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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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Electron microscopy of glycerol gradient  fractions. The 12,000 g supernatant prepared from PC12  cells was subjected to glycerol gradient centrifugation,  and the pools of fractions 1  and 2 (A) and fractions 5–9  (B) were subjected to immunoisolation using anti-synaptophysin beads followed by  fixation and processing for  electron microscopy. Note  the heterogeneous membrane structures in A and the  homogenous population of  50-nm vesicles in B. No  membranes were adsorbed  to the beads if the anti-synaptophysin antibody was  omitted (not shown). Bars,  300 nm.
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Figure 2: Electron microscopy of glycerol gradient fractions. The 12,000 g supernatant prepared from PC12 cells was subjected to glycerol gradient centrifugation, and the pools of fractions 1 and 2 (A) and fractions 5–9 (B) were subjected to immunoisolation using anti-synaptophysin beads followed by fixation and processing for electron microscopy. Note the heterogeneous membrane structures in A and the homogenous population of 50-nm vesicles in B. No membranes were adsorbed to the beads if the anti-synaptophysin antibody was omitted (not shown). Bars, 300 nm.

Mentions: To study the donor membrane from which SLMVs originate, we chose to label synaptophysin of PC12 cells by surface biotinylation, exploiting the fact that the newly synthesized form of this protein travels via the plasma membrane to SLMVs (Régnier-Vigouroux et al., 1991). We first ascertained that biotinylation of synaptophysin, which modifies lysine residues, does not interfere with its ability to be sorted to SLMVs. The appearance in SLMVs of newly synthesized synaptophysin, sulfate labeled in the trans-Golgi network, approaches a plateau after 3 h of chase (RégnierVigouroux et al., 1991). We therefore labeled PC12 cells for 5 min at 37°C by addition of the membrane-impermeant sulfo-NHS-LC–biotin to the medium, followed by a 3-h chase. After glycerol gradient centrifugation which separates SLMVs from larger membranes (Clift-O'Grady et al., 1990), two differentially sedimenting populations of biotinylated synaptophysin (determined from its binding to streptavidin–agarose) were recovered in the bottom and middle fractions of the gradient, respectively (Fig. 1, A and B), both of which also contained nonbiotinylated synaptophysin (Fig. 1 B). The bottom, but not the middle, fractions also contained biotinylated transferrin receptor (Fig. 1 A), a membrane protein constitutively cycling between the plasma membrane and early endosomes (Trowbridge et al., 1993). Electron microscopy of the membranes immunoadsorbed to beads coated with an antibody against the cytoplasmic tail of synaptophysin showed that the bottom fractions contained synaptophysin in membrane structures of various sizes (Fig. 2 A). Given the presence of transferrin receptor in these fractions, and in line with previous reports (Clift-O'Grady et al., 1990; Cameron et al., 1991), the biotinylated synaptophysin in the bottom fractions was presumably present in membranes of early endosomes. Electron microscopy of the synaptophysin-containing membranes in the middle fractions of the glycerol gradient showed that these had the expected morphology of SLMVs (Fig. 2 B), corroborating previous conclusions based on biochemical data (Clift-O'Grady et al., 1990; Cameron et al., 1991; Linstedt and Kelly, 1991). We conclude that synaptophysin biotinylated at the cell surface, like nonbiotinylated synaptophysin, is sorted to SLMVs.


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)

Electron microscopy of glycerol gradient  fractions. The 12,000 g supernatant prepared from PC12  cells was subjected to glycerol gradient centrifugation,  and the pools of fractions 1  and 2 (A) and fractions 5–9  (B) were subjected to immunoisolation using anti-synaptophysin beads followed by  fixation and processing for  electron microscopy. Note  the heterogeneous membrane structures in A and the  homogenous population of  50-nm vesicles in B. No  membranes were adsorbed  to the beads if the anti-synaptophysin antibody was  omitted (not shown). Bars,  300 nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139769&req=5

Figure 2: Electron microscopy of glycerol gradient fractions. The 12,000 g supernatant prepared from PC12 cells was subjected to glycerol gradient centrifugation, and the pools of fractions 1 and 2 (A) and fractions 5–9 (B) were subjected to immunoisolation using anti-synaptophysin beads followed by fixation and processing for electron microscopy. Note the heterogeneous membrane structures in A and the homogenous population of 50-nm vesicles in B. No membranes were adsorbed to the beads if the anti-synaptophysin antibody was omitted (not shown). Bars, 300 nm.
Mentions: To study the donor membrane from which SLMVs originate, we chose to label synaptophysin of PC12 cells by surface biotinylation, exploiting the fact that the newly synthesized form of this protein travels via the plasma membrane to SLMVs (Régnier-Vigouroux et al., 1991). We first ascertained that biotinylation of synaptophysin, which modifies lysine residues, does not interfere with its ability to be sorted to SLMVs. The appearance in SLMVs of newly synthesized synaptophysin, sulfate labeled in the trans-Golgi network, approaches a plateau after 3 h of chase (RégnierVigouroux et al., 1991). We therefore labeled PC12 cells for 5 min at 37°C by addition of the membrane-impermeant sulfo-NHS-LC–biotin to the medium, followed by a 3-h chase. After glycerol gradient centrifugation which separates SLMVs from larger membranes (Clift-O'Grady et al., 1990), two differentially sedimenting populations of biotinylated synaptophysin (determined from its binding to streptavidin–agarose) were recovered in the bottom and middle fractions of the gradient, respectively (Fig. 1, A and B), both of which also contained nonbiotinylated synaptophysin (Fig. 1 B). The bottom, but not the middle, fractions also contained biotinylated transferrin receptor (Fig. 1 A), a membrane protein constitutively cycling between the plasma membrane and early endosomes (Trowbridge et al., 1993). Electron microscopy of the membranes immunoadsorbed to beads coated with an antibody against the cytoplasmic tail of synaptophysin showed that the bottom fractions contained synaptophysin in membrane structures of various sizes (Fig. 2 A). Given the presence of transferrin receptor in these fractions, and in line with previous reports (Clift-O'Grady et al., 1990; Cameron et al., 1991), the biotinylated synaptophysin in the bottom fractions was presumably present in membranes of early endosomes. Electron microscopy of the synaptophysin-containing membranes in the middle fractions of the glycerol gradient showed that these had the expected morphology of SLMVs (Fig. 2 B), corroborating previous conclusions based on biochemical data (Clift-O'Grady et al., 1990; Cameron et al., 1991; Linstedt and Kelly, 1991). We conclude that synaptophysin biotinylated at the cell surface, like nonbiotinylated synaptophysin, is sorted to SLMVs.

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