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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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Synaptophysin biotinylated at 18°C is quantitatively  extracted from paraformaldehyde-fixed PC12 cells by Triton  X-100 (A) and is accessible to anti-synaptophysin after digitonin  permeabilization of fixed cells (B). (A) PC12 cells were incubated  without (−) or with (+) sulfo-NHS-LC–biotin for 30 min at 18°C,  chased for 5 min at 18°C in the presence of glycine, and fixed (+)  or not fixed (−), and Triton X-100 extracts were subjected to  streptavidin–agarose adsorption. Specific immunoreactivity due  to the binding of biotinylated synaptophysin to streptavidin–agarose was determined by incubating the beads without (−) or with  (+) anti-synaptophysin antibody (α-Sy) followed by HRP-conjugated goat anti–mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. (B) PC12  cells were incubated without (−) or with (+) sulfo-NHS-LC– biotin for 30 min at 18°C, chased for 5 min at 18°C in the presence  of glycine, fixed, permeabilized with digitonin, incubated without  (−) or with (+) anti-synaptophysin (α-Sy), and extracted with  Triton X-100. The Triton extracts were subjected to streptavidin– agarose adsorption, and anti-synaptophysin bound to the beads  via biotinylated synaptophysin was detected by HRP-conjugated  goat anti–mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. The lower synaptophysin immunoreactivity in B than A (compare ordinate scales)  presumably reflects incomplete accessibility of the anti-synaptophysin to its epitope when added to digitonin-permeabilized fixed  cells as compared with anti-synaptophysin addition after Triton  X-100 extraction of synaptophysin and its adsorption to streptavidin–agarose beads.
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Figure 12: Synaptophysin biotinylated at 18°C is quantitatively extracted from paraformaldehyde-fixed PC12 cells by Triton X-100 (A) and is accessible to anti-synaptophysin after digitonin permeabilization of fixed cells (B). (A) PC12 cells were incubated without (−) or with (+) sulfo-NHS-LC–biotin for 30 min at 18°C, chased for 5 min at 18°C in the presence of glycine, and fixed (+) or not fixed (−), and Triton X-100 extracts were subjected to streptavidin–agarose adsorption. Specific immunoreactivity due to the binding of biotinylated synaptophysin to streptavidin–agarose was determined by incubating the beads without (−) or with (+) anti-synaptophysin antibody (α-Sy) followed by HRP-conjugated goat anti–mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. (B) PC12 cells were incubated without (−) or with (+) sulfo-NHS-LC– biotin for 30 min at 18°C, chased for 5 min at 18°C in the presence of glycine, fixed, permeabilized with digitonin, incubated without (−) or with (+) anti-synaptophysin (α-Sy), and extracted with Triton X-100. The Triton extracts were subjected to streptavidin– agarose adsorption, and anti-synaptophysin bound to the beads via biotinylated synaptophysin was detected by HRP-conjugated goat anti–mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. The lower synaptophysin immunoreactivity in B than A (compare ordinate scales) presumably reflects incomplete accessibility of the anti-synaptophysin to its epitope when added to digitonin-permeabilized fixed cells as compared with anti-synaptophysin addition after Triton X-100 extraction of synaptophysin and its adsorption to streptavidin–agarose beads.

Mentions: By immunocytochemistry, synaptophysin has been previously shown to be colocalized with transferrin receptor in PC12 cells (Cameron et al., 1991). Given the above biochemical data, we reinvestigated the subcellular localization of synaptophysin. Confirming previous observations (Cameron et al., 1991), synaptophysin was indeed found to be colocalized with internalized transferrin when fixed PC12 cells were permeabilized with Triton X-100 and analyzed by double fluorescence using confocal microscopy (Fig. 11, C, D, c, and d). Synaptophysin immunoreactivity and fluorescent transferrin were predominantly observed in a perinuclear location. Surprisingly, a strikingly different subcellular localization of synaptophysin was observed when fixed PC12 cells were permeabilized with digitonin (Fig. 11, A, B, a, and b). In this condition, synaptophysin immunoreactivity was confined to the cell periphery (Fig. 11, A and a) and was remarkably distinct from the largely perinuclear localization of transferrin fluorescence (Fig. 11, B and b) or immunofluorescence for the transferrin receptor using an antibody against its cytoplasmic domain (data not shown). This difference in the intracellular pattern of synaptophysin immunoreactivity depending on the detergent used reflects a phenomenon that will be described in detail elsewhere (Hannah, M.J., and W.B. Huttner, manuscript in preparation) and is just briefly summarized here. Upon digitonin permeabilization of fixed PC12 cells, the anti-synaptophysin antibody has access only to synaptophysin localized at the cell periphery but not to that localized in the perinuclear area. By contrast, Triton X-100 permeabilization results in the virtually complete extraction, despite paraformaldehyde fixation, of synaptophysin from the cell periphery and allows access of the anti-synaptophysin antibody to synaptophysin in the perinuclear area. (This extration of synaptophysin from fixed cells can be demonstrated biochemically [Fig. 12 A]; Hannah, M.J., and W.B. Huttner, manuscript in preparation.)


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)

Synaptophysin biotinylated at 18°C is quantitatively  extracted from paraformaldehyde-fixed PC12 cells by Triton  X-100 (A) and is accessible to anti-synaptophysin after digitonin  permeabilization of fixed cells (B). (A) PC12 cells were incubated  without (−) or with (+) sulfo-NHS-LC–biotin for 30 min at 18°C,  chased for 5 min at 18°C in the presence of glycine, and fixed (+)  or not fixed (−), and Triton X-100 extracts were subjected to  streptavidin–agarose adsorption. Specific immunoreactivity due  to the binding of biotinylated synaptophysin to streptavidin–agarose was determined by incubating the beads without (−) or with  (+) anti-synaptophysin antibody (α-Sy) followed by HRP-conjugated goat anti–mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. (B) PC12  cells were incubated without (−) or with (+) sulfo-NHS-LC– biotin for 30 min at 18°C, chased for 5 min at 18°C in the presence  of glycine, fixed, permeabilized with digitonin, incubated without  (−) or with (+) anti-synaptophysin (α-Sy), and extracted with  Triton X-100. The Triton extracts were subjected to streptavidin– agarose adsorption, and anti-synaptophysin bound to the beads  via biotinylated synaptophysin was detected by HRP-conjugated  goat anti–mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. The lower synaptophysin immunoreactivity in B than A (compare ordinate scales)  presumably reflects incomplete accessibility of the anti-synaptophysin to its epitope when added to digitonin-permeabilized fixed  cells as compared with anti-synaptophysin addition after Triton  X-100 extraction of synaptophysin and its adsorption to streptavidin–agarose beads.
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Figure 12: Synaptophysin biotinylated at 18°C is quantitatively extracted from paraformaldehyde-fixed PC12 cells by Triton X-100 (A) and is accessible to anti-synaptophysin after digitonin permeabilization of fixed cells (B). (A) PC12 cells were incubated without (−) or with (+) sulfo-NHS-LC–biotin for 30 min at 18°C, chased for 5 min at 18°C in the presence of glycine, and fixed (+) or not fixed (−), and Triton X-100 extracts were subjected to streptavidin–agarose adsorption. Specific immunoreactivity due to the binding of biotinylated synaptophysin to streptavidin–agarose was determined by incubating the beads without (−) or with (+) anti-synaptophysin antibody (α-Sy) followed by HRP-conjugated goat anti–mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. (B) PC12 cells were incubated without (−) or with (+) sulfo-NHS-LC– biotin for 30 min at 18°C, chased for 5 min at 18°C in the presence of glycine, fixed, permeabilized with digitonin, incubated without (−) or with (+) anti-synaptophysin (α-Sy), and extracted with Triton X-100. The Triton extracts were subjected to streptavidin– agarose adsorption, and anti-synaptophysin bound to the beads via biotinylated synaptophysin was detected by HRP-conjugated goat anti–mouse IgG antibody. Data are the mean of values obtained from three coverslips; bars indicate SD. The lower synaptophysin immunoreactivity in B than A (compare ordinate scales) presumably reflects incomplete accessibility of the anti-synaptophysin to its epitope when added to digitonin-permeabilized fixed cells as compared with anti-synaptophysin addition after Triton X-100 extraction of synaptophysin and its adsorption to streptavidin–agarose beads.
Mentions: By immunocytochemistry, synaptophysin has been previously shown to be colocalized with transferrin receptor in PC12 cells (Cameron et al., 1991). Given the above biochemical data, we reinvestigated the subcellular localization of synaptophysin. Confirming previous observations (Cameron et al., 1991), synaptophysin was indeed found to be colocalized with internalized transferrin when fixed PC12 cells were permeabilized with Triton X-100 and analyzed by double fluorescence using confocal microscopy (Fig. 11, C, D, c, and d). Synaptophysin immunoreactivity and fluorescent transferrin were predominantly observed in a perinuclear location. Surprisingly, a strikingly different subcellular localization of synaptophysin was observed when fixed PC12 cells were permeabilized with digitonin (Fig. 11, A, B, a, and b). In this condition, synaptophysin immunoreactivity was confined to the cell periphery (Fig. 11, A and a) and was remarkably distinct from the largely perinuclear localization of transferrin fluorescence (Fig. 11, B and b) or immunofluorescence for the transferrin receptor using an antibody against its cytoplasmic domain (data not shown). This difference in the intracellular pattern of synaptophysin immunoreactivity depending on the detergent used reflects a phenomenon that will be described in detail elsewhere (Hannah, M.J., and W.B. Huttner, manuscript in preparation) and is just briefly summarized here. Upon digitonin permeabilization of fixed PC12 cells, the anti-synaptophysin antibody has access only to synaptophysin localized at the cell periphery but not to that localized in the perinuclear area. By contrast, Triton X-100 permeabilization results in the virtually complete extraction, despite paraformaldehyde fixation, of synaptophysin from the cell periphery and allows access of the anti-synaptophysin antibody to synaptophysin in the perinuclear area. (This extration of synaptophysin from fixed cells can be demonstrated biochemically [Fig. 12 A]; Hannah, M.J., and W.B. Huttner, manuscript in preparation.)

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