Leaflet-Dependent Distribution of PtdIns[4,5]P₂ in Supported Model Membranes

Abstract

Supported planar lipid bilayers (SLBs) prepared by spreading of unilamellar vesicles on hydrophilic substrates such as silicon dioxide are frequently used to investigate lipid−protein interactions by means of surface-sensitive methods. In recent years, the receptor lipid phosphatidylinositol-4,5-bisphosphate (PtdIns-[4,5]P 2 ) became particularly important as a significant number of proteins bind to this lipid at the inner leaflet of the plasma membrane. Here, we investigated how the lipid PtdIns[4,5]P 2 distributes between the two leaflets of an SLB on SiO 2 surfaces. We prepared SLBs on SiO 2 by spreading small unilamellar vesicles and quantified the adsorption of PtdIns[4,5]P 2 binding proteins providing information about the accessibility of PtdIns[4,5]P 2 . We compared protein binding to PtdIns[4,5]P 2 in SLBs with that in lipid monolayers on a 1,1,1-trimethyl-N-(trimethylsilyl)silanamine-functionalized SiO 2 surface using reflectometric interference spectroscopy and atomic force microscopy. Our results clearly demonstrate that the accessibility of PtdIns[4,5]P 2 for protein binding is reduced in SLBs compared to that in supported hybrid membranes, which is discussed in terms of PtdIns[4,5]P 2 distribution in the two leaflets of SLBs.

Neuroligin-2 dependent conformational activation of collybistin reconstituted in supported hybrid membranes

“…In a first step, we produced lipid membranes on a silicon support to investigate and quantify the binding capability of CB2 to PtdInsPs in the presence or absence of the intracellular NL2 C terminus . We used a previously published protocol ( 30 ) to generate lipid monolayers composed of POPC and doped with different PtdInsPs. By using silicon dioxide substrates functionalized with 1,1,1-trimethyl- N -(TMS)silanamine (HMDS), to which small unilamellar vesicles (SUVs) were fused ( Fig.…”

“…By using silicon dioxide substrates functionalized with 1,1,1-trimethyl- N -(TMS)silanamine (HMDS), to which small unilamellar vesicles (SUVs) were fused ( Fig. 1 C ), a possible asymmetric distribution of PtdInsPs between the two lipid leaflets is prevented ( 30 ). Lipid monolayers composed of POPC and doped with 3 mol % of PtdIns[3]P, PtdIns[4,5]P 2 , or PtdIns[3,4,5]P 3 on HMDS were prepared.…”

“…Previous studies led to the hypothesis that the cytosolic NL2 C terminus binds the SH3 domain of CB2 in the biological context of the postsynapse, thereby inducing a more open conformation, which allows CB to interact with PtdInsPs located at the postsynaptic membrane ( 14 , 29 ). By using SHMs, which provide a homogeneous distribution of PtdInsPs ( 30 ), we were able to establish an in vitro molecular model that allowed us to investigate directly how the interaction of CB2 with the cytosolic part of NL2 influences its ability to bind to different PtdInsPs. Our results show that, without the membrane-associated part of NL2, the WT protein CB2 SH3 does not bind to any of the three PtdInsPs tested.…”

“…Silicon substrates with a SiO 2 layer thickness of 5 μm were cleaned two times with detergent solution followed by rinsing with ultrapure water in an ultrasonic bath for 15 min each. Afterward, the substrates were treated with O 2 -plasma for 30 s and then exposed to hexamethyldisilazane (HMDS) as previously described in detail ( 30 ).…”

“…Specifically, we used supported hybrid membranes (SHMs) on hydrophobically functionalized silicon dioxide substrates via spreading of small unilamellar vesicles (SUVs). We showed recently that SHMs are superior to supported lipid bilayers as they provide a more homogeneous distribution of PtdInsPs ( 30 ). SHMs were doped with PtdInsPs serving as receptor lipids for CB2, whereas 1,2-dioleoyl- sn -glycero-3-{[ N -(5-amino-1-carboxypentyl)iminodiacetic acid]succinyl} nickel salt (DGS-NTA(Ni)) was added to specifically adsorb the intracellular domain of NL2 (His- cyt NL2) via an N -terminally fused His 6 -tag ( Fig.…”