Limits...
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.

Show MeSH

Related in: MedlinePlus

Morphology of the SLMV donor compartment. PC12 cells incubated at 18° (A–D) or 37°C (E–H) with sulfo-NHS-LC–biotin  for 30 min followed by a 5-min chase were fixed at 4°C. Ultrathin cryosections were immunogold-labeled for synaptophysin and analyzed by electron microscopy. Note the synaptophysin immunoreactivity associated with tubulo-cisternal membrane structures beneath  the plasma membrane. Little synaptophysin immunoreactivity is found at the plasma membrane itself. Bars, 100 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139769&req=5

Figure 13: Morphology of the SLMV donor compartment. PC12 cells incubated at 18° (A–D) or 37°C (E–H) with sulfo-NHS-LC–biotin for 30 min followed by a 5-min chase were fixed at 4°C. Ultrathin cryosections were immunogold-labeled for synaptophysin and analyzed by electron microscopy. Note the synaptophysin immunoreactivity associated with tubulo-cisternal membrane structures beneath the plasma membrane. Little synaptophysin immunoreactivity is found at the plasma membrane itself. Bars, 100 nm.

Mentions: Electron microscopy of immunogold-labeled ultrathin cryosections revealed synaptophysin immunoreactivity associated with a pleiomorphic subplasmalemmal membrane system in both PC12 cells biotinylated for 30 min at 18°C (Fig. 13, A–D) and cells kept at 37°C (Fig. 13, E–H). Most membranes immunoreactive for synaptophysin had a tubular or cisternal morphology and often were localized in very close proximity to the plasma membrane. Connections between these membranes and the plasmalemma could not be convincingly demonstrated, presumably because the fixation had to be carried out at 4°C to allow a comparison of synaptophysin immunoreactivity between the cells incubated at 18° and 37°C (see Materials and Methods); at 4°C, these connections become very narrow (Fig. 6 and Discussion). Little synaptophysin immunoreactivity was detected on the plasma membrane itself, consistent with the results of biotinylation at 4°C (Fig. 5 A).


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)

Morphology of the SLMV donor compartment. PC12 cells incubated at 18° (A–D) or 37°C (E–H) with sulfo-NHS-LC–biotin  for 30 min followed by a 5-min chase were fixed at 4°C. Ultrathin cryosections were immunogold-labeled for synaptophysin and analyzed by electron microscopy. Note the synaptophysin immunoreactivity associated with tubulo-cisternal membrane structures beneath  the plasma membrane. Little synaptophysin immunoreactivity is found at the plasma membrane itself. Bars, 100 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 13: Morphology of the SLMV donor compartment. PC12 cells incubated at 18° (A–D) or 37°C (E–H) with sulfo-NHS-LC–biotin for 30 min followed by a 5-min chase were fixed at 4°C. Ultrathin cryosections were immunogold-labeled for synaptophysin and analyzed by electron microscopy. Note the synaptophysin immunoreactivity associated with tubulo-cisternal membrane structures beneath the plasma membrane. Little synaptophysin immunoreactivity is found at the plasma membrane itself. Bars, 100 nm.
Mentions: Electron microscopy of immunogold-labeled ultrathin cryosections revealed synaptophysin immunoreactivity associated with a pleiomorphic subplasmalemmal membrane system in both PC12 cells biotinylated for 30 min at 18°C (Fig. 13, A–D) and cells kept at 37°C (Fig. 13, E–H). Most membranes immunoreactive for synaptophysin had a tubular or cisternal morphology and often were localized in very close proximity to the plasma membrane. Connections between these membranes and the plasmalemma could not be convincingly demonstrated, presumably because the fixation had to be carried out at 4°C to allow a comparison of synaptophysin immunoreactivity between the cells incubated at 18° and 37°C (see Materials and Methods); at 4°C, these connections become very narrow (Fig. 6 and Discussion). Little synaptophysin immunoreactivity was detected on the plasma membrane itself, consistent with the results of biotinylation at 4°C (Fig. 5 A).

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