Project Summary/Abstract Intrinsically-disordered proteins (IDPs) are drivers of intracellular self-assembly. Powered by highly multivalent interactions, IDPs organize subcellular assemblies (biomolecular condensates) governed by liquid-liquid phase separation (LLPS) dynamics. From genomic organization to synaptic plasticity, biomolecular condensates influence wide-ranging cellular mechanisms. Despite these exciting insights, the biophysical and physiological properties of the underlying IDP-assemblies remain poorly understood. This knowledge gap is pervasive because existing tools to study IDPs and their LLPS require non-physiological conditions. The major challenge is the pronounced environmental sensitivity of IDPs. Their LLPS behavior is unpredictably altered by environmental and biochemical changes, including post-translational modifications (PTMs) and molecular tagging with fluorescent proteins. New tools are needed to dissect biomolecular condensates in their native cellular environments, within tissues. Progress towards in tissue non-disruptive probing of IDP-assemblies will close the gap separating IDP biophysics and IDP-linked disease mechanisms. Crucially, while IDP-assemblies are pathological hallmarks of untreatable degenerative brain disorders, decades-old and LLPS-refined observations have failed to provide mechanistic insights. Motivated by these challenges, this proposal advances biomolecular sensors to probe and manipulate intracellular IDP-assemblies in brain-like tissues. The crucial innovation is the encoding of ultra-weak and LLPS-specific multivalent interactions into engineered IDPs equipped with fluorescent and catalytic domains. The resulting IDPs will serve as multifunctional LLPS- sensors, enabling a strategic departure from molecular tagging of native IDPs. This engineering platform builds on fluorescent LLPS-sensors recently pioneered to illuminate LLPS dynamics in skin. By catalyzing biotinylation and protein-disaggregation, next-generation LLPS-sensors will enable biomolecular dissection of IDP-assemblies and provide tools for combating neuropathological IDP-assemblies. To advance and deploy these innovations, this proposal will engineer and interrogate multifunctional LLPS-sensors in state-of-the-art brain organoid models of Alzheimer's disease, frontotemporal dementia, and amyotrophic lateral sclerosis. Combining sensor-enabled live cell imaging and proximity proteomics, the proposed experimental approaches will address long-standing key questions linking pathological IDP-assemblies and major human neurodegenerative disorders. By adding molecular tools and rigor to the modeling of neuropathology in brain organoids, this work will enable and stimulate molecular-level dissection of age-dependent human neurodegeneration. Beyond generating therapeutic insights into IDP-driven mechanisms of neurodegeneration, this proposal will advance a broadly applicable sensor-organoid platform to study biomolecular condensates acr...