An Early Stage of Membrane Fusion Mediated by the Low pH Conformation of Influenza Hemagglutinin Depends upon Membrane Lipids

Abstract

While the specificity and timing of membrane fusion in diverse physiological reactions, including virus–cell fusion, is determined by proteins, fusion always involves the merger of membrane lipid bilayers. We have isolated a lipid-dependent stage of cell–cell fusion mediated by influenza hemagglutinin and triggered by cell exposure to mildly acidic pH. This stage preceded actual membrane merger and fusion pore formation but was subsequent to a low pH–induced change in hemagglutinin conformation that is required for fusion. A low pH conformation of hemagglutinin was required to achieve this lipid-dependent stage and also, downstream of it, to drive fusion to completion. The lower the pH of the medium applied to trigger fusion and, thus, the more hemagglutinin molecules activated, the less profound was the dependence of fusion on lipids. Membrane-incorporated lipids affected fusion in a manner that correlated with their dynamic molecular shape, a characteristic that determines a lipid monolayer's propensity to bend in different directions. The lipid sensitivity of this stage, i.e., inhibition of fusion by inverted cone–shaped lysophosphatidylcholine and promotion by cone-shaped oleic acid, was consistent with the stalk hypothesis of fusion, suggesting that fusion proteins begin membrane merger by promoting the formation of a bent, lipid-involving, stalk intermediate.

Single-particle kinetics of influenza virus membrane fusion

“…Hemifusion can be detected by observing dequenching of a fluorescent probe embedded in the membrane of the impinging virion-a method also used in various bulk-phase fusion assays (13)(14)(15)17). Quenching of fluorescence from dye incorporated at high concentration is alleviated when the membrane in which it resides merges with a larger bilayer.…”

“…Conventional fusion assays provide some kinetic information by detecting ion-conducting pores or redistribution of fluorescent dye between pairs of fusing cells or between viruses and liposomes in suspension (13)(14)(15)(16)(17). The data are generally limited to averaged observations of a large number of fusion events, and information about short-lived or rare intermediate states is lost, as the underlying individual fusion events dephase over time.…”

Blebbishields, the emergency program for cancer stem cells: sphere formation and tumorigenesis after apoptosis

“…Acidic pH is known to facilitate lipid-mediated membrane fusion. 26,27 In vivo, the tumor microenvironment pH is known to be as low as 6.5 and the tumor cells are capable of avoiding intracellular acidification by proton pumps. 28,29 It has also been reported that the pH 6.5 promotes CSC sphere formation in vitro.…”

Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder?

“…In cellular membranes, PUFAs act as cone shaped lipids that induce a positive curvature of the membrane leaflet 119 , thereby enhancing membrane trafficking 118 . It is thus possible that PUFA's exert at least some of their protective effects through stabilization of disordered membrane endocytosis.…”