Shape bistability of a membrane neck: a toggle switch to control vesicle content release.

Shape dynamics and permeability of a membrane neck connecting a vesicle and plasma membrane are considered. The neck is modeled by a lipid membrane tubule extended between two parallel axisymmetric rings. Within a range of lengths, defined by system geometry and mechanical properties of the membrane, the tubule has two stable shapes: catenoidal microtubule and cylindrical nanotubule. The permeabilities of these two shapes, measured as ionic conductivity of the tubule interior, differ by up to four orders of magnitude. Near the critical length the transitions between the shapes occur within less than a millisecond. Theoretical estimates show that the shape switching is controlled by a single parameter, the tubule length. Thus the tubule connection can operate as a conductivity microswitch, toggling the release of vesicle content in such cellular processes as "kiss-and-run" exocytosis. In support of this notion, bistable behavior of membrane connections between vesicles and the cell plasma membrane in macrophages is demonstrated.

Effects of membrane potential and sphingolipid structures on fusion of Semliki Forest virus.

Cells expressing the E1 and E2 envelope proteins of Semliki Forest virus (SFV) were fused to voltage-clamped planar lipid bilayer membranes at low pH. Formation and evolution of fusion pores were electrically monitored by capacitance measurements, and membrane continuity was tracked by video fluorescence microscopy by including rhodamine-phosphatidylethanolamine in the bilayer. Fusion occurred without leakage for a negative potential applied to the trans side of the planar membrane. When a positive potential was applied, leakage was severe, obscuring the observation of any fusion. E1-mediated cell-cell fusion occurred without leakage for negative intracellular potentials but with substantial leakage for zero membrane potential. Thus, negative membrane potentials are generally required for nonleaky fusion. With planar bilayers as the target, the first fusion pore that formed almost always enlarged; pore flickering was a rare event. Similar to other target membranes, fusion required cholesterol and sphingolipids in the planar membrane. Sphingosine did not support fusion, but both ceramide, with even a minimal acyl chain (C(2)-ceramide), and lysosphingomyelin (lyso-SM) promoted fusion with the same kinetics. Thus, unrelated modifications to different parts of sphingosine yielded sphingolipids that supported fusion to the same degree. Fusion studies of pyrene-labeled SFV with cholesterol-containing liposomes showed that C(2)-ceramide supported fusion while lyso-SM did not, apparently due to its positive curvature effects. A model is proposed in which the hydroxyls of C-1 and C-3 as well as N of C-2 of the sphingosine backbone must orient so as to form multiple hydrogen bonds to amino acids of SFV E1 for fusion to proceed.