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Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles.

Richards DA, Bai J, Chapman ER - J. Cell Biol. (2005)

Bottom Line: We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons.These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis.Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons. Two populations of exocytic events were observed; small amplitude events that lose dye slowly, which made up more than half of all events, and faster, larger amplitude events with a fluorescence intensity equivalent to single stained synaptic vesicles. These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis. Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution. Kinetic analysis of the association and dissociation of FM1-43 with membranes, in combination with a simple pore permeation model, indicates that the small, slowly destaining events may be mediated by a narrow approximately 1-nm fusion pore.

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FM1-43 rapidly disassociates from liposomes and SVs. (A) Purity of the SV preparation. (left) SVs were blotted for the SV proteins synaptobrevin (syb) and synaptotagmin (syt), as well as the postsynaptic NMDA receptor (NMDAR-1), and visualized by enhanced chemiluminescence. mAbs against synaptobrevin (69.1) and NMDAR-1 (54.1) and a polyclonal antibody against the C2B domain of synaptotagmin were used. Total rat brain homogenate served as a reference to calculate the fold enrichment of synaptotagmin, synaptobrevin, and NMDAR-1 after purification and concentration procedures. (right) Blots shown in the left panel were quantified by densitometry and plotted as fold of enrichment of total protein from rat brains. SV markers, synaptotagmin and synaptobrevin, were enriched ∼300–400-fold, whereas the postsynaptic NMDA receptor was not detected in the SV preparation. (B) A significant fraction (∼20%) of SVs are flipped inside-out during purification. mAbs against the cytoplasmic domain (cd) of synaptotagmin (41.1) and synaptobrevin (69.1), as well as the luminal domain (ld) of synaptotagmin (604.1), were used for immunoprecipitation as described in Materials and methods. In the presence of Triton X-100, 41.1 and 69.1 completely immunoprecipitated synaptotagmin and synaptobrevin. However, in the absence of detergent, a small fraction (∼15–20% of total protein) of synaptotagmin and synaptobrevin were protected from immunoprecipitation by the antibodies. When antibodies against the luminal domain of synaptotagmin (604.1) were used, a small but significant fraction of synaptotagmin was immunoprecipitated in the absence of Triton X-100. (C) Departitioning kinetics of FM1-43 from membranes. 4 μM FM1-43 was mixed with model liposomes composed of 30% PE/70% PC (0.2 mM lipids), total brain lipids (0.2 mM lipids), or purified SVs, as indicated, and then loaded into a small syringe (see schematic, left). HBS was loaded into a separate, larger syringe. Rapid mixing of the contents of the two syringes resulted in a 1:11 dilution of the FM1-43 and the liposomes. Dilution led to a rapid dissociation between FM1-43 and the liposome membranes or SVs and a consequent decrease in fluorescence intensity.
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fig7: FM1-43 rapidly disassociates from liposomes and SVs. (A) Purity of the SV preparation. (left) SVs were blotted for the SV proteins synaptobrevin (syb) and synaptotagmin (syt), as well as the postsynaptic NMDA receptor (NMDAR-1), and visualized by enhanced chemiluminescence. mAbs against synaptobrevin (69.1) and NMDAR-1 (54.1) and a polyclonal antibody against the C2B domain of synaptotagmin were used. Total rat brain homogenate served as a reference to calculate the fold enrichment of synaptotagmin, synaptobrevin, and NMDAR-1 after purification and concentration procedures. (right) Blots shown in the left panel were quantified by densitometry and plotted as fold of enrichment of total protein from rat brains. SV markers, synaptotagmin and synaptobrevin, were enriched ∼300–400-fold, whereas the postsynaptic NMDA receptor was not detected in the SV preparation. (B) A significant fraction (∼20%) of SVs are flipped inside-out during purification. mAbs against the cytoplasmic domain (cd) of synaptotagmin (41.1) and synaptobrevin (69.1), as well as the luminal domain (ld) of synaptotagmin (604.1), were used for immunoprecipitation as described in Materials and methods. In the presence of Triton X-100, 41.1 and 69.1 completely immunoprecipitated synaptotagmin and synaptobrevin. However, in the absence of detergent, a small fraction (∼15–20% of total protein) of synaptotagmin and synaptobrevin were protected from immunoprecipitation by the antibodies. When antibodies against the luminal domain of synaptotagmin (604.1) were used, a small but significant fraction of synaptotagmin was immunoprecipitated in the absence of Triton X-100. (C) Departitioning kinetics of FM1-43 from membranes. 4 μM FM1-43 was mixed with model liposomes composed of 30% PE/70% PC (0.2 mM lipids), total brain lipids (0.2 mM lipids), or purified SVs, as indicated, and then loaded into a small syringe (see schematic, left). HBS was loaded into a separate, larger syringe. Rapid mixing of the contents of the two syringes resulted in a 1:11 dilution of the FM1-43 and the liposomes. Dilution led to a rapid dissociation between FM1-43 and the liposome membranes or SVs and a consequent decrease in fluorescence intensity.

Mentions: Finally, we assayed the rate of FM1-43 departitioning from three different model membranes, once again using a rapid mixing stopped-flow system (Fig. 7, A–C). FM1-43 (4 μM) was allowed to equilibrate with liposomes (0.1 mM) or SVs; then, samples were rapidly mixed with buffer. Syringes of different volumes were used to provide a 1:11 dilution in the mixing chamber. Again, mixing itself was rapid (∼1 ms dead time). Fig. 7 C shows dilution curves for synthetic lipids (70% PC/30% PE), total purified brain lipids, and SVs, respectively. In each case, the reactions are rapid, reaching completion in well under 500 ms. The resulting curves were best fitted with two exponentials: a fast component of <10 ms and a slow component of ∼100 ms (see Table I for values). If the two leaflets of SVs differed radically in the rate with which FM1-43 departitioned from them, we would have seen additional kinetic components because the SVs are ∼20% in reversed orientation (Fig. 7 B). Although there are differences between the conditions, the data suggest that lipid composition and protein complement make only modest contributions to the departitioning kinetics of FM1-43. Armed with this information, we were able to further interpret the SV destaining data presented in Figs. 1–4.


Two modes of exocytosis at hippocampal synapses revealed by rate of FM1-43 efflux from individual vesicles.

Richards DA, Bai J, Chapman ER - J. Cell Biol. (2005)

FM1-43 rapidly disassociates from liposomes and SVs. (A) Purity of the SV preparation. (left) SVs were blotted for the SV proteins synaptobrevin (syb) and synaptotagmin (syt), as well as the postsynaptic NMDA receptor (NMDAR-1), and visualized by enhanced chemiluminescence. mAbs against synaptobrevin (69.1) and NMDAR-1 (54.1) and a polyclonal antibody against the C2B domain of synaptotagmin were used. Total rat brain homogenate served as a reference to calculate the fold enrichment of synaptotagmin, synaptobrevin, and NMDAR-1 after purification and concentration procedures. (right) Blots shown in the left panel were quantified by densitometry and plotted as fold of enrichment of total protein from rat brains. SV markers, synaptotagmin and synaptobrevin, were enriched ∼300–400-fold, whereas the postsynaptic NMDA receptor was not detected in the SV preparation. (B) A significant fraction (∼20%) of SVs are flipped inside-out during purification. mAbs against the cytoplasmic domain (cd) of synaptotagmin (41.1) and synaptobrevin (69.1), as well as the luminal domain (ld) of synaptotagmin (604.1), were used for immunoprecipitation as described in Materials and methods. In the presence of Triton X-100, 41.1 and 69.1 completely immunoprecipitated synaptotagmin and synaptobrevin. However, in the absence of detergent, a small fraction (∼15–20% of total protein) of synaptotagmin and synaptobrevin were protected from immunoprecipitation by the antibodies. When antibodies against the luminal domain of synaptotagmin (604.1) were used, a small but significant fraction of synaptotagmin was immunoprecipitated in the absence of Triton X-100. (C) Departitioning kinetics of FM1-43 from membranes. 4 μM FM1-43 was mixed with model liposomes composed of 30% PE/70% PC (0.2 mM lipids), total brain lipids (0.2 mM lipids), or purified SVs, as indicated, and then loaded into a small syringe (see schematic, left). HBS was loaded into a separate, larger syringe. Rapid mixing of the contents of the two syringes resulted in a 1:11 dilution of the FM1-43 and the liposomes. Dilution led to a rapid dissociation between FM1-43 and the liposome membranes or SVs and a consequent decrease in fluorescence intensity.
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fig7: FM1-43 rapidly disassociates from liposomes and SVs. (A) Purity of the SV preparation. (left) SVs were blotted for the SV proteins synaptobrevin (syb) and synaptotagmin (syt), as well as the postsynaptic NMDA receptor (NMDAR-1), and visualized by enhanced chemiluminescence. mAbs against synaptobrevin (69.1) and NMDAR-1 (54.1) and a polyclonal antibody against the C2B domain of synaptotagmin were used. Total rat brain homogenate served as a reference to calculate the fold enrichment of synaptotagmin, synaptobrevin, and NMDAR-1 after purification and concentration procedures. (right) Blots shown in the left panel were quantified by densitometry and plotted as fold of enrichment of total protein from rat brains. SV markers, synaptotagmin and synaptobrevin, were enriched ∼300–400-fold, whereas the postsynaptic NMDA receptor was not detected in the SV preparation. (B) A significant fraction (∼20%) of SVs are flipped inside-out during purification. mAbs against the cytoplasmic domain (cd) of synaptotagmin (41.1) and synaptobrevin (69.1), as well as the luminal domain (ld) of synaptotagmin (604.1), were used for immunoprecipitation as described in Materials and methods. In the presence of Triton X-100, 41.1 and 69.1 completely immunoprecipitated synaptotagmin and synaptobrevin. However, in the absence of detergent, a small fraction (∼15–20% of total protein) of synaptotagmin and synaptobrevin were protected from immunoprecipitation by the antibodies. When antibodies against the luminal domain of synaptotagmin (604.1) were used, a small but significant fraction of synaptotagmin was immunoprecipitated in the absence of Triton X-100. (C) Departitioning kinetics of FM1-43 from membranes. 4 μM FM1-43 was mixed with model liposomes composed of 30% PE/70% PC (0.2 mM lipids), total brain lipids (0.2 mM lipids), or purified SVs, as indicated, and then loaded into a small syringe (see schematic, left). HBS was loaded into a separate, larger syringe. Rapid mixing of the contents of the two syringes resulted in a 1:11 dilution of the FM1-43 and the liposomes. Dilution led to a rapid dissociation between FM1-43 and the liposome membranes or SVs and a consequent decrease in fluorescence intensity.
Mentions: Finally, we assayed the rate of FM1-43 departitioning from three different model membranes, once again using a rapid mixing stopped-flow system (Fig. 7, A–C). FM1-43 (4 μM) was allowed to equilibrate with liposomes (0.1 mM) or SVs; then, samples were rapidly mixed with buffer. Syringes of different volumes were used to provide a 1:11 dilution in the mixing chamber. Again, mixing itself was rapid (∼1 ms dead time). Fig. 7 C shows dilution curves for synthetic lipids (70% PC/30% PE), total purified brain lipids, and SVs, respectively. In each case, the reactions are rapid, reaching completion in well under 500 ms. The resulting curves were best fitted with two exponentials: a fast component of <10 ms and a slow component of ∼100 ms (see Table I for values). If the two leaflets of SVs differed radically in the rate with which FM1-43 departitioned from them, we would have seen additional kinetic components because the SVs are ∼20% in reversed orientation (Fig. 7 B). Although there are differences between the conditions, the data suggest that lipid composition and protein complement make only modest contributions to the departitioning kinetics of FM1-43. Armed with this information, we were able to further interpret the SV destaining data presented in Figs. 1–4.

Bottom Line: We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons.These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis.Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
We have examined the kinetics by which FM1-43 escapes from individual synaptic vesicles during exocytosis at hippocampal boutons. Two populations of exocytic events were observed; small amplitude events that lose dye slowly, which made up more than half of all events, and faster, larger amplitude events with a fluorescence intensity equivalent to single stained synaptic vesicles. These populations of destaining events are distinct in both brightness and kinetics, suggesting that they result from two distinct modes of exocytosis. Small amplitude events show tightly clustered rate constants of dye release, whereas larger events have a more scattered distribution. Kinetic analysis of the association and dissociation of FM1-43 with membranes, in combination with a simple pore permeation model, indicates that the small, slowly destaining events may be mediated by a narrow approximately 1-nm fusion pore.

Show MeSH
Related in: MedlinePlus