Combinatorial Polarized TIRF-AFM Study of Membrane Reorganization by Alpha-Synuclein and Human Prion Protein

Abstract

Many neurodegenerative diseases are caused by aggregation or fibrillation of membrane-associated proteins. Alpha-Synuclein (aS) is associated with Parkinson's Disease as a major component of Lewy bodies, and misfolded aggregates of human Prion Protein (PrP) are associated with spongiform encephalopathies. Here we study the influence of these proteins on the organization and topography of canonical raft-mimetic supported lipid bilayers (DSPC/DOPC/cholesterol at 35/35/30 mol% on mica) using a correlated polarized Total Internal Reflectance Fluorescence-Atomic Force Microscopy (pTIRF-AFM) platform. pTIRF allows for high-speed measurements of dynamic processes at diffraction-limited spatial resolution, while AFM gives time-averaged nm-scale spatial resolution. Using pTIRF and an oriented lipophilic dye (DiI-C18) we determine the local order in the membrane by measuring the ratio of fluorescence parallel and perpendicular to the membrane plane, while simultaneously measuring membrane topography using AFM. Upon addition of aS to our phase-segregated membrane system, liquid-ordered and liquid-disordered domains were mixed and the local order increased such that lipid headgroups were oriented more parallel to the membrane plane. Moreover, AFM topography revealed the formation of long, closed-loop tubules. Addition of the active segment PrP(106-126) to these raft-mimetic membranes resulted in a similar mixing of domains and an increase of local order, while AFM revealed the formation of peptide fibrils in and around cholesterol-rich domains. This study provides direct evidence of membrane reorganization and restructuring by aS and PrP, and the effect of membrane chemistry on aS and PrP self-association. These results portend future detailed investigations into the molecular-level factors responsible for these effects.