Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Poster Abstracts

34-POS Board 9 PHOSPHOINOSITIDE DOMAIN FORMATION AND PROPERTIES IN ASYMMETRIC LIPID BILAYERS Trevor A. Paratore 1 ; Alonzo H Ross 1 ; Arne Gericke 1 ; 1 Worcester Polytechnic Institute, Department of Chemistry and Biochemistry, Worcester, MA, USA Phosphoinositides (PIPs) have been shown to mediate a broad range of cellular events by attracting proteins to specific cellular sites. This spatiotemporal control of protein function is often associated with the formation of phosphoinositide enriched patches (domains) or gradients. The plasma membrane lipid composition is different (asymmetric) in the two leaflets of the bilayer. It has been suggested that outer leaflet lipid raft domains affect inner leaflet phosphoinositide domains and hence PIP mediated signaling events. Phosphoinositides exhibit predominantly a stearoyl-arachidonoyl acyl chain composition and PIP accumulation in a liquid- ordered membrane environment is expected to be unfavorable. Therefore, the question arises how outer leaflet lipid rafts can affect inner leaflet phosphoinositide distribution and function. To address this question, we adapted the hemifusion method introduced by Enoki and Feigenson (Biophys. J. 117 (2019) 103) to fabricate asymmetric giant unilamellar vesicles (aGUVs). This process involves the Ca 2+ initiated fusion of a symmetric GUV with a solid supported lipid bilayer (SLB), which contains in our case the phosphoinositide component. We find a large variability of the PIP concentration in the resulting aGUV population. We attribute this to the formation of micro-sized domains in the SLB, leading to the fusion of the sGUVs with regions rich in PIPs and other areas that lack them. To overcome this issue, we use 1 mM Mg 2+ instead of Ca 2+ as the initiator for the fusion process since in the presence of Mg 2+ PIPs don’t form macroscopic domains. We obtain a narrow PIP concentration distribution across the aGUV population. In the next step we investigate PIP domains in the presence of a lipid raft mixture in the opposing leaflet.

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