethylrhodamine dextran and the calcium ionophore A23187 was focally applied to the egg with a micropipette. This resulted in local cortical granule exocytosis manifested by focal elevation of the fertilization envelope. When eggs were subsequently perfused with sea water to remove extracellular fluid phase marker and imaged by fluorescent microscopy, large red fluorescent inclusions were observed only at the sites where cortical granule exocytosis had occurred (Fig. 3). Other eggs in the field were not activated and had no fluorescent inclusions. Fluid phase uptake evoked in this manner was completely blocked by ω-agatoxin TK (which blocks the calcium influx required for endocytotic membrane retrieval [Vogel et al. 1999]), yet focal elevation of the fertilization envelope was still observed (data not shown). It is known that ionophore-mediated calcium influx does not rescue retrieval activity in agatoxin-treated cells in normal seawater (with 9.3 mM calcium; Vogel et al. 1999). Calcium ionophores did rescue retrieval activity when the extracellular calcium concentration was elevated to >12 mM (Vogel et al. 1999). Therefore, in this experiment where we use normal sea water, focal cortical granule exocytosis was triggered by ionophore-mediated calcium influx, while focal membrane retrieval was triggered by P-channel–mediated calcium influx. Since the depolarization required to open channels is presumed to be uniform across the egg surface, these observations are consistent with the notion that cortical granule exocytosis is required for subsequent compensatory endocytosis.


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 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.
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Related In: Results  -  Collection

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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.
Mentions: By definition, compensatory endocytosis is preceded by exocytosis, and in the sea urchin egg exocytosis is the physiologically relevant trigger for membrane retrieval. Nonetheless, the mechanism of compensatory endocytosis may not inherently require prior exocytosis. For example, retrieval might be triggered by a plasma-membrane flacidity sensor, or by the binding of secreted ligands to cell surface receptors. Activation of the hypothetical cell surface sensors would then activate a signaling pathway to initiate membrane retrieval. If such a coupling mechanism was operant in the sea urchin egg, one could imagine that activation of the appropriate signaling pathway might trigger retrieval even in the absence of exocytotic activity. Clearly, it is important to test if exocytosis is required for retrieval. Specific retrieval of granule components suggests that prior exocytosis is required for compensatory endocytosis. If prior cortical granule exocytosis is an absolute requirement for compensatory endocytosis, (a) membrane retrieval should occur only at sites where exocytosis has occurred, and (b) depolarization-triggered gating of P-type calcium channels should only trigger retrieval when granule components are present on the cell surface. We exploited the ability to trigger exocytosis focally (Lawson et al. 1978; Chambers and Hinkley 1979) to test the first prediction. We used focal application of a calcium ionophore to trigger exocytosis focally (rather than globally). Eggs were placed in sea water containing the fluid phase marker tetramethylrhodamine dextran and the calcium ionophore A23187 was focally applied to the egg with a micropipette. This resulted in local cortical granule exocytosis manifested by focal elevation of the fertilization envelope. When eggs were subsequently perfused with sea water to remove extracellular fluid phase marker and imaged by fluorescent microscopy, large red fluorescent inclusions were observed only at the sites where cortical granule exocytosis had occurred (Fig. 3). Other eggs in the field were not activated and had no fluorescent inclusions. Fluid phase uptake evoked in this manner was completely blocked by ω-agatoxin TK (which blocks the calcium influx required for endocytotic membrane retrieval [Vogel et al. 1999]), yet focal elevation of the fertilization envelope was still observed (data not shown). It is known that ionophore-mediated calcium influx does not rescue retrieval activity in agatoxin-treated cells in normal seawater (with 9.3 mM calcium; Vogel et al. 1999). Calcium ionophores did rescue retrieval activity when the extracellular calcium concentration was elevated to >12 mM (Vogel et al. 1999). Therefore, in this experiment where we use normal sea water, focal cortical granule exocytosis was triggered by ionophore-mediated calcium influx, while focal membrane retrieval was triggered by P-channel–mediated calcium influx. Since the depolarization required to open channels is presumed to be uniform across the egg surface, these observations are consistent with the notion that cortical granule exocytosis is required for subsequent compensatory endocytosis.

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