All-or-none versus graded: single-vesicle analysis reveals lipid composition effects on membrane permeabilization.
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.
Unidad de Biofísica, Consejo Superior de Investigaciones Científicas/University of the Basque Country, Bilbao, Spain.
Beatriz
B
Apellániz
José L
JL
Nieva
Petra
P
Schwille
Ana J
AJ
García-Sáez
D002784:Cholesterol / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D007700:Kinetics; D008055:Lipids / Q000737:chemistry*; D008567:Membranes, Artificial; D010455:Peptides / Q000494:pharmacology; D010539:Permeability / Q000187:drug effects; D016062:Porosity / Q000187:drug effects; D053835:Unilamellar Liposomes / Q000737:chemistry*
D005456:Fluorescent Dyes; D008055:Lipids; D008567:Membranes, Artificial; D010455:Peptides; D053835:Unilamellar Liposomes; D002784:Cholesterol