Single molecule biomolecular condensate analysis in neurons

University Of Queensland, Brisbane

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Abstract

PROJECT SUMMARY/ABSTRACT Biomolecular condensates (BMCs) define small membraneless cellular compartments that segregate specific proteins and nucleic acids to generate cellular functions. Fluorescence recovery after photobleaching (FRAP) is currently the method of choice to characterize BMCs in vitro and in vivo and derive the average diffusion and trapping of molecules trapped in these condensates. Although FRAP is a good way to retrieve these important metrics, it greatly limits our understanding of BMCs nanoscale organization and dynamics in live cells and precludes the analysis of nanoscale BMCs (below the diffraction of light ~200 nm). Single-molecule imaging super-resolution microscopy allows direct imaging of molecules trapped in BMCs with much greater spatiotemporal resolution both in vitro and in live neurons, and has the potential to reveal nanoscale BMCs and their evolution in the context of ageing and Alzheimer’s disease (AD). However, there are currently no bioinformatic tools to specifically analyze BMCs at the single-molecule level. We have developed a novel tool, named Nanoscale Spatiotemporal Indexing Clustering (NASTIC), (Wallis et al., bioRxiv, 2021, ref. 7), which offers unprecedented insights into the dynamics of proteins undergoing liquid-liquid phase separation/transition in large and nanoscale BMCs, in live neurons. We identified Tau and synuclein as candidates for their ability to generate synaptic BMCs in hippocampal neurons. NASTIC analysis reveals that Tau molecules can indeed form small nanoclusters in live hippocampal neurons in which they exhibit very low mobility and sensitivity to 1,6-hexanediol, an aliphatic alcohol used to inhibit weak molecular interactions that mediate BMCs. NASTIC will be at the core of our ability to make a significant contribution to the understanding of BMCs in neurons because it offers unprecedented opportunities to examine the spatiotemporal behaviour of molecules in these condensates. NASTIC will be developed further to encompass two-color single-molecule analysis. This will be instrumental to assess the spatiotemporal relationship of these nanoscale BMCs with their cellular environment in 2 and 3D. We will validate our two-color NASTIC implementation with the pairwise dual imaging of Tau wildtype and AD mutant P301L, and a-synuclein. This will allow us to decipher the co-clustering dynamics and examine potential hierarchical dependency of one nanoBMC upon the other allowing refined understanding of the generation of nanoscale BMCs in synapses. We anticipate that the P301L mutation will largely increase the size and lifetime of the Tau nanoBMCs and alter their relationship with other synaptic molecules. The outcome of this grant will be the development and validation of an analytical pipeline that will enable exploration of the spatiotemporal relationship of Tau and a-synuclein nanoBMCs in live hippocampal neurons. Our project will therefore generate data and a much-needed technology opening new avenues for our understanding of BMCs in neuronal function and AD.