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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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Characterization of FM1-43–membrane interactions. (A) Titration of liposomes (0.2 mM lipid composed of 70% PC/30% PE) with FM1-43. Thin lines are fluorescence spectra of FM1-43 with increasing concentrations of FM1-43 (0.25, 1.0, 2.5, 5.0, 8.0, 14.0, and 18.0 μM), and the dashed line is 18 μM FM1-43 without liposomes. (B) Quantification of spectra from A expressed as integrated fluorescence (500–700 nm). Data are the mean of three separate experiments plus SEM. (C) The ratio of fluorescence intensity of FM1-43 with and without liposomes, expressed as fold enhancement. (D) Titration of 4 μM FM1-43 with liposomes. From bottom to top the lipid concentrations are 0.01, 0.1, 0.3, 0.6, 0.8, and 0.9 mM. (E) Quantification of D with error bars showing SEM. (F) Two representative traces from stopped-flow experiments where FM1-43 and liposomes are mixed rapidly (∼1 ms dead time). In each case both the data and single exponential fits are shown. (G) Exponential fits from stopped-flow data provide kobs, which is plotted against lipid concentration for both synthetic liposomes (70% PC/30% PE) and liposomes formed from purified total brain lipid. Assuming pseudo first order kinetics, kon and koff were calculated as detailed in Materials and methods.
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fig6: Characterization of FM1-43–membrane interactions. (A) Titration of liposomes (0.2 mM lipid composed of 70% PC/30% PE) with FM1-43. Thin lines are fluorescence spectra of FM1-43 with increasing concentrations of FM1-43 (0.25, 1.0, 2.5, 5.0, 8.0, 14.0, and 18.0 μM), and the dashed line is 18 μM FM1-43 without liposomes. (B) Quantification of spectra from A expressed as integrated fluorescence (500–700 nm). Data are the mean of three separate experiments plus SEM. (C) The ratio of fluorescence intensity of FM1-43 with and without liposomes, expressed as fold enhancement. (D) Titration of 4 μM FM1-43 with liposomes. From bottom to top the lipid concentrations are 0.01, 0.1, 0.3, 0.6, 0.8, and 0.9 mM. (E) Quantification of D with error bars showing SEM. (F) Two representative traces from stopped-flow experiments where FM1-43 and liposomes are mixed rapidly (∼1 ms dead time). In each case both the data and single exponential fits are shown. (G) Exponential fits from stopped-flow data provide kobs, which is plotted against lipid concentration for both synthetic liposomes (70% PC/30% PE) and liposomes formed from purified total brain lipid. Assuming pseudo first order kinetics, kon and koff were calculated as detailed in Materials and methods.

Mentions: To further interpret our data on vesicle destaining, we performed a detailed analysis of FM1-43 binding and unbinding to and from model membranes. Equilibrium measurements (Fig. 6, A–E) revealed that FM1-43 fluorescence increases ∼40-fold on binding to liposomes (compared with the ∼350-fold enhancement seen on addition of detergent; Henkel et al., 1996), whereas at concentrations above ∼4 μM, the enhancement seen on addition of lipid diminishes sharply. Next, we examined the kinetics of this reaction using a rapid mixing stopped-flow method. FM1-43 concentration was kept constant (4 μM), whereas the liposome concentration was varied from 0.1–0.5 mM. Liposomes composed of synthetic lipids (70% 1,2-dioleoyl-sn-glycero-3-phosphocholine [PC]/30% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [PE]) and liposomes formed from purified total brain lipid were compared, and lipid composition was found to have a minimal influence on the kon and koff (Fig. 6, F and G).


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)

Characterization of FM1-43–membrane interactions. (A) Titration of liposomes (0.2 mM lipid composed of 70% PC/30% PE) with FM1-43. Thin lines are fluorescence spectra of FM1-43 with increasing concentrations of FM1-43 (0.25, 1.0, 2.5, 5.0, 8.0, 14.0, and 18.0 μM), and the dashed line is 18 μM FM1-43 without liposomes. (B) Quantification of spectra from A expressed as integrated fluorescence (500–700 nm). Data are the mean of three separate experiments plus SEM. (C) The ratio of fluorescence intensity of FM1-43 with and without liposomes, expressed as fold enhancement. (D) Titration of 4 μM FM1-43 with liposomes. From bottom to top the lipid concentrations are 0.01, 0.1, 0.3, 0.6, 0.8, and 0.9 mM. (E) Quantification of D with error bars showing SEM. (F) Two representative traces from stopped-flow experiments where FM1-43 and liposomes are mixed rapidly (∼1 ms dead time). In each case both the data and single exponential fits are shown. (G) Exponential fits from stopped-flow data provide kobs, which is plotted against lipid concentration for both synthetic liposomes (70% PC/30% PE) and liposomes formed from purified total brain lipid. Assuming pseudo first order kinetics, kon and koff were calculated as detailed in Materials and methods.
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fig6: Characterization of FM1-43–membrane interactions. (A) Titration of liposomes (0.2 mM lipid composed of 70% PC/30% PE) with FM1-43. Thin lines are fluorescence spectra of FM1-43 with increasing concentrations of FM1-43 (0.25, 1.0, 2.5, 5.0, 8.0, 14.0, and 18.0 μM), and the dashed line is 18 μM FM1-43 without liposomes. (B) Quantification of spectra from A expressed as integrated fluorescence (500–700 nm). Data are the mean of three separate experiments plus SEM. (C) The ratio of fluorescence intensity of FM1-43 with and without liposomes, expressed as fold enhancement. (D) Titration of 4 μM FM1-43 with liposomes. From bottom to top the lipid concentrations are 0.01, 0.1, 0.3, 0.6, 0.8, and 0.9 mM. (E) Quantification of D with error bars showing SEM. (F) Two representative traces from stopped-flow experiments where FM1-43 and liposomes are mixed rapidly (∼1 ms dead time). In each case both the data and single exponential fits are shown. (G) Exponential fits from stopped-flow data provide kobs, which is plotted against lipid concentration for both synthetic liposomes (70% PC/30% PE) and liposomes formed from purified total brain lipid. Assuming pseudo first order kinetics, kon and koff were calculated as detailed in Materials and methods.
Mentions: To further interpret our data on vesicle destaining, we performed a detailed analysis of FM1-43 binding and unbinding to and from model membranes. Equilibrium measurements (Fig. 6, A–E) revealed that FM1-43 fluorescence increases ∼40-fold on binding to liposomes (compared with the ∼350-fold enhancement seen on addition of detergent; Henkel et al., 1996), whereas at concentrations above ∼4 μM, the enhancement seen on addition of lipid diminishes sharply. Next, we examined the kinetics of this reaction using a rapid mixing stopped-flow method. FM1-43 concentration was kept constant (4 μM), whereas the liposome concentration was varied from 0.1–0.5 mM. Liposomes composed of synthetic lipids (70% 1,2-dioleoyl-sn-glycero-3-phosphocholine [PC]/30% 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine [PE]) and liposomes formed from purified total brain lipid were compared, and lipid composition was found to have a minimal influence on the kon and koff (Fig. 6, F and G).

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