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Synaptic-like microvesicles of neuroendocrine cells originate from a novel compartment that is continuous with the plasma membrane and devoid of transferrin receptor.

Schmidt A, Hannah MJ, Huttner WB - J. Cell Biol. (1997)

Bottom Line: We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate.The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100.We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Heidelberg, Germany.

ABSTRACT
We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18 degrees C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18 degrees C with the membrane-impermeant, cleavable sulfo-NHS-SS-biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37 degrees C originated exclusively from the membranes containing the MesNa-accessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37 degrees C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18 degrees or 37 degrees C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18 degrees C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.

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SLMV biogenesis  involves the MesNa-sensitive population of synaptophysin molecules biotinylated  at 18°C. PC12 cells were incubated with sulfo-NHS-SS–biotin for 30 min at 18°C, chased  for 5 min at 18°C in the presence of glycine, incubated at  4°C in the absence (Control)  or presence of extracellularly added MesNa, and chased for 10  min at 37°C. The 66,000-g supernatants prepared from the cells  were subjected to glycerol gradient centrifugation, and fractions  were analyzed for biotinylated synaptophysin by streptavidin– agarose adsorption followed by immunoblotting of bound material with anti-synaptophysin antibodies. 11% and 4% of the total  synaptophysin present in the sum of the 66,000 g pellet plus supernatant bound to streptavidin–agarose without and with MesNa  addition before the 37°C chase, respectively.
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Figure 7: SLMV biogenesis involves the MesNa-sensitive population of synaptophysin molecules biotinylated at 18°C. PC12 cells were incubated with sulfo-NHS-SS–biotin for 30 min at 18°C, chased for 5 min at 18°C in the presence of glycine, incubated at 4°C in the absence (Control) or presence of extracellularly added MesNa, and chased for 10 min at 37°C. The 66,000-g supernatants prepared from the cells were subjected to glycerol gradient centrifugation, and fractions were analyzed for biotinylated synaptophysin by streptavidin– agarose adsorption followed by immunoblotting of bound material with anti-synaptophysin antibodies. 11% and 4% of the total synaptophysin present in the sum of the 66,000 g pellet plus supernatant bound to streptavidin–agarose without and with MesNa addition before the 37°C chase, respectively.

Mentions: Studies on the endocytosis of clathrin-coated vesicles in nonneuroendocrine cells (Carter et al., 1993) have revealed the existence of an intermediate, referred to as a deeply invaginated coated pit, which is thought to still be connected to the plasma membrane via a narrow neck, because the lumen of the pit is not accessible to the 68,000-D protein avidin but is sensitive to the small (150 D) membrane-impermeant, thiol-reducing agent MesNa. Given this finding and the inaccessibility of the synaptophysin biotinylated at 18°C to avidin, we investigated whether or not this synaptophysin was sensitive to MesNa added to the medium. For this purpose, we used sulfo-NHS-SS–biotin in which the biotin moiety after crosslinking to protein can be cleaved off by thiol reduction. As was the case for sulfoNHS-LC–biotin, labeling of synaptophysin with sulfo-NHSSS–biotin did not interfere with its ability to be sorted to SLMVs (data not shown; see Fig. 7). When PC12 cells biotinylated for 30 min at 18°C with sulfo-NHS-SS–biotin and chased for 5 min at 18°C were treated with MesNa at 4°C, ∼70% of the biotinylated synaptophysin was found to be MesNa sensitive, as indicated by the decrease in binding to streptavidin–agarose compared to the control (Fig. 5 C). In contrast, transferrin receptor biotinylated in the same condition was completely protected from MesNa (Fig. 5 D). These results show that the majority of synaptophysin biotinylated at 18°C is internalized into a compartment that has a narrow connection to the plasma membrane allowing the passage of MesNa but not avidin and in which there is no detectable transferrin receptor.


Synaptic-like microvesicles of neuroendocrine cells originate from a novel compartment that is continuous with the plasma membrane and devoid of transferrin receptor.

Schmidt A, Hannah MJ, Huttner WB - J. Cell Biol. (1997)

SLMV biogenesis  involves the MesNa-sensitive population of synaptophysin molecules biotinylated  at 18°C. PC12 cells were incubated with sulfo-NHS-SS–biotin for 30 min at 18°C, chased  for 5 min at 18°C in the presence of glycine, incubated at  4°C in the absence (Control)  or presence of extracellularly added MesNa, and chased for 10  min at 37°C. The 66,000-g supernatants prepared from the cells  were subjected to glycerol gradient centrifugation, and fractions  were analyzed for biotinylated synaptophysin by streptavidin– agarose adsorption followed by immunoblotting of bound material with anti-synaptophysin antibodies. 11% and 4% of the total  synaptophysin present in the sum of the 66,000 g pellet plus supernatant bound to streptavidin–agarose without and with MesNa  addition before the 37°C chase, respectively.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139769&req=5

Figure 7: SLMV biogenesis involves the MesNa-sensitive population of synaptophysin molecules biotinylated at 18°C. PC12 cells were incubated with sulfo-NHS-SS–biotin for 30 min at 18°C, chased for 5 min at 18°C in the presence of glycine, incubated at 4°C in the absence (Control) or presence of extracellularly added MesNa, and chased for 10 min at 37°C. The 66,000-g supernatants prepared from the cells were subjected to glycerol gradient centrifugation, and fractions were analyzed for biotinylated synaptophysin by streptavidin– agarose adsorption followed by immunoblotting of bound material with anti-synaptophysin antibodies. 11% and 4% of the total synaptophysin present in the sum of the 66,000 g pellet plus supernatant bound to streptavidin–agarose without and with MesNa addition before the 37°C chase, respectively.
Mentions: Studies on the endocytosis of clathrin-coated vesicles in nonneuroendocrine cells (Carter et al., 1993) have revealed the existence of an intermediate, referred to as a deeply invaginated coated pit, which is thought to still be connected to the plasma membrane via a narrow neck, because the lumen of the pit is not accessible to the 68,000-D protein avidin but is sensitive to the small (150 D) membrane-impermeant, thiol-reducing agent MesNa. Given this finding and the inaccessibility of the synaptophysin biotinylated at 18°C to avidin, we investigated whether or not this synaptophysin was sensitive to MesNa added to the medium. For this purpose, we used sulfo-NHS-SS–biotin in which the biotin moiety after crosslinking to protein can be cleaved off by thiol reduction. As was the case for sulfoNHS-LC–biotin, labeling of synaptophysin with sulfo-NHSSS–biotin did not interfere with its ability to be sorted to SLMVs (data not shown; see Fig. 7). When PC12 cells biotinylated for 30 min at 18°C with sulfo-NHS-SS–biotin and chased for 5 min at 18°C were treated with MesNa at 4°C, ∼70% of the biotinylated synaptophysin was found to be MesNa sensitive, as indicated by the decrease in binding to streptavidin–agarose compared to the control (Fig. 5 C). In contrast, transferrin receptor biotinylated in the same condition was completely protected from MesNa (Fig. 5 D). These results show that the majority of synaptophysin biotinylated at 18°C is internalized into a compartment that has a narrow connection to the plasma membrane allowing the passage of MesNa but not avidin and in which there is no detectable transferrin receptor.

Bottom Line: We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate.The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100.We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Heidelberg, Germany.

ABSTRACT
We have characterized the compartment from which synaptic-like microvesicles (SLMVs), the neuroendocrine counterpart of neuronal synaptic vesicles, originate. For this purpose we have exploited the previous observation that newly synthesized synaptophysin, a membrane marker of synaptic vesicles and SLMVs, is delivered to the latter organelles via the plasma membrane and an internal compartment. Specifically, synaptophysin was labeled by cell surface biotinylation of unstimulated PC12 cells at 18 degrees C, a condition which blocked the appearance of biotinylated synaptophysin in SLMVs and in which there appeared to be no significant exocytosis of SLMVs. The majority of synaptophysin labeled at 18 degrees C with the membrane-impermeant, cleavable sulfo-NHS-SS-biotin was still accessible to extracellularly added MesNa, a 150-D membrane-impermeant thiol-reducing agent, but not to the 68,000-D protein avidin. The SLMVs generated upon reversal of the temperature to 37 degrees C originated exclusively from the membranes containing the MesNa-accessible rather than the MesNa-protected population of synaptophysin molecules. Biogenesis of SLMVs from MesNa-accessible membranes was also observed after a short (2 min) biotinylation of synaptophysin at 37 degrees C followed by chase. In contrast to synaptophysin, transferrin receptor biotinylated at 18 degrees or 37 degrees C became rapidly inaccessible to MesNa. Immunofluorescence and immunogold electron microscopy of PC12 cells revealed, in addition to the previously described perinuclear endosome in which synaptophysin and transferrin receptor are colocalized, a sub-plasmalemmal tubulocisternal membrane system distinct from caveolin-positive caveolae that contained synaptophysin but little, if any, transferrin receptor. The latter synaptophysin was selectively visualized upon digitonin permeabilization and quantitatively extracted, despite paraformaldehyde fixation, by Triton X-100. Synaptophysin biotinylated at 18 degrees C was present in these subplasmalemmal membranes. We conclude that SLMVs originate from a novel compartment that is connected to the plasma membrane via a narrow membrane continuity and lacks transferrin receptor.

Show MeSH
Related in: MedlinePlus