Ordered Lipid Domains Assemble via Concerted Recruitment of Constituents from Both Membrane Leaflets

Lipid rafts serve as anchoring platforms for membrane proteins. Thus far they escaped direct observation by light microscopy due to their small size. Here we used differently colored dyes as reporters for the registration of both ordered and disordered lipids from the two leaves of a freestanding bilayer. Photoswitchable lipids dissolved or reformed the domains. Measurements of domain mobility indicated the presence of 120 nm wide ordered and 40 nm wide disordered domains. These sizes are in line with the predicted roles of line tension and membrane undulation as driving forces for alignment.


Apparent domain diameter da and true domain diameter d differ by a size independent
term δ δ has two constituents:

S1
where dd and ds are the apparent increments in d due to diffraction limitations and domain diffusion, respectively. Both dd and ds do not depend on d in a wide range of domain diameters, as we show below, thereby justifying the assumption that δ varies negligibly, i.e. d = da -δ for all domains.

The invariance of δs
Domain displacement during the time td of its image acquisition gives rise to a seemingly larger domain size. The effect scales with the diffusion coefficient D of the domain:

S2
td depends on the apparent domain diameter da, more precisely on the number of lines NL that the microscope has to scan in order to acquire the domain image. For the diameter of a single pixel dp (~220 nm) we find: . S3 Since acquisition of 1024 lines takes about 1.7 seconds, we may write for td: where c1= 0.0075 s µm -1 . The lateral displacement of the diffusing domain during acquisition of its image can be estimated as: where the experimentally determined product Dda for da < 1.7 µm is equal to (i) 1.32 µm 3/2 s -1/2 for LODs in LDDs and (ii) 0.67 µm 3/2 s -1/2 for LDDs in LODs (SFig. 1). Accordingly, Eq. S5 returns for the respective values of δs: 0.22 µm and 0.12 µm.

Supplementary Figure 1. The difference between apparent domain diameter (da) and true domain diameter (d) due to non-instantaneous image acquisition is independent of domain size.
We measure (i) a smaller diffusion coefficient D and (ii) a longer scanning time (proportional to da) for larger domains. As a result, does not depend on da. This is true both for LODs diffusing in LDDs (black circles) and LDDs diffusing in LODs (white circles). The horizontal dotted lines correspond to the best fit of the dependencies.

The invariance of δd
Diffraction limitations let domain diameter d appear larger by δd. δd does not depend on d. Rather, it is a function of both pixel size dp and diameter of the focal area. In our case, the latter is roughly twice the size of the former (the exact ratio depending on the wavelength). Since we are taking into account only domains that are well separated in space and since every pixel contains several of fluorescent dye molecules, δd is equal to the distance at which fluorescence intensity (i) drops from the brightness of an LDD domain to that of an LOD domain or (ii) increases from the brightness of an LOD domain to that of an LDD domain. This distance may vary between dp and 2dp depending on the positioning of the domain boarder (red circle in SFig. 2). have size da, because fluorescence brightness between the dimmer LOD regions (dark gray) and brighter LDD regions (light gray) does not change instantaneously, but at least over the distance dp of one pixel (gray).

Selection of domain trajectories
In order to decide whether traces of single domain diffusion should enter the analysis, we calculated the straightness of the diffusion trajectory, S: where N is a number of consecutive two dimensional positions of a tracked particle (domain), xi is the radius-vector of the particle center of mass at the i-th step of the trajectory [32]. S is a build in criteria of the software (Image J). We required S ≤ 0.2. Its use for the exclusion of events that are not compatible with simple diffusion is appealing, because S also excludes events with rather short trajectories.