Fig 1.
Various lipidic platforms used in this work to study the mobility and interaction of lipids and proteins within membranes.
Supported lipid bilayers are formed by the Langmuir-Blodgett deposition technique, which allows the formation of asymmetrical membranes (inner and outer monolayers can be of different lipid compositions). Giant liposomes are free standing membranes of 10–100 μm diameter. Sponge phases consist of interconnected bilayers forming aqueous channels whose diameter can be varied from 6 to 30 nm.
Table 1.
Advantages and disadvantages of the various in vitro membrane platforms available to study FRAP by confocal microscopy.
Fig 2.
Fluorescence recovery of (A) DOPE-Rho or (B) DOPE-NBD lipids in the outer monolayer of a supported DOPC lipid bilayer following photobleaching of (A) a fluorescent disk of d = 20 μm diameter or (B) a pattern of fluorescent fringes separated by f = 20 μm. The recovery curve (result of 1 bleaching experiment on the same region in A and average of 3 bleaching experiments on the same region in B) can be fitted with (A) a Bessel function or (B) an Exponential function. (C) The contribution of the intrinsic photobleaching occurring during fluorescence reading (bleach) in the case of DOPE-NBD lipids can be removed in the fringe system by using the average of fluorescence gain in the dark fringes and fluorescence loss in the bright fringes. No intrinsic photobleaching was observed with DOPE-Rho lipids.
Fig 3.
Diffusion coefficient (D) of DOPE-Rho lipids in the outer leaflet of a supported DOPC lipid bilayer deduced by varying the area of the bleached region (disks of diameters d = 2, 5, 10 or 20 μm in A; networks of fringes with an interfringe f = 5, 10 or 20 μm in B). The linear relationship between the bleaching area s2 (d2 or f2) and the recovery time (τ) proves that lipid diffusion is controlled by Brownian motion. The diffusion coefficient (D) is calculated from the slope of this straight line: D = d2 / 16 τ in the case of the disk system, and D = f2 / 4π2 τ in the case of the fringe system. Each data point in A or B is the average of N = 3 independent bleaching experiments on different regions of the same bilayer, and the error bars correspond to the standard deviations from these averages. These experiments were reproduced with 4 independent (freshly prepared) bilayers using either the disk- or the fringe-shaped bleaching geometry, leading to D = (1.9 ± 0.3) μm2/s.
Table 2.
Diffusion coefficient of various headgroup-labeled DOPE lipids in different artificial membrane environments.
Two-sample t-tests: supported lipid bilayers vs. giant liposomes (p<0.01); supported lipid bilayers vs. sponge phases (p<0.001); giant liposomes vs. sponge phases (non-significant).
Fig 4.
Experimental set-up used to perform FRAP experiments on (A) giant liposomes or (B) sponge phases. (A) To prevent liposome movement during FRAP measurements, giant liposomes are confined in a closed chamber of ~500 μm height (not shown here) or ideally (as shown here) held through micromanipulation. Fluorescence bleaching and recovery measurements are performed at the top (pole) of the giant liposome, which appears as a fluorescent disk in the confocal microscope. In this system, FRAP measurements are performed using the disk-shaped geometry. To ensure that the bleached spherical cap can be treated as a disk, the diameter of the bleaching disk should not exceed 25% of the giant liposome diameter. The confocal picture on the right shows the result of bleaching a 7 μm diameter fluorescent disk in the membrane of a DOPC:DOPE-Rho (99:1) giant liposome of 45 μm diameter. (B) Sponge phases (3 μL solution) are sandwiched between two glass surfaces (1.5 cm diameter) to form a liquid layer of ~20 μm height; this allows bleaching at all z-values within the sponge phase and thus measuring 2-dimensional fluorescence recovery. Here, bleaching is performed using the fringe-shaped geometry (interfringe f = 12 μm). The two systems were used to study the mobility of DOPE-Rho lipids. In both cases, DOPE-Rho lipids diffused faster than in supported lipid bilayers (D = 3.7 ± 0.5 μm2/s in the membrane of giant liposomes and D = 4.1 ± 0.4 μm2/s in sponge phases), which we attribute to the reduction of friction forces between the outer and the inner leaflets when the membrane is not linked to a solid substrate.
Fig 5.
Interaction between SNARE proteins in a sponge phase.
The full length Syn1A protein and the cytoplasmic domain of FITC-labeled VAMP2 protein (cdVAMP2*) were reconstituted into two separate sponge phases (at the respective lipid-to-protein molar ratios of 20,000 and 80,000). The Syn1A sponge was pre-incubated for 1 hour at room temperature with or without Munc18-1 protein (1:1 molar ratio between Syn1A and Munc18-1). The Syn1A ± Munc18-1 sponge and the cdVAMP2* sponge were then mixed and allowed to interact for 1 hour at room temperature. In the absence of Munc18-1 (blue fitting curve), cdVAMP2* displays two diffusion coefficients: a slow diffusion coefficient (2.0 ± 0.1 μm2/s) corresponding to cdVAMP2* bound to Syn1A in the sponge membrane and a fast diffusion coefficient (9.7 ± 1.4 μm2/s) corresponding to free (unbound) cdVAMP2* in the sponge channels. In the presence of Munc18-1 (red fitting curve), cdVAMP2* displays a single, fast, diffusion coefficient (6.4 ± 1.1 μm2/s), as observed when it is added to a protein-free sponge phase, showing that Munc18-1 inhibits the interaction between Syn1A and cdVAMP2* (see also Table 3).
Table 3.
Interaction parameters (diffusion coefficients and mobile fractions) of Syn1A and cdVAMP2 in a sponge phase, in the absence or presence of Munc18-1.
FITC-labeled molecules are indicated with a star. Two-sample t-tests on fast diffusion components: cdVAMP2* vs. Syn1A+cdVAMP2* (p<0.05); cdVAMP2* vs. Syn1A+cdVAMP2*+Munc18 (non-significant); Syn1A+cdVAMP2* vs. Syn1A+cdVAMP2*+Munc18 (p<0.05). Two-sample t-test on slow diffusion components: Syn1A* vs. Syn1A+cdVAMP2* (non-significant).