BPS2026 – Exploring the phase behavior of model membranes composed of branched-chain bacterial lipids and hopanoid

Lipids and proteins in bacterial plasma membranes (PMs) are thought to organize into functional membrane microdomains (FMMs) analogous to lipid rafts in eukaryotic cell membranes. Studies of simplified three-component model membranes have proven extremely valuable for elucidating the lipid-lipid interactions that drive raft formation in eukaryotic plasma membranes (PM). Here, we examine how bacterial lipids influence phase behavior in ternary mixtures containing a low-melting lipid, a high-melting lipid, and either cholesterol (Chol) or the hopanoid diplopterol (Dip), a cholesterol analog in bacteria hypothesized to exert a membrane ordering effect similar to cholesterol. Using DPPC/DOPC/Chol as a baseline model for the eukaryotic PM outer leaflet, we replaced DOPC with anteiso-15:0-iso-15:0 PC (ai15i15PC), a low-melting lipid whose branched chain structure is used by some bacterial species to maintain fluidity. Interestingly, confocal fluorescence microscopy of GUVs revealed that the large region of liquid-ordered + liquid-disordered phase coexistence observed in DPPC/DOPC/Chol was eliminated in vesicles composed of DPPC/ai15i15PC/Chol. Additionally, when we replaced Chol with Dip, we found that GUVs retained gel-fluid characteristics to high Dip concentration. Di4 generalized polarization (GP) measurements showed that Dip exerts a membrane-ordering effect of lower magnitude compared to Chol. Förster resonance energy transfer (FRET) revealed nanoscopic lateral heterogeneity in membranes containing DPPC/ai15i15PC (1:1 and 2:1 ratio) with 10, 15, and 20 mol% Chol. Furthermore, cryo-EM revealed uniform mixing for DPPC/ai15i15PC (1:1) with either 20 mol% Chol or Dip.