Abstract
The core component of a biological membrane is a fluid–lipid bilayer held together by interfacial–hydrophobic and van der Waals interactions, which are balanced for the most part by acyl chain entropy confinement. If biomembranes are subjected to persistent tensions, an unstable (nanoscale) hole will emerge at some time to cause rupture. Because of the large energy required to create a hole, thermal activation appears to be requisite for initiating a hole and the activation energy is expected to depend significantly on mechanical tension. Although models exist for the kinetic process of hole nucleation in tense membranes, studies of membrane survival have failed to cover the ranges of tension and lifetime needed to critically examine nucleation theory. Hence, rupturing giant (∼20 μm) membrane vesicles ultra-slowly to ultra-quickly with slow to fast ramps of tension, we demonstrate a method to directly quantify kinetic rates at which unstable holes form in fluid membranes, at the same time providing a range of kinetic rates from <0.01 to >100 s−1. Measuring lifetimes of many hundreds of vesicles, each tensed by precision control of micropipette suction, we have determined the rates of failure for vesicles made from several synthetic phospholipids plus 1:1 mixtures of phospho- and sphingo-lipids with cholesterol, all of which represent prominent constituents of eukaryotic cell membranes. Plotted on a logarithmic scale, the failure rates for vesicles are found to rise dramatically with an increase in tension. Converting the experimental profiles of kinetic rates into changes of activation energy versus tension, we show that the results closely match expressions for thermal activation derived from a combination of meso-scale theory and molecular-scale simulations of hole formation. Moreover, we demonstrate a generic approach to transform analytical fits of activation energies obtained from rupture experiments into energy landscapes characterizing the process of hole nucleation along the reaction coordinate defined by hole size.
Highlights
A century ago, physiologists found that cells rupture quickly when subjected to large osmotic pressure differences yet remain sealed much longer before rupturing when subjected to smaller osmotic stresses
Values we present for edge energies and the length scales describing precursor structures will differ significantly from the simulation results, the models used to correlate the experimental data for activation energies in Fig. 9 will mirror the shapes of energy landscapes obtained in the coarse-grained molecular dynamics and illustrated by the right panels in Fig. 10b. [Note: distinct from pore size, the apparent hole radius begins with the outward/inward radial displacements of material due to thinning/thickening of the membrane at the site of pore nucleation, which is followed by opening of the pore lumen.]
Turning to the intriguing experiments in which kinetic rates were found to depend on ramp speed as well as tension, we have found that the activation energies obtained from logarithms of kinetic rates for rupture of diC18:2 PC and C18:0/1 PC vesicles at the various ramp speeds can be collapsed to generic dependences on tension
Summary
Physiologists found that cells rupture quickly (lyse) when subjected to large osmotic pressure differences yet remain sealed much longer before rupturing when subjected to smaller osmotic stresses (cf. classic book on red blood cell hemolysis by Ponder [1]). In contrast to transient permeation and conductance in supported membranes, studies of cell and vesicle membrane poration have for the most part been focussed on the measuring the stresses (lateral tension or electro-compression) needed to break the membranes, yet neglecting the time dependence of membrane survival on the history of stress For this reason, a few years ago we developed an approach called dynamic tension spectroscopy (DTS, [13]) to quantify the most frequent tensions σ* at which giant (~20 μm) membrane vesicles rupture when subjected to ramps of tension, σ(t) = rσ t, and the most frequent membrane lifetimes under increasing tension, t*. Using these correlations as examples, we end by demonstrating how analytical fits to the measured dependencies of barrier energy on tension can be transformed into quantitative images of the energy landscapes governing thermal activation along a scalar reaction coordinate defined by hole size
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