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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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Melittin as a model for efflux of FM1-43 though nanometer size pores. We investigated the rate of FM1-43 efflux through pores by preforming the pores in the presence of 4 μM FM1-43 for 5 min, so that inner and outer leaflets were in equilibrium. (A) An outline of the experimental procedure. Rapid dilution caused the departitioning of FM1-43 from both leaflets—rapidly from the outer leaflet (τ = ∼8 ms; see Fig. 7) and with a slower rate from the inner leaflet that was dependent on the pore density. (B) Hand-mixing experiments. The baseline trace shows the fluorescence from liposomes diluted into buffer that contained 4 μM FM1-43; this trace provides a reference for the maximum signal from these samples. The blank dilution sample was diluted with buffer alone (i.e., no FM1-43 or melittin); the drop in fluorescence compared with the baseline is due to departitioning of the dye from outer leaflet and provides the reference for the fluorescence from FM1-43 in the inner leaflet of the liposomes. In samples preincubated with melittin, a slow phase of fluorescence decline was observed (B), reflecting the rate of dye efflux through fully assembled melittin pores. (C) Rapid mixing stopped-flow measurements of FM1-43 efflux though pores. The destaining kinetics mediated by melittin at 1:300, 1:60, and 1:20 peptide/lipid ratios are plotted from top to bottom. The time course of dye loss from liposomes with multiple pores require multi-exponential fits, and so for simplicity we provide τ values for 1:300 only. When melittin/lipid is 1:20, liposomes contain many pores on average, resulting in rapid destaining kinetics (t1/2 = 0.1 s). At a melittin to lipid ratio of 1:60, a large proportion of liposomes still possess greater than 1 pore per vesicle. At 1:300 almost half the liposomes have no pores (and therefore do not destain), and the remaining ones have approximately one pore per vesicle. This last case is most equivalent to the situation in SVs where exocytosis proceeds through a single fusion pore. Under these conditions, a single exponential fit is adequate and has a τ of 6.7 s (after bleach correction), a value that is similar to the rate of dye loss from slow destaining events from synaptic boutons (τ = 3–7 s; see Fig. 5).
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fig9: Melittin as a model for efflux of FM1-43 though nanometer size pores. We investigated the rate of FM1-43 efflux through pores by preforming the pores in the presence of 4 μM FM1-43 for 5 min, so that inner and outer leaflets were in equilibrium. (A) An outline of the experimental procedure. Rapid dilution caused the departitioning of FM1-43 from both leaflets—rapidly from the outer leaflet (τ = ∼8 ms; see Fig. 7) and with a slower rate from the inner leaflet that was dependent on the pore density. (B) Hand-mixing experiments. The baseline trace shows the fluorescence from liposomes diluted into buffer that contained 4 μM FM1-43; this trace provides a reference for the maximum signal from these samples. The blank dilution sample was diluted with buffer alone (i.e., no FM1-43 or melittin); the drop in fluorescence compared with the baseline is due to departitioning of the dye from outer leaflet and provides the reference for the fluorescence from FM1-43 in the inner leaflet of the liposomes. In samples preincubated with melittin, a slow phase of fluorescence decline was observed (B), reflecting the rate of dye efflux through fully assembled melittin pores. (C) Rapid mixing stopped-flow measurements of FM1-43 efflux though pores. The destaining kinetics mediated by melittin at 1:300, 1:60, and 1:20 peptide/lipid ratios are plotted from top to bottom. The time course of dye loss from liposomes with multiple pores require multi-exponential fits, and so for simplicity we provide τ values for 1:300 only. When melittin/lipid is 1:20, liposomes contain many pores on average, resulting in rapid destaining kinetics (t1/2 = 0.1 s). At a melittin to lipid ratio of 1:60, a large proportion of liposomes still possess greater than 1 pore per vesicle. At 1:300 almost half the liposomes have no pores (and therefore do not destain), and the remaining ones have approximately one pore per vesicle. This last case is most equivalent to the situation in SVs where exocytosis proceeds through a single fusion pore. Under these conditions, a single exponential fit is adequate and has a τ of 6.7 s (after bleach correction), a value that is similar to the rate of dye loss from slow destaining events from synaptic boutons (τ = 3–7 s; see Fig. 5).

Mentions: To determine the influence of the pore on dye efflux, we allowed the pores to fully assemble, and then observed the loss of FM1-43 after rapid dilution. Liposomes harboring FM1-43 in both internal and external leaflets were incubated with melittin for 5 min to allow pore formation. The resultant liposomes with melittin pores contained FM1-43 in equilibrium with the external solution (4 μM FM1-43). Liposomes were then rapidly diluted into buffer within a cuvette and the fluorescence was monitored (the experimental procedure is outlined in Fig. 9 A). The loss in fluorescence has two components: a rapid phase of ∼8 ms (i.e., the time course of dye departitioning from the outer leaflet of membranes; Fig. 7), which is too fast for observation in these experiments, and a slower phase that reflects the rate of dye leaving the liposomes via a small pore. After dilution, a slow phase of fluorescence decline was observed (Fig. 9 B), which reflects the rate of dye efflux through fully assembled melittin pores. As the ratio of melittin to lipid was increased from 1:300 to 1:60 and 1:20, the rate of dye loss also increased. This increase in rate is due to an increase in the total number of pores and/or an increase in the diameter of the pores.


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)

Melittin as a model for efflux of FM1-43 though nanometer size pores. We investigated the rate of FM1-43 efflux through pores by preforming the pores in the presence of 4 μM FM1-43 for 5 min, so that inner and outer leaflets were in equilibrium. (A) An outline of the experimental procedure. Rapid dilution caused the departitioning of FM1-43 from both leaflets—rapidly from the outer leaflet (τ = ∼8 ms; see Fig. 7) and with a slower rate from the inner leaflet that was dependent on the pore density. (B) Hand-mixing experiments. The baseline trace shows the fluorescence from liposomes diluted into buffer that contained 4 μM FM1-43; this trace provides a reference for the maximum signal from these samples. The blank dilution sample was diluted with buffer alone (i.e., no FM1-43 or melittin); the drop in fluorescence compared with the baseline is due to departitioning of the dye from outer leaflet and provides the reference for the fluorescence from FM1-43 in the inner leaflet of the liposomes. In samples preincubated with melittin, a slow phase of fluorescence decline was observed (B), reflecting the rate of dye efflux through fully assembled melittin pores. (C) Rapid mixing stopped-flow measurements of FM1-43 efflux though pores. The destaining kinetics mediated by melittin at 1:300, 1:60, and 1:20 peptide/lipid ratios are plotted from top to bottom. The time course of dye loss from liposomes with multiple pores require multi-exponential fits, and so for simplicity we provide τ values for 1:300 only. When melittin/lipid is 1:20, liposomes contain many pores on average, resulting in rapid destaining kinetics (t1/2 = 0.1 s). At a melittin to lipid ratio of 1:60, a large proportion of liposomes still possess greater than 1 pore per vesicle. At 1:300 almost half the liposomes have no pores (and therefore do not destain), and the remaining ones have approximately one pore per vesicle. This last case is most equivalent to the situation in SVs where exocytosis proceeds through a single fusion pore. Under these conditions, a single exponential fit is adequate and has a τ of 6.7 s (after bleach correction), a value that is similar to the rate of dye loss from slow destaining events from synaptic boutons (τ = 3–7 s; see Fig. 5).
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Related In: Results  -  Collection

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fig9: Melittin as a model for efflux of FM1-43 though nanometer size pores. We investigated the rate of FM1-43 efflux through pores by preforming the pores in the presence of 4 μM FM1-43 for 5 min, so that inner and outer leaflets were in equilibrium. (A) An outline of the experimental procedure. Rapid dilution caused the departitioning of FM1-43 from both leaflets—rapidly from the outer leaflet (τ = ∼8 ms; see Fig. 7) and with a slower rate from the inner leaflet that was dependent on the pore density. (B) Hand-mixing experiments. The baseline trace shows the fluorescence from liposomes diluted into buffer that contained 4 μM FM1-43; this trace provides a reference for the maximum signal from these samples. The blank dilution sample was diluted with buffer alone (i.e., no FM1-43 or melittin); the drop in fluorescence compared with the baseline is due to departitioning of the dye from outer leaflet and provides the reference for the fluorescence from FM1-43 in the inner leaflet of the liposomes. In samples preincubated with melittin, a slow phase of fluorescence decline was observed (B), reflecting the rate of dye efflux through fully assembled melittin pores. (C) Rapid mixing stopped-flow measurements of FM1-43 efflux though pores. The destaining kinetics mediated by melittin at 1:300, 1:60, and 1:20 peptide/lipid ratios are plotted from top to bottom. The time course of dye loss from liposomes with multiple pores require multi-exponential fits, and so for simplicity we provide τ values for 1:300 only. When melittin/lipid is 1:20, liposomes contain many pores on average, resulting in rapid destaining kinetics (t1/2 = 0.1 s). At a melittin to lipid ratio of 1:60, a large proportion of liposomes still possess greater than 1 pore per vesicle. At 1:300 almost half the liposomes have no pores (and therefore do not destain), and the remaining ones have approximately one pore per vesicle. This last case is most equivalent to the situation in SVs where exocytosis proceeds through a single fusion pore. Under these conditions, a single exponential fit is adequate and has a τ of 6.7 s (after bleach correction), a value that is similar to the rate of dye loss from slow destaining events from synaptic boutons (τ = 3–7 s; see Fig. 5).
Mentions: To determine the influence of the pore on dye efflux, we allowed the pores to fully assemble, and then observed the loss of FM1-43 after rapid dilution. Liposomes harboring FM1-43 in both internal and external leaflets were incubated with melittin for 5 min to allow pore formation. The resultant liposomes with melittin pores contained FM1-43 in equilibrium with the external solution (4 μM FM1-43). Liposomes were then rapidly diluted into buffer within a cuvette and the fluorescence was monitored (the experimental procedure is outlined in Fig. 9 A). The loss in fluorescence has two components: a rapid phase of ∼8 ms (i.e., the time course of dye departitioning from the outer leaflet of membranes; Fig. 7), which is too fast for observation in these experiments, and a slower phase that reflects the rate of dye leaving the liposomes via a small pore. After dilution, a slow phase of fluorescence decline was observed (Fig. 9 B), which reflects the rate of dye efflux through fully assembled melittin pores. As the ratio of melittin to lipid was increased from 1:300 to 1:60 and 1:20, the rate of dye loss also increased. This increase in rate is due to an increase in the total number of pores and/or an increase in the diameter of the pores.

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