Establishing Force Spectrscopy with Lipid Vesicle Probes towards the Investigation of Membrane Fusion

Abstract

The interaction and fusion of phospholipid membranes plays an important role in many essential physiological processes such as neurotransmitter release at the synapse. Many of those fusion processes are facilitated by SNARE complexes. In spite of the physiological importance, it is still not fully understood how this SNARE-mediated process establishes a fusion pore between two membranes. Especially vague is the understanding about the role of the transmembrane domains (TMDs) of the SNARE proteins for the establishment of hemi- and full fusion states. Therefore, we propose a new experimental system to investigate the fusogenic role of SNARE TMDs in model lipid membranes. The system consist of an atomic force microscopy (AFM) cantilever with an integrated microfluidic channel, that is used to firstly attach a giant unilamellar vesicle (GUV) to the tip of the cantilever by applying negative pressure inside of the channel, and to subsequently bring this GUV into force-controlled contact with a supported lipid bilayer or a surface immobilized GUV. Using fluorescence measurements we are able to observe fusion states dependent on the contact force, contact time, and other parameters like membrane tension and ion concentration. By bringing two lipid membranes into close and force-controlled proximity, the presented experimental system is able to mimic the zippering function of the cytosolic domains of the SNARE complex. Therefore, the next step will be the inclusion of SNARE TMDs into the phospholipid membranes in varying concentrations and combinations to gain insights into their function within the fusion machinery. Consequently, the proposed experimental system will facilitate new insights into SNARE-mediated fusion and will shed light on the basic principles of neuronal signal transmission. Additionally, this system can also be used to investigate other phenomena related to the interaction of lipid membranes.