FRAP to Characterize Molecular Diffusion and Interaction in Various Membrane Environments

Abstract

Fluorescence recovery after photobleaching (FRAP) is a standard method used to study the dynamics of lipids and proteins in artificial and cellular membrane systems. The advent of confocal microscopy two decades ago has made quantitative FRAP easily available to most laboratories. Usually, a single bleaching pattern/area is used and the corresponding recovery time is assumed to directly provide a diffusion coefficient, although this is only true in the case of unrestricted Brownian motion. Here, we propose some general guidelines to perform FRAP experiments under a confocal microscope with different bleaching patterns and area, allowing the experimentalist to establish whether the molecules undergo Brownian motion (free diffusion) or whether they have restricted or directed movements. Using in silico simulations of FRAP measurements, we further indicate the data acquisition criteria that have to be verified in order to obtain accurate values for the diffusion coefficient and to be able to distinguish between different diffusive species. Using this approach, we compare the behavior of lipids in three different membrane platforms (supported lipid bilayers, giant liposomes and sponge phases), and we demonstrate that FRAP measurements are consistent with results obtained using other techniques such as Fluorescence Correlation Spectroscopy (FCS) or Single Particle Tracking (SPT). Finally, we apply this method to show that the presence of the synaptic protein Munc18-1 inhibits the interaction between the synaptic vesicle SNARE protein, VAMP2, and its partner from the plasma membrane, Syn1A.

Highly Reproducible Physiological Asymmetric Membrane with Freely Diffusing Embedded Proteins in a 3D‐Printed Microfluidic Setup

“…We have used a fluorescently labeled protein and performed FRAP experiments to check the mobility of the t‐SNARE in the membrane. The proportionality between r 2 and τ shows that t‐SNAREs movement follows standard free diffusion with a diffusion coefficient of ≈4 µm 2 s −1 (Figure c) of the same order as what was previously measured with giant liposomes and sponge phase . Finally, t‐SNAREs are functional as they specifically bind the CTD of the fluorescently labeled cognate partner, vesicular SNARE (v‐SNARE, Figure d).…”

“…The proportionality between r 2 and τ shows that t-SNAREs movement follows standard free diffusion with a diffusion coefficient of ≈4 µm 2 s −1 (Figure 6c) of the same order as what was previously measured with giant liposomes and sponge phase. [37] Finally, t-SNAREs are functional as they specifically bind the CTD of the fluorescently labeled cognate partner, vesicular SNARE (v-SNARE, Figure 7d). Hence, it is possible to successfully insert transmembrane proteins while keeping their mobility, controlled orientation, and functionality in this microfluidic setup.…”

Effects of Pore Size and Tethering on the Diffusivity of Lipids Confined in Mesoporous Silica

“…This effect is similar to comparing diffusivities in planar bilayers and bicontinuous lipid mesophases, where similar diffusivities are observed using FRAP. [46] The diffusivity of supported bilayers on the surface of particles varies with pore diameter, with mobility increasing with increasing pore size (Figure 6a). Bilayers on nonporous particles and SBAS-3.0 enveloped the external particle surface, so measurements were only made at the particle cap, where the measured diffusivity is 1.79 × 10 −4 ± 0.61 × 10 −4 and 0.44 × 10 −4 ± 0.17 × 10 −4 µm 2 s −1 , respectively.…”

Hypothesis – buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission

“…This is to be compared to the section perpendicular to the lateral movement of a protein with a single transmembrane domain like SNAREs; this section spans both hydrophobic leaflets (5 nm) and has a width of ~ 1 nm which means it covers 5 nm². Thus, the diffusion coefficient of the loops will be similar to that is of individual SNAREs which is D  ~ 1 μm²/s 104. Using this value for the diffusion coefficient of the loops will provide a good approximation of the insertion time.…”

“…The average zippering force of the last layers of the SNAREpin is obtained from the energy landscape in Fig. A1D, ~ 30  k B T are released over 3 nm, that is, F  ~ 40 pN and D  ~ 1 μm²·s −1 104. The initial angle depends on the exact location of the transmembrane domains but is at most β init  = 30°.…”