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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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Biotinylated synaptophysin does not appear in  SLMVs at 18°C. PC12 cells  were incubated with sulfoNHS-LC–biotin either for 30  min at 37°C (A), for 30 min at  18°C (B–E), or for 30 min at  18°C followed by a 30-min  chase at 37°C (F). The 12,000 g  (A–D) or 66,000 g (E and F)  supernatants prepared from  the cells were subjected to  glycerol gradient centrifugation, and the fractions were  analyzed for biotinylated (BSy; A, B, and F) and nonbiotinylated (NB-Sy; D and E)  synaptophysin and biotinylated transferrin receptor (BTfR; C) by streptavidin–agarose adsorption followed by  immunoblotting of bound  (A–C and F) and unbound  (D and E) material with the  respective antibodies. The  immunoblots shown in (B) and (C) were obtained from the same  filter. 16% (A) and 15% (F) of the total biotinylated synaptophysin was recovered in the SLMV-containing fractions (A, n  5–8; F, n 4–8); the immunoblot shown in F is a longer exposure  relative to that shown in A. Note that the ratio of SLMVs to the  larger membranes recovered in the bottom fractions of the gradient is greater in E than D, because a 66,000 g and a 12,000 g supernatant, respectively, was subjected to glycerol gradient centrifugation.
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Figure 4: Biotinylated synaptophysin does not appear in SLMVs at 18°C. PC12 cells were incubated with sulfoNHS-LC–biotin either for 30 min at 37°C (A), for 30 min at 18°C (B–E), or for 30 min at 18°C followed by a 30-min chase at 37°C (F). The 12,000 g (A–D) or 66,000 g (E and F) supernatants prepared from the cells were subjected to glycerol gradient centrifugation, and the fractions were analyzed for biotinylated (BSy; A, B, and F) and nonbiotinylated (NB-Sy; D and E) synaptophysin and biotinylated transferrin receptor (BTfR; C) by streptavidin–agarose adsorption followed by immunoblotting of bound (A–C and F) and unbound (D and E) material with the respective antibodies. The immunoblots shown in (B) and (C) were obtained from the same filter. 16% (A) and 15% (F) of the total biotinylated synaptophysin was recovered in the SLMV-containing fractions (A, n 5–8; F, n 4–8); the immunoblot shown in F is a longer exposure relative to that shown in A. Note that the ratio of SLMVs to the larger membranes recovered in the bottom fractions of the gradient is greater in E than D, because a 66,000 g and a 12,000 g supernatant, respectively, was subjected to glycerol gradient centrifugation.

Mentions: Glycerol gradient analysis of PC12 cells incubated for 30 min at 18°C with sulfo-NHS-LC–biotin showed that the reduced temperature indeed blocked the appearance of biotinylated synaptophysin in SLMVs. In contrast to an incubation at 37°C after which biotinylated synaptophysin was found in both bottom and middle fractions of the gradient (Fig. 4 A), synaptophysin biotinylated at 18°C was found in the bottom fractions of the gradient (Fig. 4 B) containing early endosomes, as indicated by the presence of biotinylated transferrin receptor (Fig. 4 C), but not in the middle fractions (Fig. 4 B) containing SLMVs formed before biotinylation, as indicated by the presence of nonbiotinylated synaptophysin (Fig. 4, D and E; two distinct procedures of differential centrifugation before the glycerol gradient; see figure legend). A chase for 30 min at 37°C, performed after the 30 min biotinylation at 18°C, resulted in the appearance of biotinylated synaptophysin in SLMVs (∼15% of the total biotinylated synaptophysin; Fig. 4 F), showing that the temperature block was reversible. Our observations are consistent with the results of Desnos et al. (1995) reported while the present work was in progress, which show that an epitope-tagged VAMP/synaptobrevin mutant labeled by an antibody bound to its extracellular domain does not appear in SLMVs at 15°C but does so at 37°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)

Biotinylated synaptophysin does not appear in  SLMVs at 18°C. PC12 cells  were incubated with sulfoNHS-LC–biotin either for 30  min at 37°C (A), for 30 min at  18°C (B–E), or for 30 min at  18°C followed by a 30-min  chase at 37°C (F). The 12,000 g  (A–D) or 66,000 g (E and F)  supernatants prepared from  the cells were subjected to  glycerol gradient centrifugation, and the fractions were  analyzed for biotinylated (BSy; A, B, and F) and nonbiotinylated (NB-Sy; D and E)  synaptophysin and biotinylated transferrin receptor (BTfR; C) by streptavidin–agarose adsorption followed by  immunoblotting of bound  (A–C and F) and unbound  (D and E) material with the  respective antibodies. The  immunoblots shown in (B) and (C) were obtained from the same  filter. 16% (A) and 15% (F) of the total biotinylated synaptophysin was recovered in the SLMV-containing fractions (A, n  5–8; F, n 4–8); the immunoblot shown in F is a longer exposure  relative to that shown in A. Note that the ratio of SLMVs to the  larger membranes recovered in the bottom fractions of the gradient is greater in E than D, because a 66,000 g and a 12,000 g supernatant, respectively, was subjected to glycerol gradient centrifugation.
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Figure 4: Biotinylated synaptophysin does not appear in SLMVs at 18°C. PC12 cells were incubated with sulfoNHS-LC–biotin either for 30 min at 37°C (A), for 30 min at 18°C (B–E), or for 30 min at 18°C followed by a 30-min chase at 37°C (F). The 12,000 g (A–D) or 66,000 g (E and F) supernatants prepared from the cells were subjected to glycerol gradient centrifugation, and the fractions were analyzed for biotinylated (BSy; A, B, and F) and nonbiotinylated (NB-Sy; D and E) synaptophysin and biotinylated transferrin receptor (BTfR; C) by streptavidin–agarose adsorption followed by immunoblotting of bound (A–C and F) and unbound (D and E) material with the respective antibodies. The immunoblots shown in (B) and (C) were obtained from the same filter. 16% (A) and 15% (F) of the total biotinylated synaptophysin was recovered in the SLMV-containing fractions (A, n 5–8; F, n 4–8); the immunoblot shown in F is a longer exposure relative to that shown in A. Note that the ratio of SLMVs to the larger membranes recovered in the bottom fractions of the gradient is greater in E than D, because a 66,000 g and a 12,000 g supernatant, respectively, was subjected to glycerol gradient centrifugation.
Mentions: Glycerol gradient analysis of PC12 cells incubated for 30 min at 18°C with sulfo-NHS-LC–biotin showed that the reduced temperature indeed blocked the appearance of biotinylated synaptophysin in SLMVs. In contrast to an incubation at 37°C after which biotinylated synaptophysin was found in both bottom and middle fractions of the gradient (Fig. 4 A), synaptophysin biotinylated at 18°C was found in the bottom fractions of the gradient (Fig. 4 B) containing early endosomes, as indicated by the presence of biotinylated transferrin receptor (Fig. 4 C), but not in the middle fractions (Fig. 4 B) containing SLMVs formed before biotinylation, as indicated by the presence of nonbiotinylated synaptophysin (Fig. 4, D and E; two distinct procedures of differential centrifugation before the glycerol gradient; see figure legend). A chase for 30 min at 37°C, performed after the 30 min biotinylation at 18°C, resulted in the appearance of biotinylated synaptophysin in SLMVs (∼15% of the total biotinylated synaptophysin; Fig. 4 F), showing that the temperature block was reversible. Our observations are consistent with the results of Desnos et al. (1995) reported while the present work was in progress, which show that an epitope-tagged VAMP/synaptobrevin mutant labeled by an antibody bound to its extracellular domain does not appear in SLMVs at 15°C but does so at 37°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