Studying protein-reconstituted proteoliposome fusion with content indicators in vitro

Abstract

In vitro reconstitution assays are commonly used to study biological membrane fusion. However, to date, most ensemble and single-vesicle experiments involving SNARE proteins have been performed only with lipid-mixing, but not content-mixing indicators. Through simultaneous detection of lipid and small content-mixing indicators, we found that lipid mixing often occurs seconds prior to content mixing, or without any content mixing at all, during a 50-seconds observation period, for Ca(2+) -triggered fusion with SNAREs, full-length synaptotagmin-1, and complexin. Our results illustrate the caveats of commonly used bulk lipid-mixing fusion experiments. We recommend that proteoliposome fusion experiments should always employ content-mixing indicators in addition to, or in place of, lipid-mixing indicators.

Fig. 1

Single vesicle-vesicle fusion assay. A: synaptic vesicle mimics: unlabeled t-vesicle with syntaxin-1A & SNAP-25A, lipid/content dye dual-labelled v-vesicle reconstituted with synaptobrevin-2 (VAMP2) & synaptotagmin-1, and complexin-1. B: Experimental schema. The left panel shows the recently developed protocol: t-vesicles are immobilized, v-vesicles are docked/primed at low concentration, and then Ca²⁺ is injected [8]. Between the steps, buffer exchanges are performed with at least 200 μl vesicle buffer (90 mM NaCl, 20 mM HEPES, 20 μM EGTA, 1% β-mercaptoethanol, pH 7.4). The right panel shows the previous incubation method [5,13]: v-vesicles are docked at high concentration for a short period, followed by buffer exchange, and then an incubation stage for a longer period. For both methods, Ca²⁺-triggered fusion events are monitored by observing changes in the fluorescence intensities of the content and lipid dyes, respectively.

Fig. 2

Typical data obtained from the single vesicle-vesicle fusion assay. A: Traces of fluorescence intensity against time for a typical lipid dye (red) and a content dye (green). Full fusion is defined as a content mixing fluorescence increase, while hemifusion corresponds to a step increase in lipid fluorescence in the absence of content mixing. Corresponding membrane states are shown above. B: Histograms of the occurrence of Ca²⁺-triggered lipid- (top) and content- (bottom) mixing events for the system consisting of neuronal SNAREs and synaptotagmin-1 using 500 msec time binning for one of multiple experiments (histograms were generated from 321 individual traces). Events during a 7.5-sec period before Ca²⁺ injection are also shown (if any). Time t = 0 corresponds to the instance of 500 μM Ca²⁺ injection as determined by the appearance of cascade-blue fluorescence. Histograms are normalized with respect to the number of total events of lipid mixing.

Fig. 3

Automated step-increase detection. An example of a content dye fluorescence intensity time trace is shown. Two red lines indicate a step increase greater than a set threshold as determined by the local noise level. The two blue lines represent false-positives that are automatically excluded by the Wald-Wolfowitz test [31].

Fig. 4

Imaging of vesicle-vesicle morphologies by cryo-electron microscopy. Shown are mixtures of v- (with reconstituted synaptobrevin-2) and t-vesicles (with reconstituted syntaxin-1A and SNAP-25A), which were imaged in the holes of the substrate carbon film, visible as the darker areas in the image, in conditions that clearly show the lipid bilayers. Point contacts, (i.e. appositions with small (1-5 nm) separation between vesicles but without merger or deformation of membranes) between docked vesicles were observed (large black arrows) along with hemifused diaphragms (small white/black arrows). The scale bar is 100 nm.

Fig. 5

Possible Ca²⁺-concentration profiles. Shown are a one-step function, a multi-step function, a step function with increasing concentration, a short pulse, and multiple short pulses.