Biomolecular condensates (BMCs) can form membraneless intracellular compartments via liquid-liquid phase separation (LLPS). Several synaptic proteins such as synapsin-1 can undergo LLPS suggesting that a synaptic BMC could control synaptic functions (1-3). Further, LLPS have made the headlines with the demonstration that Tau, TDP-43, FUS and α-synuclein could form toxic condensates in the context of neurodegenerative diseases.
Despite these exciting advances, the field is still in great need for reliable techniques to allow the quantification of BMC dynamics in live cells. Upon LLPS, molecules involved in generating BMCs undergo a dramatic drop in their mobility, which can be measured indirectly via fluorescent recovery after photobleaching (FRAP). However, using FRAP to bleach large and visible BMCs precludes analysis of nanoscale BMCs (nanoBMCs) which are smaller than the diffraction limit of visible light and likely to be present at the synapse (3). NanoBMCs could be critically important as they are likely to initiate the generation of larger BMCs by acting as “seeds”. The regulation of the growth phase from these seeds may be important as Tau BMCs can evolve into amyloids and play deleterious roles in neurodegenerative disorders such as Alzheimer’s diseases.
Synapses are usually very small which therefore precludes the use of FRAP for visualizing synaptic BMCs. Single-molecule super-resolution microscopy allows direct visualization and tracking of individual proteins of interest in live cells, neurons and their synapses. As such, this technique is perfectly suited to study BMCs at nanoscale level as molecules undergoing LLPS can be tracked with unprecedented resolution in space and time.
In this presentation I will present our latest attempt to identify and characterize of synaptic nanoBMCs formed by key synaptic proteins involved in neurodegenerative diseases and design a new analytic platform (4) to unveil their formation dynamics.
References:
1 Bademosi, A. T. et al. In vivo single-molecule imaging of syntaxin1A reveals polyphosphoinositide- and activity-dependent trapping in presynaptic nanoclusters. Nat Commun 8, 13660, doi:10.1038/ncomms13660 (2017).
2 Joensuu, M. et al. Visualizing endocytic recycling and trafficking in live neurons by subdiffractional tracking of internalized molecules. Nat Protoc 12, 2590-2622, doi:10.1038/nprot.2017.116 (2017).
3 Padmanabhan, P., Martinez-Marmol, R., Xia, D., Gotz, J. & Meunier, F. A. Frontotemporal dementia mutant Tau promotes aberrant Fyn nanoclustering in hippocampal dendritic spines. Elife 8, doi:10.7554/eLife.45040 (2019)
4 Wallis, T. P. et al. MOLECULAR VIDEOGAMING: SUPER-RESOLVED TRAJECTORY-BASED NANOCLUSTERING ANALYSIS USING SPATIO-TEMPORAL INDEXING. bioRxiv, 2021.2009.2008.459552, doi:10.1101/2021.09.08.459552 (2021).