PROJECT SUMMARY The crowded conditions within cells present a complex physicochemical challenge for maintaining the solubility and fluidity of thousands of distinct macromolecules. High macromolecular densities promote assembly into a variety of distinct structures, which in turn can lead to the formation of insoluble solid aggregates. Though solidified structures could indeed serve protective purposes or as quiescent storage sites, they are more generally considered to be toxic to the cell. The physical constraints and properties generated by crowded cellular environments, which contribute significantly to the functional behaviors of the resident molecules, are often difficult to probe experimentally. In addition to membrane-bound compartments, which define the primary structural organization of cells, numerous membraneless organelles (MLOs) populate the cytoplasm and the nucleoplasm providing additional complexity. These biomolecular condensates (BMCs) form via liquid-liquid phase separation (LLPS), a process that concentrates the resident macromolecules, but which in turn also can promote additional phase states, including solid aggregates. Multiple BMC-resident proteins that undergo LLPS in vitro are the dominant proteins in cytoplasmic inclusions found in patients with a range of devastating neurological disorders. In one such example, the fused in sarcoma (FUS) protein accumulates in MLOs termed stress granules (SGs), which form as a natural response to various cellular stress conditions. The high molecular density of FUS in SGs is potentially an essential early step toward the formation of FUS-containing solid inclusions linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Directly observing the formation and growth of such inclusions within a cellular context is challenged by the lack of appropriate tools. While genetic approaches to attach fluorescent tags are commonly used to determine spatiotemporal behaviors, their attachment can often perturb function, there are generally only a few reasonable attachment points, and such probes have inferior photophysical properties. Recent improvements in genetic code expansion to incorporate non-canonical amino acids (ncAAs) for in cellulo labeling promises to significantly extend the types of questions that may be addressed. This is particularly relevant for the application of multiple single molecule biophysical strategies. The Specific Aim of the proposed work is to identify and characterize solid aggregates within live cells. To accomplish this, designer MLOs will be used to specifically modify FUS via ncAA-mediated fluorescent labeling in cells. Microsecond-scale single molecule rotational diffusion (µs-SiMRoD) microscopy will then be used to probe for local environmental constraints such as increased viscosity or structural confinement. The target application will be identifying and monitoring solidified FUS-containing aggregates. This strategy is expected to be i...