Direct observation of heterogeneous formation of amyloid spherulites in real-time by super-resolution microscopy.

Protein misfolding in the form of fibrils or spherulites is involved in a spectrum of pathological abnormalities. Our current understanding of protein aggregation mechanisms has primarily relied on the use of spectrometric methods to determine the average growth rates and diffraction-limited microscopes with low temporal resolution to observe the large-scale morphologies of intermediates. We developed a REal-time kinetics via binding and Photobleaching LOcalization Microscopy (REPLOM) super-resolution method to directly observe and quantify the existence and abundance of diverse aggregate morphologies of human insulin, below the diffraction limit and extract their heterogeneous growth kinetics. Our results revealed that even the growth of microscopically identical aggregates, e.g., amyloid spherulites, may follow distinct pathways. Specifically, spherulites do not exclusively grow isotropically but, surprisingly, may also grow anisotropically, following similar pathways as reported for minerals and polymers. Combining our technique with machine learning approaches, we associated growth rates to specific morphological transitions and provided energy barriers and the energy landscape at the level of single aggregate morphology. Our unifying framework for the detection and analysis of spherulite growth can be extended to other self-assembled systems characterized by a high degree of heterogeneity, disentangling the broad spectrum of diverse morphologies at the single-molecule level.

Live-cell super-resolution microscopy reveals a primary role for diffusion in polyglutamine-driven aggresome assembly.

The mechanisms leading to self-assembly of misfolded proteins into amyloid aggregates have been studied extensively in the test tube under well-controlled conditions. However, to what extent these processes are representative of those in the cellular environment remains unclear. Using super-resolution imaging of live cells, we show here that an amyloidogenic polyglutamine-containing protein first forms small, amorphous aggregate clusters in the cytosol, chiefly by diffusion. Dynamic interactions among these clusters limited their elongation and led to structures with a branched morphology, differing from the predominantly linear fibrils observed in vitro Some of these clusters then assembled via active transport at the microtubule-organizing center and thereby initiated the formation of perinuclear aggresomes. Although it is widely believed that aggresome formation is entirely governed by active transport along microtubules, here we demonstrate, using a combined approach of advanced imaging and mathematical modeling, that diffusion is the principal mechanism driving aggresome expansion. We found that the increasing surface area of the expanding aggresome increases the rate of accretion caused by diffusion of cytosolic aggregates and that this pathway soon dominates aggresome assembly. Our findings lead to a different view of aggresome formation than that proposed previously. We also show that aggresomes mature over time, becoming more compacted as the structure grows. The presence of large perinuclear aggregates profoundly affects the behavior and health of the cell, and our super-resolution imaging results indicate that aggresome formation and development are governed by highly dynamic processes that could be important for the design of potential therapeutic strategies.

Fluorescence Self-Quenching from Reporter Dyes Informs on the Structural Properties of Amyloid Clusters Formed in Vitro and in Cells.

The characterization of the aggregation kinetics of protein amyloids and the structural properties of the ensuing aggregates are vital in the study of the pathogenesis of many neurodegenerative diseases and the discovery of therapeutic targets. In this article, we show that the fluorescence lifetime of synthetic dyes covalently attached to amyloid proteins informs on the structural properties of amyloid clusters formed both in vitro and in cells. We demonstrate that the mechanism behind such a "lifetime sensor" of protein aggregation is based on fluorescence self-quenching and that it offers a good dynamic range to report on various stages of aggregation without significantly perturbing the process under investigation. We show that the sensor informs on the structural density of amyloid clusters in a high-throughput and quantitative manner and in these aspects the sensor outperforms super-resolution imaging techniques. We demonstrate the power and speed of the method, offering capabilities, for example, in therapeutic screenings that monitor biological self-assembly. We investigate the mechanism and advantages of the lifetime sensor in studies of the K18 protein fragment of the Alzheimer's disease related protein tau and its amyloid aggregates formed in vitro. Finally, we demonstrate the sensor in the study of aggregates of polyglutamine protein, a model used in studies related to Huntington's disease, by performing correlative fluorescence lifetime imaging microscopy and structured-illumination microscopy experiments in cells.