All-or-None versus Graded: Single-Vesicle Analysis Reveals Lipid Composition Effects on Membrane Permeabilization

Abstract

We report a single-vesicle approach to compare the all-or-none and graded mechanisms of lipid bilayer permeabilization by CpreTM and NpreTM, two peptides derived from the membrane-proximal external region of the HIV fusion glycoprotein gp41 subunit. According to bulk requenching assays, these peptides permeabilize large unilamellar vesicles via all-or-none and graded mechanisms, respectively. Visualization of the process using giant unilamellar vesicles shows that the permeabilization of individual liposomes by these two peptides differs in kinetics, degree of dye filling, and stability of the permeabilized state. All-or-none permeabilization by CpreTM is characterized by fast and total filling of the individual vesicles. This process is usually accompanied by the formation of stably open pores, as judged from the capacity of the vesicles to incorporate a second dye added after several hours. In contrast, graded permeabilization by NpreTM is transient and exhibits slower kinetics, which leads to partial filling of the individual liposomes. Of importance, quantitative analysis of vesicle population distribution allowed the identification of mixed mechanisms of membrane permeabilization and the assessment of cholesterol effects. Specifically, the presence of this viral envelope lipid increased the stability of the permeating structures, which may have implications for the fusogenic activity of gp41.

Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2)-dependent Oligomerization of Fibroblast Growth Factor 2 (FGF2) Triggers the Formation of a Lipidic Membrane Pore Implicated in Unconventional Secretion

“…Following incubation of PI(4,5)P 2 containing GUVs with FGF2-Y82pCMF, a substantial fraction of about 40% of total GUVs was characterized by high luminal fluorescence (Ն80% compared with the surroundings), indicating that the fluorescent tracer had equilibrated between the lumen and the surroundings of the vesicles. This behavior is typical for an "all-or-none" mechanism of protein-dependent membrane activity (42). Quantification of control experiments using GUVs either lacking PI(4,5)P 2 or containing the Ni-NTA lipid revealed that, following incubation with FGF2-Y82pCMF, only a minor population contained the fluorescent tracer (Fig.…”

“…Analysis of PI(4,5)P 2 and Tyrosine Phosphorylation-dependent Membrane Activity of FGF2 Oligomers Employing Giant Unilamellar Vesicles and Confocal Microscopy-To directly visualize the membrane activity of FGF2-Y82pCMF, we employed GUVs combined with confocal microscopy (42). Three types of GUVs with a plasma membrane-like lipid composition either lacking PI(4,5)P 2 , containing PI(4,5)P 2 (plasma membrane ϩ PIP 2 ), or containing the Ni-NTA lipid (plasma membrane ϩ nickel) were generated (Fig.…”

Negatively Charged Lipids as a Potential Target for New Amphiphilic Aminoglycoside Antibiotics

“…2A), we observed a dose-dependent calcein release from LUVs incubated with 3Ј,6-dinonyl neamine at concentrations ranging from 0.25 to 1.7 M. After the addition of 3Ј,6-dinonyl neamine at 1 M, Ͼ80% of calcein was released in the presence of cardiolipin, whereas only around 50% was released when cardiolipin was absent. These differences could reflect differences in mechanism (all-or-none versus graded mechanisms) (49), but further studies are required to confirm this. Fig.…”

Cytochrome c causes pore formation in cardiolipin-containing membranes

“…Fig. 2 shows representative plots for GUVs treated with cyt c. An abrupt change of the interior plot from zero or minimal slope to a steep slope indicates the instant when leakage begins; this observation is interpreted as the opening of a lipid pore, corresponding to an all-or-none type of leakage described in literature (27). Behavior and event timing varied for GUVs within a single sample, implying that pore formation is a stochastic process, whereas initial binding of cyt c to the membrane was fast.…”