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Thy-1 is a component common to multiple populations of synaptic vesicles.

Jeng CJ, McCarroll SA, Martin TF, Floor E, Adams J, Krantz D, Butz S, Edwards R, Schweitzer ES - J. Cell Biol. (1998)

Bottom Line: Thy-1 is also present in synaptic vesicles in rat central nervous system.Experiments on permeabilized PC12 cells demonstrate that antibodies against Thy-1 inhibit the regulated release of neurotransmitter; this inhibition appears to be independent of any effect on the Ca2+ channel.These findings suggest Thy-1 is an integral component of many types of regulated secretory vesicles, and plays an important role in the regulated vesicular release of neurotransmitter at the synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90095-1763, USA.

ABSTRACT
Thy-1, a glycosylphosphatidylinositol-linked integral membrane protein of the immunoglobulin superfamily, is a component of both large dense-core and small clear vesicles in PC12 cells. A majority of this protein, formerly recognized only on the plasma membrane of neurons, is localized to regulated secretory vesicles. Thy-1 is also present in synaptic vesicles in rat central nervous system. Experiments on permeabilized PC12 cells demonstrate that antibodies against Thy-1 inhibit the regulated release of neurotransmitter; this inhibition appears to be independent of any effect on the Ca2+ channel. These findings suggest Thy-1 is an integral component of many types of regulated secretory vesicles, and plays an important role in the regulated vesicular release of neurotransmitter at the synapse.

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Immunofluorescent staining of nonpermeabilized PC12 cells with 7C8. NGF-treated PC12 cells were incubated with 7C8  antibodies at 0°C for 1 h before fixation. After fixation, cells were incubated with biotinylated sheep anti–mouse antibodies, followed  by Texas red-streptavidin. Bar, 10 μm. A and  B represent the phase contrast and fluorescence images of the same cells; C represents a  single confocal optical section through a different cluster of PC12 cells.
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Figure 6: Immunofluorescent staining of nonpermeabilized PC12 cells with 7C8. NGF-treated PC12 cells were incubated with 7C8 antibodies at 0°C for 1 h before fixation. After fixation, cells were incubated with biotinylated sheep anti–mouse antibodies, followed by Texas red-streptavidin. Bar, 10 μm. A and B represent the phase contrast and fluorescence images of the same cells; C represents a single confocal optical section through a different cluster of PC12 cells.

Mentions: Because Thy-1 has previously been considered to be only a marker for the plasma membrane, it was important to ask why we did not observe 7C8 binding to the cell surface as well as to vesicles. It was also essential to determine unambiguously that the Thy-1 we observed as a component of vesicles was not contamination from the plasma membrane. To address the former question, we carried out immunofluorescent staining of nonfixed, nonpermeabilized PC12 cells to determine whether we could observe surface staining. As shown in Fig. 6, binding of the antibody to the cells before fixation and detergent treatment resulted in intense staining around the periphery of the cell, consistent with staining of the plasma membrane. Our standard procedure for immunofluorescence involved fixing cells and then permeabilizing with 0.1% Triton X-100 for 2 min. Although we occasionally observed such surface staining with conventionally fixed and permeabilized cells (data not shown), we routinely observed only the vesicular staining illustrated in Figs. 2 and 3. Why the plasma membrane Thy-1 is preferentially lost during standard fixation and permeabilization, even as the intracellular staining is retained, is not completely clear at this point. However, perhaps because of its GPI anchor, Thy-1 is known to present unusual difficulties for morphological localization (Morris and Grosveld, 1989).


Thy-1 is a component common to multiple populations of synaptic vesicles.

Jeng CJ, McCarroll SA, Martin TF, Floor E, Adams J, Krantz D, Butz S, Edwards R, Schweitzer ES - J. Cell Biol. (1998)

Immunofluorescent staining of nonpermeabilized PC12 cells with 7C8. NGF-treated PC12 cells were incubated with 7C8  antibodies at 0°C for 1 h before fixation. After fixation, cells were incubated with biotinylated sheep anti–mouse antibodies, followed  by Texas red-streptavidin. Bar, 10 μm. A and  B represent the phase contrast and fluorescence images of the same cells; C represents a  single confocal optical section through a different cluster of PC12 cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140167&req=5

Figure 6: Immunofluorescent staining of nonpermeabilized PC12 cells with 7C8. NGF-treated PC12 cells were incubated with 7C8 antibodies at 0°C for 1 h before fixation. After fixation, cells were incubated with biotinylated sheep anti–mouse antibodies, followed by Texas red-streptavidin. Bar, 10 μm. A and B represent the phase contrast and fluorescence images of the same cells; C represents a single confocal optical section through a different cluster of PC12 cells.
Mentions: Because Thy-1 has previously been considered to be only a marker for the plasma membrane, it was important to ask why we did not observe 7C8 binding to the cell surface as well as to vesicles. It was also essential to determine unambiguously that the Thy-1 we observed as a component of vesicles was not contamination from the plasma membrane. To address the former question, we carried out immunofluorescent staining of nonfixed, nonpermeabilized PC12 cells to determine whether we could observe surface staining. As shown in Fig. 6, binding of the antibody to the cells before fixation and detergent treatment resulted in intense staining around the periphery of the cell, consistent with staining of the plasma membrane. Our standard procedure for immunofluorescence involved fixing cells and then permeabilizing with 0.1% Triton X-100 for 2 min. Although we occasionally observed such surface staining with conventionally fixed and permeabilized cells (data not shown), we routinely observed only the vesicular staining illustrated in Figs. 2 and 3. Why the plasma membrane Thy-1 is preferentially lost during standard fixation and permeabilization, even as the intracellular staining is retained, is not completely clear at this point. However, perhaps because of its GPI anchor, Thy-1 is known to present unusual difficulties for morphological localization (Morris and Grosveld, 1989).

Bottom Line: Thy-1 is also present in synaptic vesicles in rat central nervous system.Experiments on permeabilized PC12 cells demonstrate that antibodies against Thy-1 inhibit the regulated release of neurotransmitter; this inhibition appears to be independent of any effect on the Ca2+ channel.These findings suggest Thy-1 is an integral component of many types of regulated secretory vesicles, and plays an important role in the regulated vesicular release of neurotransmitter at the synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology and Brain Research Institute, UCLA School of Medicine, Los Angeles, California 90095-1763, USA.

ABSTRACT
Thy-1, a glycosylphosphatidylinositol-linked integral membrane protein of the immunoglobulin superfamily, is a component of both large dense-core and small clear vesicles in PC12 cells. A majority of this protein, formerly recognized only on the plasma membrane of neurons, is localized to regulated secretory vesicles. Thy-1 is also present in synaptic vesicles in rat central nervous system. Experiments on permeabilized PC12 cells demonstrate that antibodies against Thy-1 inhibit the regulated release of neurotransmitter; this inhibition appears to be independent of any effect on the Ca2+ channel. These findings suggest Thy-1 is an integral component of many types of regulated secretory vesicles, and plays an important role in the regulated vesicular release of neurotransmitter at the synapse.

Show MeSH
Related in: MedlinePlus