Direct Observation of Anisotropy-Driven Formation of Amyloid Protein Core-Shell Structures in Real-time by Super-resolution Microscopy
Proteins misfolding and aggregation in the form of fibrils or amyloid containing spherulite-like structures, are involved in a spectrum of degenerative diseases. Current understanding of protein aggregation mechanism primarily relies on conventional spectrometric methods reporting the average growth rates and microscopy readouts of final structures, consequently masking the morphological and growth heterogeneity of the aggregates. Here we developed REal-time kinetics via binding and Photobleaching LOcalization Microscopy (REPLOM) super resolution method to observe directly and quantify the existence and abundance of diverse aggregation morphologies as well as the heterogeneous growth kinetics of each of them. Our results surprisingly revealed insulin aggregation is not exclusively isotropic, but it may also occur anisotropically. Combined with Machine learning we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape for each aggregation morphology. Our unifying framework of detection and analysis of spherulite growth can be extended to other protein systems and reveal their aggregation processes at single molecule level.