Time-resolved fluorescence characterization of phase-separating proteins involved in neurodegeneration

Abstract

Dementias represent the most prevalent class of diseases worldwide, marked by debilitating symptoms such as motor impairment and cognitive decline, ultimately leading to death. Despite extensive research, the mechanisms underlying disease onset remain incompletely understood. A hallmark of many dementias is the accumulation of aggregated proteins, which are now recognized to be predominantly intrinsically disordered in their monomeric form, lacking a defined tertiary structure. The microtubule-associated protein Tau is one of these intrinsically disordered proteins and has been reported to predominantly adopt a compact “paperclip-like” conformation. Importantly, Tau is implicated in several dementias, including Alzheimer’s disease, Pick’s disease, frontotemporal dementia, and corticobasal degeneration, where it aggregates into neurofibrillary tangles. Within these fibrils, individual Tau molecules exhibit a rigid core formed by the microtubule-binding region, which adopts characteristic C-shaped architectures. These observations lead us to hypothesize that aggregation is driven by conformational transitions within the monomeric protein. One suggested pathway towards aggregation is liquid-liquid phase separation, a process by which proteins partition in a dilute and a dense phase. Physiologically this process allows for higher localized protein concentration, as well as response to cellular stress. In disease, it might provide an environment which promotes conformational transitions towards disease associated structures. In this work, we aimed at studying the effect of the Tau protein integration in a crowded environment in its conformation. We applied single-molecule Förster Resonance Energy Transfer (smFRET) to investigate the structure and dynamics of the microtubule-binding region (MTBR) of Tau. In its monomeric state, the MTBR predominantly samples a compact conformation, however, it remains intrinsically disordered and transitions between extended and compact states on the microsecond timescale. The terminal domains occupy larger distances relative to the MTBR and exhibit faster conformational exchange. We also sought to characterize the physical properties of biocondensates and the conformational behavior of proteins within them. Due to their high density, biocondensates sediment to the bottom of the imaging chamber, making diffusion-based confocal methods difficult to implement. To probe the internal microenvironment, we employed raster imaging correlation spectroscopy (RICS), which quantifies molecular diffusion from confocal images. Molecules within droplets experienced markedly higher viscosity, with diffusion coefficients approximately ten-fold lower than in the dilute phase. Because smFRET inside droplets is technically challenging and time-consuming, we turned to fluorescence lifetime imaging microscopy (FLIM) to assess ensemble conformations. FLIM revealed that the MTBR adopts more compact conformations within dense phases compared to the dilute phase, consistent with structural features reported for fibril cores. To overcome sedimentation, we developed a microfluidic strategy enabling biocondensates to flow through a stationary confocal detection volume one at a time. Flow characteristics of the microfluidic chip were validated using fluorescence correlation spectroscopy, showing close agreement with simulated velocity profiles, including accelerated flow at the channel center. Droplet flow produced distinct intensity spikes corresponding to individual condensates, allowing us to determine their size distribution. Importantly, flow speed could be tuned to extend observation times, offering a promising avenue for future single-molecule studies of intradroplet conformational dynamics.