Experimental Measurement of the Activation Energy of Phospholipid Membrane Fusion

Abstract

Organisms and cells are divided in different compartments which have their own function but nevertheless need to communicate with one another. This communication relies on intracellular vesicular traffic, in which vesicles bud from a donor compartment and are transported to an acceptor compartment where they fuse and release their content. Thus, membrane fusion is a most important process in living organisms.In vivo, fusion must not occur spontaneously in order to maintain the integrity of the compartments. This is why this process requires a large activation energy that is overcome thanks to the action of multiple proteins. This activation energy probably depends on many parameters such as membrane composition, curvature, surface tension.Even though biological fusion is very complex and involves proteins, the result is the coalescence of both lipid bilayers that constitute the cores of the involved membranes. This is why the activation energy that is necessary to disrupt the leaflet arrangement during lipid bilayer fusion should be similar to that of in vivo membrane fusion. Hence, we established a protocol to experimentally measure the activation energy for phospholipid membrane fusion, in order to approach the biological membrane fusion activation energy. Theoretical studies and simulations predict an activation energy in the range of 40-150 kT. To our knowledge, no experimental measurement has been done yet.In our experimental setup, the frequency of the rare spontaneous fusion events is investigated at various temperatures, which allows determining the activation energy thanks to Arrhenius’ law. We will present our protocol and show the results for DOPC and POPC membranes containing increasing amounts of DOPE, a phospholipid known to promote hemifusion. We will also present results concerning more physiological compositions, mimicking the fusion of the synaptic vesicle with the plasma membrane.