Amyloidogenic Self-Assembly of Insulin Aggregates Probed by High Resolution Atomic Force Microscopy

As the application of high-resolution atomic force microscopy (AFM) has led us recently to the discovery of a unique pressure-induced circular amyloid, we used the same approach to examine morphological events accompanying insulin aggregation under ambient conditions. This study presents the multistage, hierarchical character of the spontaneous fibrillation of insulin at low pH and at 60 and 70°C, and—due to the marked enhancement of image resolution achieved—brings new clues as to the fibrils’ ultrastructure and mechanisms of its assembly. Specifically, focusing on the prefibrillar amorphous aggregates occurring 30s after elevating temperature to the nucleation-enhancing 60°C, revealed the tendency of the globule-shaped oligomers to queue and assembly into elongated forms. This suggests that the shape of the nuclei itself predetermines—in part—the fibrillar architecture of the amyloid. Among first fibrillar features, short but relatively thick (8-nm) seedlike forms appeared on a very short timescale within the first minute of incubation. It has been shown that such fibrils are likely to act as lateral scaffolds for the growth of amyloid. By using phase-image AFM as a nanometer-resolved probe of visco-elastic surface properties, we were able to show that bundles of early protofilaments associated into parallel fibrils are capable of a cooperative transformation into twisted, highly ordered superhelices of the mature amyloid. Independently from producing evidence for the step-resolved character of the process, intermediate and morphologically heterogeneous forms were trapped and characterized, which yields direct evidence for the multipathway character of the amyloidogenesis of insulin. Apart from the faster kinetics, the increased temperature of 70°C leads to a higher degree of morphological variability: along straight rods, twisted ribbonlike structures, rod bundles, and ropelike structures become prominent in the corresponding AFM data.