Supported lipid bilayers (SLBs) are broadly used as minimal membrane models and commonly produced by vesicle fusion (VF) on solid supports. Despite its advantages, VF does not allow the controlled formation of bilayers that mimic the leaflet asymmetry in lipid composition normally found in biological systems. Here we present a simple, quick, and versatile method to produce SLBs with a desired asymmetric lipid composition which is stable for ca. 4 h. We apply methyl-β-cyclodextrin mediated lipid exchange to SLBs formed by VF to enrich the upper leaflet of the bilayer with sphingomyelin. The bilayer asymmetry is assessed by fluorescence correlation spectroscopy, measuring the lipid mobility separately in each leaflet. To check the compatibility of the method with the most common protein reconstitution approaches, we report the production of asymmetric SLBs (aSLBs) in the presence of a glycosylphosphatidylinositol-anchored protein, reconstituted in the bilayer both, via direct protein insertion, and via proteoliposomes fusion. We finally apply aSLBs to study phase separation and transbilayer lipid movement of raft-mimicking lipid mixtures. The observed differences in terms of phase separation in symmetric and asymmetric SLBs with the same overall lipid composition provide further experimental evidence that the transversal lipid distribution affects the overall lipid miscibility and allow to temporally investigate leaflet mixing.

The RhoGTPases are characterized as membrane-associated molecular switches that cycle between active, GTP-bound and inactive, GDP-bound states. However, 90–95% of RhoGTPases are maintained in a soluble form by RhoGDI, which is generally viewed as a passive shuttle for inactive RhoGTPases. Our current understanding of RhoGTPase:RhoGDI dynamics has been limited by two experimental challenges: direct visualization of the RhoGTPases in vivo and reconstitution of the cycle in vitro. We developed methods to directly image vertebrate RhoGTPases in vivo or on lipid bilayers in vitro. Using these methods, we identified pools of active and inactive RhoGTPase associated with the membrane, found that RhoGDI can extract both inactive and active RhoGTPases, and found that extraction of active RhoGTPase contributes to their spatial regulation around cell wounds. These results indicate that RhoGDI directly contributes to the spatiotemporal patterning of RhoGTPases by removing active RhoGTPases from the plasma membrane.

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20The RhoGTPases are characterized as membrane-associated molecular switches cycling 21 between active, GTP-bound and inactive, GDP-bound states. However, 90-95% of RhoGTPases 22 are maintained in a soluble form by RhoGDI, which is generally viewed as a passive shuttle for 23 inactive RhoGTPases. Our current understanding of RhoGTPase:RhoGDI dynamics has been 24 limited by two experimental challenges: direct visualization of the RhoGTPases in vivo and 25 reconstitution of the cycle in vitro. We developed methods to directly image vertebrate 26 RhoGTPases in vivo or on lipid bilayers in vitro. Using these tools, we identified pools of active 27 and inactive RhoGTPase associated with the membrane, showed that RhoGDI can actively 28 extract both inactive and active RhoGTPases, and that the extraction of active RhoGTPase 29 contributes to their spatial regulation around wounds. In contrast to the textbook model of the 30 RhoGTPase cycle, these results indicate that RhoGDI actively contributes to spatiotemporal 31 patterning by removing active RhoGTPases from the plasma membrane. 32 33 34 35 36 37 38 39 40 41

Recently, a new and versatile assay to determine the partitioning coefficient KP as a measure for the affinity of peripheral membrane proteins for lipid bilayers was presented in the research article entitled, “Introducing a fluorescence-based standard to quantify protein partitioning into membranes” [1]. Here, the well-characterized binding of hexahistidine-tag (His6) to NTA(Ni) was utilized. Complementarily, this data article reports the average diffusion coefficient D of His6-tagged enhanced green fluorescent protein (eGFP-His6) and the fluorescent lipid analog ATTO‐647N‐DOPE in giant unilamellar vesicles (GUVs) containing different amounts of NTA(Ni) lipids. In addition, dissociation constants Kd of the NTA(Ni)/eGFP-His6 system are reported. Further, a conversion between Kd and KP is provided.