Limits...
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.

Show MeSH

Related in: MedlinePlus

Mechanisms of FM1-43 efflux from exocytosing SVs. (A) Schematic diagram illustrating possible modes by which FM1-43 might leave SVs during exocytosis. If vesicles fully collapse into the plasma membrane, FM1-43 could escape by direct departitioning into the bulk solution. Alternatively, dye could escape through even a tiny fusion pore if the pore was lipidic and allowed mixing of vesicular and plasma membrane components, as a result of diffusion within the bilayer. The final possibility is that a small aqueous pore opens, the constituents of which form a barrier to diffusion of FM1-43 within the membrane. (B) Examination of the kinetics of fast destaining events, using the averaged values from Fig. 5 D. Because the fastest events occur at a rate similar to the sampling frequency, we cannot be certain when the drop in fluorescence actually begins. Consequently, the data do not fall all that far from a τ of ∼120 ms, which is the rate of the slow component of departitioning we observed in our stopped-flow experiments. As a result of the limitations of the imaging frequency, we cannot readily distinguish between FM1-43 leaving vesicles by diffusion within the membrane and leaving by direct departitioning. (C) Examination of the kinetics of slow destaining events. The averaged curves are replotted from Fig. 5 D. The slow component of membrane departitioning is much too slow to account for the kinetics by which FM1-43 escapes from vesicles. Instead, we have compared the rate of efflux of FM1-43 with simple diffusion-limited permeation through an aqueous pore with an opening of 1 nm. There is a fairly close agreement between this and the experimental data, although this model does not take into account any electrostatic interactions or geometric constraints, which might tend to slow the dye efflux. Error bars are SEM.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171786&req=5

fig8: Mechanisms of FM1-43 efflux from exocytosing SVs. (A) Schematic diagram illustrating possible modes by which FM1-43 might leave SVs during exocytosis. If vesicles fully collapse into the plasma membrane, FM1-43 could escape by direct departitioning into the bulk solution. Alternatively, dye could escape through even a tiny fusion pore if the pore was lipidic and allowed mixing of vesicular and plasma membrane components, as a result of diffusion within the bilayer. The final possibility is that a small aqueous pore opens, the constituents of which form a barrier to diffusion of FM1-43 within the membrane. (B) Examination of the kinetics of fast destaining events, using the averaged values from Fig. 5 D. Because the fastest events occur at a rate similar to the sampling frequency, we cannot be certain when the drop in fluorescence actually begins. Consequently, the data do not fall all that far from a τ of ∼120 ms, which is the rate of the slow component of departitioning we observed in our stopped-flow experiments. As a result of the limitations of the imaging frequency, we cannot readily distinguish between FM1-43 leaving vesicles by diffusion within the membrane and leaving by direct departitioning. (C) Examination of the kinetics of slow destaining events. The averaged curves are replotted from Fig. 5 D. The slow component of membrane departitioning is much too slow to account for the kinetics by which FM1-43 escapes from vesicles. Instead, we have compared the rate of efflux of FM1-43 with simple diffusion-limited permeation through an aqueous pore with an opening of 1 nm. There is a fairly close agreement between this and the experimental data, although this model does not take into account any electrostatic interactions or geometric constraints, which might tend to slow the dye efflux. Error bars are SEM.

Mentions: Multiple modes can be considered for the escape of FM1-43 from SVs. The simplest case is that the dye rapidly diffuses out in the plane of the membrane once the bilayers fuse, as recently described by Zenisek et al. (2002) in experiments using goldfish retinal bipolar neurons. Equally simple in kinetic terms is the situation where departitioning of the dye from the membrane dominates. In contrast, a mode of exocytosis mediated solely by a lipid impermeant fusion pore relies on two kinetic steps: unbinding of FM1-43 from the membrane balanced by the flux of aqueous FM dye through the fusion pore. All three modes are illustrated in Fig. 8 A.


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)

Mechanisms of FM1-43 efflux from exocytosing SVs. (A) Schematic diagram illustrating possible modes by which FM1-43 might leave SVs during exocytosis. If vesicles fully collapse into the plasma membrane, FM1-43 could escape by direct departitioning into the bulk solution. Alternatively, dye could escape through even a tiny fusion pore if the pore was lipidic and allowed mixing of vesicular and plasma membrane components, as a result of diffusion within the bilayer. The final possibility is that a small aqueous pore opens, the constituents of which form a barrier to diffusion of FM1-43 within the membrane. (B) Examination of the kinetics of fast destaining events, using the averaged values from Fig. 5 D. Because the fastest events occur at a rate similar to the sampling frequency, we cannot be certain when the drop in fluorescence actually begins. Consequently, the data do not fall all that far from a τ of ∼120 ms, which is the rate of the slow component of departitioning we observed in our stopped-flow experiments. As a result of the limitations of the imaging frequency, we cannot readily distinguish between FM1-43 leaving vesicles by diffusion within the membrane and leaving by direct departitioning. (C) Examination of the kinetics of slow destaining events. The averaged curves are replotted from Fig. 5 D. The slow component of membrane departitioning is much too slow to account for the kinetics by which FM1-43 escapes from vesicles. Instead, we have compared the rate of efflux of FM1-43 with simple diffusion-limited permeation through an aqueous pore with an opening of 1 nm. There is a fairly close agreement between this and the experimental data, although this model does not take into account any electrostatic interactions or geometric constraints, which might tend to slow the dye efflux. Error bars are SEM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171786&req=5

fig8: Mechanisms of FM1-43 efflux from exocytosing SVs. (A) Schematic diagram illustrating possible modes by which FM1-43 might leave SVs during exocytosis. If vesicles fully collapse into the plasma membrane, FM1-43 could escape by direct departitioning into the bulk solution. Alternatively, dye could escape through even a tiny fusion pore if the pore was lipidic and allowed mixing of vesicular and plasma membrane components, as a result of diffusion within the bilayer. The final possibility is that a small aqueous pore opens, the constituents of which form a barrier to diffusion of FM1-43 within the membrane. (B) Examination of the kinetics of fast destaining events, using the averaged values from Fig. 5 D. Because the fastest events occur at a rate similar to the sampling frequency, we cannot be certain when the drop in fluorescence actually begins. Consequently, the data do not fall all that far from a τ of ∼120 ms, which is the rate of the slow component of departitioning we observed in our stopped-flow experiments. As a result of the limitations of the imaging frequency, we cannot readily distinguish between FM1-43 leaving vesicles by diffusion within the membrane and leaving by direct departitioning. (C) Examination of the kinetics of slow destaining events. The averaged curves are replotted from Fig. 5 D. The slow component of membrane departitioning is much too slow to account for the kinetics by which FM1-43 escapes from vesicles. Instead, we have compared the rate of efflux of FM1-43 with simple diffusion-limited permeation through an aqueous pore with an opening of 1 nm. There is a fairly close agreement between this and the experimental data, although this model does not take into account any electrostatic interactions or geometric constraints, which might tend to slow the dye efflux. Error bars are SEM.
Mentions: Multiple modes can be considered for the escape of FM1-43 from SVs. The simplest case is that the dye rapidly diffuses out in the plane of the membrane once the bilayers fuse, as recently described by Zenisek et al. (2002) in experiments using goldfish retinal bipolar neurons. Equally simple in kinetic terms is the situation where departitioning of the dye from the membrane dominates. In contrast, a mode of exocytosis mediated solely by a lipid impermeant fusion pore relies on two kinetic steps: unbinding of FM1-43 from the membrane balanced by the flux of aqueous FM dye through the fusion pore. All three modes are illustrated in Fig. 8 A.

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