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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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SV2 is accessible to  MesNa after biotinylation 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, and  incubated at 4°C in the absence (control) or presence  of extracellularly added MesNa. Transferrin receptor, synaptophysin, and SV2 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 transferrin receptor, synaptophysin, and  SV2 present in the postnuclear supernatant were calculated as  percentage of total (sum of streptavidin-bound plus streptavidinunbound transferrin receptor, synaptophysin, and SV2, respectively), and the individual values obtained after MesNa treatment were expressed as percentage of control. Data are the  mean of three independent experiments; bars indicate SD. The  mean values of biotinylated protein for the control condition  were 25.3% (transferrin receptor), 7.5% (synaptophysin), and  3.1% (SV2).
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Figure 8: SV2 is accessible to MesNa after biotinylation 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, and incubated at 4°C in the absence (control) or presence of extracellularly added MesNa. Transferrin receptor, synaptophysin, and SV2 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 transferrin receptor, synaptophysin, and SV2 present in the postnuclear supernatant were calculated as percentage of total (sum of streptavidin-bound plus streptavidinunbound transferrin receptor, synaptophysin, and SV2, respectively), and the individual values obtained after MesNa treatment were expressed as percentage of control. Data are the mean of three independent experiments; bars indicate SD. The mean values of biotinylated protein for the control condition were 25.3% (transferrin receptor), 7.5% (synaptophysin), and 3.1% (SV2).

Mentions: If the MesNa-accessible compartment gives rise to SLMVs, one would expect it to contain SLMV-specific membrane proteins other than synaptophysin. We investigated this issue by analyzing whether one such protein, SV2, showed a similar accessibility to MesNa after cell surface biotinylation for 30 min at 18°C. As shown in Fig. 8, MesNa accessibility of biotinylated SV2 was indeed comparable to that of synaptophysin (the latter confirming the data shown in Fig. 5 C) and in marked contrast to the almost complete MesNa inaccessibility of the transferrin receptor (confirming the data shown in Fig. 5 D).


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)

SV2 is accessible to  MesNa after biotinylation 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, and  incubated at 4°C in the absence (control) or presence  of extracellularly added MesNa. Transferrin receptor, synaptophysin, and SV2 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 transferrin receptor, synaptophysin, and  SV2 present in the postnuclear supernatant were calculated as  percentage of total (sum of streptavidin-bound plus streptavidinunbound transferrin receptor, synaptophysin, and SV2, respectively), and the individual values obtained after MesNa treatment were expressed as percentage of control. Data are the  mean of three independent experiments; bars indicate SD. The  mean values of biotinylated protein for the control condition  were 25.3% (transferrin receptor), 7.5% (synaptophysin), and  3.1% (SV2).
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Related In: Results  -  Collection

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Figure 8: SV2 is accessible to MesNa after biotinylation 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, and incubated at 4°C in the absence (control) or presence of extracellularly added MesNa. Transferrin receptor, synaptophysin, and SV2 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 transferrin receptor, synaptophysin, and SV2 present in the postnuclear supernatant were calculated as percentage of total (sum of streptavidin-bound plus streptavidinunbound transferrin receptor, synaptophysin, and SV2, respectively), and the individual values obtained after MesNa treatment were expressed as percentage of control. Data are the mean of three independent experiments; bars indicate SD. The mean values of biotinylated protein for the control condition were 25.3% (transferrin receptor), 7.5% (synaptophysin), and 3.1% (SV2).
Mentions: If the MesNa-accessible compartment gives rise to SLMVs, one would expect it to contain SLMV-specific membrane proteins other than synaptophysin. We investigated this issue by analyzing whether one such protein, SV2, showed a similar accessibility to MesNa after cell surface biotinylation for 30 min at 18°C. As shown in Fig. 8, MesNa accessibility of biotinylated SV2 was indeed comparable to that of synaptophysin (the latter confirming the data shown in Fig. 5 C) and in marked contrast to the almost complete MesNa inaccessibility of the transferrin receptor (confirming the data shown in Fig. 5 D).

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