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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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Differential sensitivity to extracellular probes  of synaptophysin and transferrin receptor biotinylated  at 18°C. PC12 cells were incubated with sulfo-NHS-LC– biotin (A and B) or sulfoNHS-SS–biotin (C and D)  for 60 min at 4°C (A) or for  30 min at 18°C (B–D), chased  for 5 min at 18°C in the presence of glycine (B–D) or not  chased (A), and incubated at  4°C in the absence (−) or  presence (+) of extracellularly  added avidin (A and B) or MesNa (C and D). Synaptophysin and transferrin receptor in the postnuclear supernatants were analyzed for  binding to streptavidin–agarose by immunoblotting of bound and unbound material with the respective antibodies. Streptavidin-bound  biotinylated synaptophysin and transferrin receptor present in the postnuclear supernatant is expressed as percentage of total (sum of  streptavidin-bound and streptavidin-unbound synaptophysin and transferrin receptor, respectively). (A) Data are the mean of two independent experiments; bars indicate the variation of the individual values from the mean. (B–D) Data are the mean of three independent  experiments; bars indicate SD.
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Figure 5: Differential sensitivity to extracellular probes of synaptophysin and transferrin receptor biotinylated at 18°C. PC12 cells were incubated with sulfo-NHS-LC– biotin (A and B) or sulfoNHS-SS–biotin (C and D) for 60 min at 4°C (A) or for 30 min at 18°C (B–D), chased for 5 min at 18°C in the presence of glycine (B–D) or not chased (A), and incubated at 4°C in the absence (−) or presence (+) of extracellularly added avidin (A and B) or MesNa (C and D). Synaptophysin and transferrin receptor in the postnuclear supernatants were analyzed for binding to streptavidin–agarose by immunoblotting of bound and unbound material with the respective antibodies. Streptavidin-bound biotinylated synaptophysin and transferrin receptor present in the postnuclear supernatant is expressed as percentage of total (sum of streptavidin-bound and streptavidin-unbound synaptophysin and transferrin receptor, respectively). (A) Data are the mean of two independent experiments; bars indicate the variation of the individual values from the mean. (B–D) Data are the mean of three independent experiments; bars indicate SD.

Mentions: Given that synaptophysin biotinylated at the cell surface did not appear in SLMVs at 18°C, we next investigated whether it was internalized at this temperature. For this purpose, we studied the accessibility of biotinylated synaptophysin to extracellularly added avidin. As a positive control, PC12 cells were rapidly cooled from 37° to 4°C (a temperature preventing further membrane traffic), biotinylated for 60 min at 4°C, and then exposed to avidin at 4°C. In this condition one would expect to biotinylate only those synaptophysin molecules that are exposed at the cell surface; these should be accessible to avidin and, hence, not be able to bind to streptavidin–agarose subsequently. Only ∼2% of the total synaptophysin was biotinylated in this condition, in line with previous observations showing that only very little of the total synaptophysin is present at the surface of PC12 cells (Johnston et al., 1989), and most of the biotinylated synaptophysin was accessible to avidin (Fig. 5 A). In contrast, when PC12 cells biotinylated for 30 min at 18°C and chased for 5 min at 18°C were exposed to avidin at 4°C, the amount of biotinylated synaptophysin that subsequently could be adsorbed to streptavidin–agarose was not reduced compared to control (Fig. 5 B). Furthermore, the same amount of synaptophysin bound to streptavidin–agarose when PC12 cells biotinylated for 15 min at 18°C were exposed to avidin at either 18° or 4°C (data not shown). These results indicated that the synaptophysin biotinylated at 18°C was no longer exposed at the cell surface.


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)

Differential sensitivity to extracellular probes  of synaptophysin and transferrin receptor biotinylated  at 18°C. PC12 cells were incubated with sulfo-NHS-LC– biotin (A and B) or sulfoNHS-SS–biotin (C and D)  for 60 min at 4°C (A) or for  30 min at 18°C (B–D), chased  for 5 min at 18°C in the presence of glycine (B–D) or not  chased (A), and incubated at  4°C in the absence (−) or  presence (+) of extracellularly  added avidin (A and B) or MesNa (C and D). Synaptophysin and transferrin receptor in the postnuclear supernatants were analyzed for  binding to streptavidin–agarose by immunoblotting of bound and unbound material with the respective antibodies. Streptavidin-bound  biotinylated synaptophysin and transferrin receptor present in the postnuclear supernatant is expressed as percentage of total (sum of  streptavidin-bound and streptavidin-unbound synaptophysin and transferrin receptor, respectively). (A) Data are the mean of two independent experiments; bars indicate the variation of the individual values from the mean. (B–D) Data are the mean of three independent  experiments; bars indicate SD.
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Related In: Results  -  Collection

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Figure 5: Differential sensitivity to extracellular probes of synaptophysin and transferrin receptor biotinylated at 18°C. PC12 cells were incubated with sulfo-NHS-LC– biotin (A and B) or sulfoNHS-SS–biotin (C and D) for 60 min at 4°C (A) or for 30 min at 18°C (B–D), chased for 5 min at 18°C in the presence of glycine (B–D) or not chased (A), and incubated at 4°C in the absence (−) or presence (+) of extracellularly added avidin (A and B) or MesNa (C and D). Synaptophysin and transferrin receptor in the postnuclear supernatants were analyzed for binding to streptavidin–agarose by immunoblotting of bound and unbound material with the respective antibodies. Streptavidin-bound biotinylated synaptophysin and transferrin receptor present in the postnuclear supernatant is expressed as percentage of total (sum of streptavidin-bound and streptavidin-unbound synaptophysin and transferrin receptor, respectively). (A) Data are the mean of two independent experiments; bars indicate the variation of the individual values from the mean. (B–D) Data are the mean of three independent experiments; bars indicate SD.
Mentions: Given that synaptophysin biotinylated at the cell surface did not appear in SLMVs at 18°C, we next investigated whether it was internalized at this temperature. For this purpose, we studied the accessibility of biotinylated synaptophysin to extracellularly added avidin. As a positive control, PC12 cells were rapidly cooled from 37° to 4°C (a temperature preventing further membrane traffic), biotinylated for 60 min at 4°C, and then exposed to avidin at 4°C. In this condition one would expect to biotinylate only those synaptophysin molecules that are exposed at the cell surface; these should be accessible to avidin and, hence, not be able to bind to streptavidin–agarose subsequently. Only ∼2% of the total synaptophysin was biotinylated in this condition, in line with previous observations showing that only very little of the total synaptophysin is present at the surface of PC12 cells (Johnston et al., 1989), and most of the biotinylated synaptophysin was accessible to avidin (Fig. 5 A). In contrast, when PC12 cells biotinylated for 30 min at 18°C and chased for 5 min at 18°C were exposed to avidin at 4°C, the amount of biotinylated synaptophysin that subsequently could be adsorbed to streptavidin–agarose was not reduced compared to control (Fig. 5 B). Furthermore, the same amount of synaptophysin bound to streptavidin–agarose when PC12 cells biotinylated for 15 min at 18°C were exposed to avidin at either 18° or 4°C (data not shown). These results indicated that the synaptophysin biotinylated at 18°C was no longer exposed at the cell surface.

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