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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 appearance of biotinylated synaptophysin in SLMVs. PC12  cells were incubated with  sulfo-NHS-LC–biotin for 5  min at 37°C and chased at  37°C for various times as indicated (A) or for 3 min (B)  and 180 min (C). The 66,000 g  (A) or 12,000 g (B and C) supernatants prepared from  the cells were subjected to  glycerol gradient centrifugation, and the fractions were  analyzed for biotinylated  synaptophysin by streptavidin–agarose adsorption followed by immunoblotting of  bound material with antisynaptophysin. (A) Synaptophysin immunoreactivity in the SLMV-containing fractions is expressed as percent of total biotinylated synaptophysin. Data are  the mean of two (0, 10, 180 min) or three (60 min) independent  experiments; bars indicate the variation of individual values from  the mean or the standard error, respectively. (B and C) Immunoblots; 2.5% (B) and 11% (C) of the total biotinylated synaptophysin was recovered in the SLMV-containing fractions (B, n 5–8;  C, n 4–8).
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Figure 3: Time course of appearance of biotinylated synaptophysin in SLMVs. PC12 cells were incubated with sulfo-NHS-LC–biotin for 5 min at 37°C and chased at 37°C for various times as indicated (A) or for 3 min (B) and 180 min (C). The 66,000 g (A) or 12,000 g (B and C) supernatants prepared from the cells were subjected to glycerol gradient centrifugation, and the fractions were analyzed for biotinylated synaptophysin by streptavidin–agarose adsorption followed by immunoblotting of bound material with antisynaptophysin. (A) Synaptophysin immunoreactivity in the SLMV-containing fractions is expressed as percent of total biotinylated synaptophysin. Data are the mean of two (0, 10, 180 min) or three (60 min) independent experiments; bars indicate the variation of individual values from the mean or the standard error, respectively. (B and C) Immunoblots; 2.5% (B) and 11% (C) of the total biotinylated synaptophysin was recovered in the SLMV-containing fractions (B, n 5–8; C, n 4–8).

Mentions: We next determined the kinetics of appearance of cell surface–biotinylated synaptophysin in SLMVs. PC12 cells were biotinylated for 5 min, chased for various times, and analyzed by glycerol gradient centrifugation to separate SLMVs from other membranes. This showed that cell surface–biotinylated synaptophysin appeared very rapidly in SLMVs. Already without chase, i.e., at the end of the 5 min biotinylation, a small proportion (∼2%) of the biotinylated synaptophysin was detectable in the SLMV-containing fractions of the glycerol gradient (Fig. 3 A). A similar observation was made after 3 min of chase (Fig. 3 B; 2.5% of total biotinylated synaptophysin recovered in SLMV fractions). Within 10 min of chase, the appearance of biotinylated synaptophysin in SLMVs approached a value corresponding to ∼13% of the total biotinylated synaptophysin, which remained constant for at least 3 h (Fig. 3, A and 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 appearance of biotinylated synaptophysin in SLMVs. PC12  cells were incubated with  sulfo-NHS-LC–biotin for 5  min at 37°C and chased at  37°C for various times as indicated (A) or for 3 min (B)  and 180 min (C). The 66,000 g  (A) or 12,000 g (B and C) supernatants prepared from  the cells were subjected to  glycerol gradient centrifugation, and the fractions were  analyzed for biotinylated  synaptophysin by streptavidin–agarose adsorption followed by immunoblotting of  bound material with antisynaptophysin. (A) Synaptophysin immunoreactivity in the SLMV-containing fractions is expressed as percent of total biotinylated synaptophysin. Data are  the mean of two (0, 10, 180 min) or three (60 min) independent  experiments; bars indicate the variation of individual values from  the mean or the standard error, respectively. (B and C) Immunoblots; 2.5% (B) and 11% (C) of the total biotinylated synaptophysin was recovered in the SLMV-containing fractions (B, n 5–8;  C, n 4–8).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139769&req=5

Figure 3: Time course of appearance of biotinylated synaptophysin in SLMVs. PC12 cells were incubated with sulfo-NHS-LC–biotin for 5 min at 37°C and chased at 37°C for various times as indicated (A) or for 3 min (B) and 180 min (C). The 66,000 g (A) or 12,000 g (B and C) supernatants prepared from the cells were subjected to glycerol gradient centrifugation, and the fractions were analyzed for biotinylated synaptophysin by streptavidin–agarose adsorption followed by immunoblotting of bound material with antisynaptophysin. (A) Synaptophysin immunoreactivity in the SLMV-containing fractions is expressed as percent of total biotinylated synaptophysin. Data are the mean of two (0, 10, 180 min) or three (60 min) independent experiments; bars indicate the variation of individual values from the mean or the standard error, respectively. (B and C) Immunoblots; 2.5% (B) and 11% (C) of the total biotinylated synaptophysin was recovered in the SLMV-containing fractions (B, n 5–8; C, n 4–8).
Mentions: We next determined the kinetics of appearance of cell surface–biotinylated synaptophysin in SLMVs. PC12 cells were biotinylated for 5 min, chased for various times, and analyzed by glycerol gradient centrifugation to separate SLMVs from other membranes. This showed that cell surface–biotinylated synaptophysin appeared very rapidly in SLMVs. Already without chase, i.e., at the end of the 5 min biotinylation, a small proportion (∼2%) of the biotinylated synaptophysin was detectable in the SLMV-containing fractions of the glycerol gradient (Fig. 3 A). A similar observation was made after 3 min of chase (Fig. 3 B; 2.5% of total biotinylated synaptophysin recovered in SLMV fractions). Within 10 min of chase, the appearance of biotinylated synaptophysin in SLMVs approached a value corresponding to ∼13% of the total biotinylated synaptophysin, which remained constant for at least 3 h (Fig. 3, A and 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