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.
Khan et al. (Sun,) studied this question.