(56g) Alpha-Synuclein-Mediated Membrane Remodeling
Equilibrium binding between aSyn and lipid bilayers, and the structure of membrane-bound aSyn, has been extensively studied. In contrast, investigation of aSyn-mediated membrane remodeling has been hampered by limitations on experimental techniques. Virtually all data come from two types of assays: vesicle clearance, and electron microscopy. The vesicle clearance assay is simple to perform, but provides no quantitative information on change in size, shape, or number of lipid vesicles caused by aSyn. EM provides important insightsinto the nature of structural changes, as the lipid vesicles can be directly imaged before and after addition of aSyn, but the technique is not quantitative and not amenable to obtaining kinetic data.
In this talk we describe the use of nanoparticle tracking analysis (NTA) as a tool to quantify aSyn-mediated membrane remodeling. Briefly, in NTA, diffusing particles are tracked over time to obtain a hydrodynamic diameter. Because individual particles are counted, the technique allows measurement of a particle-by-particle size distribution and a total particle number concentration. Furthermore, the scattered intensity of each particle is obtained. We used NTA to interrogate the interaction of aSyn with unilamellar vesicles of DOPS (1,2-dioleoyl-sn-glycerol-3-phospho-L-serine) and DLPS (1,2-dilauroyl-sn-glycero-3-phospho-L-serine). These two lipids were chosen because phosphatidylserine is the most abundant anionic lipid in eukaryotic membranes and because aSyn is well-known to interact best with charged polar head groups. DOPS has similar physical properties as natural brain lipids whereas DLPS has saturated but shorter acyl chains. Immediately upon addition of aSyn to DOPS, we observed a small but measurable increase in the hydrodynamic diameter of liposomes, but no change in number concentration even after several hours. In sharp contrast, with DLPS we observed a decrease in mean liposome diameter and a decrease in liposome number concentration that continued over a 5-h period. Furthermore, we saw a significant drop in the scattered intensity of particles, after correcting for hydrodynamic diameter. We hypothesized that the decrease in scattered intensity was due to a radical change in particle shape, because the intensity is a function of both particle size and shape. Indeed, by TEM, we observed that aSyn induced substantial tubulation and large-scale membrane restructuring in DLPS but not DOPS. Molecular dynamic simulations showed that aSyn strongly induces negative curvature in DLPS but not DOPS, consistent with tubulation and membrane restructuring. This work demonstrates the novel use of NTA to quantify the kinetics and nature of protein-induced membrane restructuring. The results contribute to our understanding of how the physical properties and composition of the lipid bilayer influence the mechanism by which aSyn regulates membrane remodeling, vesicle release, or vesicle fusion.