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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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Time course of accumulation of biotinylated  synaptophysin and transferrin receptor at 18°C. PC12  cells were incubated with  (open and closed circles) or  without (closed triangles)  sulfo-NHS-LC–biotin at 18°C  for the indicated times. The  cells incubated without sulfoNHS-LC–biotin at 18°C were  subsequently biotinylated for  30 min at 4°C (closed triangles). Postnuclear supernatants prepared  from the cells were analyzed for biotinylated and nonbiotinylated synaptophysin and transferrin receptor by streptavidin– agarose adsorption, followed by immunoblotting of bound and  unbound material with the respective antibodies. Biotinylated  synaptophysin and transferrin receptor is expressed as percent of  total (sum of biotinylated plus nonbiotinylated synaptophysin  and transferrin receptor, respectively). Data points without error  bars represent single determinations. Data points with error bars  represent the mean of three (transferrin receptor), four (synaptophysin at 10 and 30 min), or two (synaptophysin at 20 min) independent determinations; bars indicate SD or the variation of the  individual values from the mean.
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Figure 6: Time course of accumulation of biotinylated synaptophysin and transferrin receptor at 18°C. PC12 cells were incubated with (open and closed circles) or without (closed triangles) sulfo-NHS-LC–biotin at 18°C for the indicated times. The cells incubated without sulfoNHS-LC–biotin at 18°C were subsequently biotinylated for 30 min at 4°C (closed triangles). Postnuclear supernatants prepared from the cells were analyzed for biotinylated and nonbiotinylated synaptophysin and transferrin receptor by streptavidin– agarose adsorption, followed by immunoblotting of bound and unbound material with the respective antibodies. Biotinylated synaptophysin and transferrin receptor is expressed as percent of total (sum of biotinylated plus nonbiotinylated synaptophysin and transferrin receptor, respectively). Data points without error bars represent single determinations. Data points with error bars represent the mean of three (transferrin receptor), four (synaptophysin at 10 and 30 min), or two (synaptophysin at 20 min) independent determinations; bars indicate SD or the variation of the individual values from the mean.

Mentions: The proportion of the total synaptophysin biotinylated after 30 min at 18°C (∼10% in Fig. 5 B) was at least five times that biotinylated for 60 min at 4°C (Fig. 5 A). Biotinylation at the latter temperature for 30 min yielded the same value (∼2% of the total synaptophysin) as that carried out for 60 min (data not shown), showing that within 30 min at 4°C, every synaptophysin molecule accessible to sulfo-NHS-LC–biotin was biotinylated to an extent sufficient to allow binding to streptavidin. This in turn suggested that the greater proportion of biotinylated synaptophysin obtained at 18° as opposed to 4°C reflected the delivery, at 18°C, either of unbiotinylated synaptophysin molecules from an intracellular pool to the site where biotinylation occurred, or of sulfo-NHS-LC–biotin to an intracellular site where synaptophysin resided. This conclusion was supported by studying the time course of biotinylation of synaptophysin at 18°C, which revealed a continuous increase in the amount of biotinylated synaptophysin over time (Fig. 6, closed circles). Together with the accessibility of the majority of this synaptophysin to MesNa (Fig. 5 C), these results indicate that upon incubation at 18°C, biotinylated synaptophysin accumulated in a membrane compartment connected to the plasma membrane. Interestingly, when PC12 cells incubated for various times at 18°C were subsequently biotinylated for 30 min at 4°C, only ∼2% of the total synaptophysin was biotinylated, irrespective of the incubation time at 18°C (Fig. 6, closed triangles). This suggested that upon cooling the cells to 4°C, the lumen of this membrane compartment became inaccessible to sulfo-NHS-LC–biotin, as was the case for avidin (Fig. 5 B) but not MesNa (Fig. 5 C). Biotinylation of the transferrin receptor at 18°C also increased over time, reaching a value of ∼35% of total by the end of the 30-min incubation (Fig. 6). Given the inaccessibility of the transferrin receptor to MesNa (Fig. 5 D), this implied its continuous delivery to the plasma membrane and endocytosis at 18°C.


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)

Time course of accumulation of biotinylated  synaptophysin and transferrin receptor at 18°C. PC12  cells were incubated with  (open and closed circles) or  without (closed triangles)  sulfo-NHS-LC–biotin at 18°C  for the indicated times. The  cells incubated without sulfoNHS-LC–biotin at 18°C were  subsequently biotinylated for  30 min at 4°C (closed triangles). Postnuclear supernatants prepared  from the cells were analyzed for biotinylated and nonbiotinylated synaptophysin and transferrin receptor by streptavidin– agarose adsorption, followed by immunoblotting of bound and  unbound material with the respective antibodies. Biotinylated  synaptophysin and transferrin receptor is expressed as percent of  total (sum of biotinylated plus nonbiotinylated synaptophysin  and transferrin receptor, respectively). Data points without error  bars represent single determinations. Data points with error bars  represent the mean of three (transferrin receptor), four (synaptophysin at 10 and 30 min), or two (synaptophysin at 20 min) independent determinations; bars indicate SD or the variation of the  individual values from the mean.
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Figure 6: Time course of accumulation of biotinylated synaptophysin and transferrin receptor at 18°C. PC12 cells were incubated with (open and closed circles) or without (closed triangles) sulfo-NHS-LC–biotin at 18°C for the indicated times. The cells incubated without sulfoNHS-LC–biotin at 18°C were subsequently biotinylated for 30 min at 4°C (closed triangles). Postnuclear supernatants prepared from the cells were analyzed for biotinylated and nonbiotinylated synaptophysin and transferrin receptor by streptavidin– agarose adsorption, followed by immunoblotting of bound and unbound material with the respective antibodies. Biotinylated synaptophysin and transferrin receptor is expressed as percent of total (sum of biotinylated plus nonbiotinylated synaptophysin and transferrin receptor, respectively). Data points without error bars represent single determinations. Data points with error bars represent the mean of three (transferrin receptor), four (synaptophysin at 10 and 30 min), or two (synaptophysin at 20 min) independent determinations; bars indicate SD or the variation of the individual values from the mean.
Mentions: The proportion of the total synaptophysin biotinylated after 30 min at 18°C (∼10% in Fig. 5 B) was at least five times that biotinylated for 60 min at 4°C (Fig. 5 A). Biotinylation at the latter temperature for 30 min yielded the same value (∼2% of the total synaptophysin) as that carried out for 60 min (data not shown), showing that within 30 min at 4°C, every synaptophysin molecule accessible to sulfo-NHS-LC–biotin was biotinylated to an extent sufficient to allow binding to streptavidin. This in turn suggested that the greater proportion of biotinylated synaptophysin obtained at 18° as opposed to 4°C reflected the delivery, at 18°C, either of unbiotinylated synaptophysin molecules from an intracellular pool to the site where biotinylation occurred, or of sulfo-NHS-LC–biotin to an intracellular site where synaptophysin resided. This conclusion was supported by studying the time course of biotinylation of synaptophysin at 18°C, which revealed a continuous increase in the amount of biotinylated synaptophysin over time (Fig. 6, closed circles). Together with the accessibility of the majority of this synaptophysin to MesNa (Fig. 5 C), these results indicate that upon incubation at 18°C, biotinylated synaptophysin accumulated in a membrane compartment connected to the plasma membrane. Interestingly, when PC12 cells incubated for various times at 18°C were subsequently biotinylated for 30 min at 4°C, only ∼2% of the total synaptophysin was biotinylated, irrespective of the incubation time at 18°C (Fig. 6, closed triangles). This suggested that upon cooling the cells to 4°C, the lumen of this membrane compartment became inaccessible to sulfo-NHS-LC–biotin, as was the case for avidin (Fig. 5 B) but not MesNa (Fig. 5 C). Biotinylation of the transferrin receptor at 18°C also increased over time, reaching a value of ∼35% of total by the end of the 30-min incubation (Fig. 6). Given the inaccessibility of the transferrin receptor to MesNa (Fig. 5 D), this implied its continuous delivery to the plasma membrane and endocytosis at 18°C.

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