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. 2013 Jan;8(1):1-16.
doi: 10.1038/nprot.2012.134. Epub 2012 Dec 6.

Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system

Minjoung Kyoung  1 , Yunxiang Zhang, Jiajie Diao, Steven Chu, Axel T Brunger
Affiliations

Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system

Minjoung Kyoung et al. Nat Protoc. 2013 Jan.

Abstract

This protocol describes a single vesicle-vesicle microscopy system to study Ca(2+)-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and synaptosomal-associated protein 25 (SNAP-25), and they are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at a zero-Ca(2+) concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca(2+) injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-100-millisecond time scale. Other factors such as complexin can be easily added. Our system is unique in that it monitors both content and lipid mixing and starts from a metastable state of interacting vesicle pairs before Ca(2+) injection.

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Figures

Figure 1
Figure 1
Schematic of the single vesicle-vesicle microscopy system. (a) Donor vesicles labeled with self-quenched sulforhodamine B (content dye) and DiD (lipid dye) along with complexin are added to the sample chamber containing acceptor vesicles immobilized on a cover glass surface via biotin-neutravidin interactions. (b) During the first, short, incubation period (typically 10 to 30 seconds), donor vesicles are allowed to interact with acceptor vesicles. Excess donor vesicles are then rinsed away by buffer exchange. (c) A second, longer, incubation period (typically 30 mins) follows. (d) Ca2+ buffer, along with cascade blue dye is injected. Content and lipid mixing events are monitored by observing changes in fluorescence intensity of the content and lipid dyes, respectively. The observation period typically starts 5 seconds prior to the Ca2+ injection and can typically last 30 to 50 seconds (limited by photobleaching).
Figure 2
Figure 2
Schematic diagram of the objective-based TIR setup. The three-color excitation and emission paths are depicted. The evanescent field is created at the interface of the cover glass and buffer in the sample chamber. Abbreviations: M: mirror, DM: dichroic mirror, F: filter, L: lens, Mp: periscope mirror.
Figure 3
Figure 3
Sample chamber and outlet ports. (a)-(f) Showing the preparation of the sample chamber. Inlet and outlet holes are initially covered by double-sided tape. (a-c) A rectangular piece of tape is cut out around the pairs of inlet and outlet holes to establish the area of the sample chamber. (d) A cover slip is added onto the top of the double-sided tape. (e) A total of 12 sample chambers with each ~ 2 μl volume can be accommodated with a single glass slide and cover slip. (g) A picture showing the outlet port of one of the sample chambers.
Figure 4
Figure 4
Representative time traces of content and lipid mixing events for a fluorescent spot that corresponds to a single interacting vesicle pair. Green (lower) and red (upper) lines are the fluorescence intensity time traces of content and lipid dyes, respectively. Time point 0 indicates the instance of Ca2+ buffer injection. The time binning used in this particular experiment was 200 msec. (a) Immediate full fusion. (b) Delayed full fusion. (c) Hemifusion only.
Figure 5
Figure 5
Testing non-specific binding to the surface. The captured image shows a PEG-coated surface that was prepared by the protocol described in steps 1-14. The PEG-coated surface was incubated with donor vesicles for the same amount time as for experiments with acceptor vesicle-tethered surfaces and then washed with Vesicle Buffer (content mixing channel on the left side and lipid mixing channel on the right side). If the procedures of the PEG coating and surface preparation are successful one expects to only observe very rare non-specific binding of donor vesicles to the surface, and only background fluorescence, as shown in this figure.
Figure 6
Figure 6
Screenshot of an image series (movie) loaded into our custom IDL program.
Figure 7
Figure 7
Display of an example of a fluorescence intensity time trace.
Figure 8
Figure 8
Display of time points of content and lipid mixing events.
Figure 9
Figure 9
Image of loaded beads.
Figure 10
Figure 10
Selection of pairs of beads.
Figure 11
Figure 11
Generation of the bead mapping coefficient.
Figure 12
Figure 12
Examples of content and lipid mixing histograms (the fraction of vesicles that show a jump in fluorescence intensity (“occurrence”) in a particular time bin) vs. time. (a) Content mixing histogram, and (b) lipid mixing histogram with 200 msecs time binning. Inserts represent histograms from -2 sec. to 10 sec. Time point 0 corresponds to Ca2+ injection. The black lines are fitted exponential decay functions (f (t) = 0.03 + 30.3 e–4.4t + 2.0 e–0.5t and f (t) = 0.003 + 69.4 e–6.7t + 7.5 e–1.5t for the content and lipid mixing histograms, respectively). Insets are close-up views of the histograms around time point 0. The histograms are normalized with respect to the number of docked vesicles.
See this image and copyright information in PMC

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