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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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Model for the biogenesis of SLMVs from a subplasmalemmal compartment connected to the plasma membrane. T,  transferrin receptor; S, synaptophysin. Segregation of synaptophysin from the transferrin receptor occurs at the plasma membrane. The transferrin receptor is internalized via endocytic vesicles  to endosomes located predominantly in the perinuclear region  (MesNa protected), from which it recycles to the plasma membrane. Both endocytosis and recycling occur at 18°C. Synaptophysin, but not the transferrin receptor, moves into the SLMV  donor compartment via lateral mobility and/or membrane invagination. The SLMV donor compartment, located at the periphery  of the cell, is connected with the plasma membrane via a narrow  membrane continuity allowing entry of MesNa, but not avidin, at  4°C. From the SLMV donor compartment, a minor proportion of  synaptophysin (10–15%) is incorporated into SLMVs. This process is blocked at 18°C. The majority of synaptophysin is either  delivered to perinuclear, MesNa-protected endosomes from which  it recycles, like the transferrin receptor, to the plasma membrane  (broken arrows) or moves to MesNa-inaccessible membranes  connected to the SLMV donor compartment (not illustrated).  For further details, see Discussion.
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Figure 14: Model for the biogenesis of SLMVs from a subplasmalemmal compartment connected to the plasma membrane. T, transferrin receptor; S, synaptophysin. Segregation of synaptophysin from the transferrin receptor occurs at the plasma membrane. The transferrin receptor is internalized via endocytic vesicles to endosomes located predominantly in the perinuclear region (MesNa protected), from which it recycles to the plasma membrane. Both endocytosis and recycling occur at 18°C. Synaptophysin, but not the transferrin receptor, moves into the SLMV donor compartment via lateral mobility and/or membrane invagination. The SLMV donor compartment, located at the periphery of the cell, is connected with the plasma membrane via a narrow membrane continuity allowing entry of MesNa, but not avidin, at 4°C. From the SLMV donor compartment, a minor proportion of synaptophysin (10–15%) is incorporated into SLMVs. This process is blocked at 18°C. The majority of synaptophysin is either delivered to perinuclear, MesNa-protected endosomes from which it recycles, like the transferrin receptor, to the plasma membrane (broken arrows) or moves to MesNa-inaccessible membranes connected to the SLMV donor compartment (not illustrated). For further details, see Discussion.

Mentions: We have characterized the compartment from which SLMVs originate, using cell surface–biotinylated synaptophysin and SV2 as markers. Our results lead to a modification of the concept about the biogenesis of SLMVs in neuroendocrine cells, which is illustrated by the model shown in Fig. 14. According to our results, SLMVs originate from a subplasmalemmal tubulo-cisternal membrane system, referred to as SLMV donor compartment, that is (a) distinct from the transferrin receptor–containing endosome and (b) connected to the plasmalemma via a narrow membrane continuity.


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)

Model for the biogenesis of SLMVs from a subplasmalemmal compartment connected to the plasma membrane. T,  transferrin receptor; S, synaptophysin. Segregation of synaptophysin from the transferrin receptor occurs at the plasma membrane. The transferrin receptor is internalized via endocytic vesicles  to endosomes located predominantly in the perinuclear region  (MesNa protected), from which it recycles to the plasma membrane. Both endocytosis and recycling occur at 18°C. Synaptophysin, but not the transferrin receptor, moves into the SLMV  donor compartment via lateral mobility and/or membrane invagination. The SLMV donor compartment, located at the periphery  of the cell, is connected with the plasma membrane via a narrow  membrane continuity allowing entry of MesNa, but not avidin, at  4°C. From the SLMV donor compartment, a minor proportion of  synaptophysin (10–15%) is incorporated into SLMVs. This process is blocked at 18°C. The majority of synaptophysin is either  delivered to perinuclear, MesNa-protected endosomes from which  it recycles, like the transferrin receptor, to the plasma membrane  (broken arrows) or moves to MesNa-inaccessible membranes  connected to the SLMV donor compartment (not illustrated).  For further details, see Discussion.
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Related In: Results  -  Collection

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

Figure 14: Model for the biogenesis of SLMVs from a subplasmalemmal compartment connected to the plasma membrane. T, transferrin receptor; S, synaptophysin. Segregation of synaptophysin from the transferrin receptor occurs at the plasma membrane. The transferrin receptor is internalized via endocytic vesicles to endosomes located predominantly in the perinuclear region (MesNa protected), from which it recycles to the plasma membrane. Both endocytosis and recycling occur at 18°C. Synaptophysin, but not the transferrin receptor, moves into the SLMV donor compartment via lateral mobility and/or membrane invagination. The SLMV donor compartment, located at the periphery of the cell, is connected with the plasma membrane via a narrow membrane continuity allowing entry of MesNa, but not avidin, at 4°C. From the SLMV donor compartment, a minor proportion of synaptophysin (10–15%) is incorporated into SLMVs. This process is blocked at 18°C. The majority of synaptophysin is either delivered to perinuclear, MesNa-protected endosomes from which it recycles, like the transferrin receptor, to the plasma membrane (broken arrows) or moves to MesNa-inaccessible membranes connected to the SLMV donor compartment (not illustrated). For further details, see Discussion.
Mentions: We have characterized the compartment from which SLMVs originate, using cell surface–biotinylated synaptophysin and SV2 as markers. Our results lead to a modification of the concept about the biogenesis of SLMVs in neuroendocrine cells, which is illustrated by the model shown in Fig. 14. According to our results, SLMVs originate from a subplasmalemmal tubulo-cisternal membrane system, referred to as SLMV donor compartment, that is (a) distinct from the transferrin receptor–containing endosome and (b) connected to the plasmalemma via a narrow membrane continuity.

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