Amyotrophic lateral sclerosis (ALS) is a debilitating and terminal disease. A common hallmark of its familial and sporadic forms is the accumulation of insoluble protein aggregates in the cytoplasm of motor neurons. FUS RNA binding protein (FUS) is found in cytoplasmic protein aggregates of post-mortem spinal cords and brains in patients with ALS and frontotemporal lobar degeneration. Importantly, mutation in FUS is causal in a subset of ALS cases. The progression of FUS to various aggregated states (facilitated by mutation or stress) and how this process impacts on key biological functions in neurons remain poorly understood, leaving an important gap in our understanding of the defects leading to ALS pathogenesis. We hypothesize that localization of FUS to the cytosol and its association with specific proteins induce aberrant changes in neuronal functions, leading to its subsequent aggregation and dysfunction in ALS. This hypothesis is supported by recent reports that identified several interacting proteins that mitigate the aggregation and toxicity of cytosolic FUS. In this project, we will use an unbiased proximity-dependent biotinylation technique called BioID to ascertain which protein interactors are gained and lost when mutant FUS localizes to the cytoplasm and how age-related stress conditions alter these interactions. We will determine the interactions that modulate FUS dynamics by performing aggregation assays following the overexpression and depletion of candidate proteins in cells and in vitro. Finally, we will investigate how changes in these interactions influence the cytosolic functions of FUS in human motor neurons derived from the induced pluripotent stem cells of healthy donors and patients with ALS expressing FUS mutants. Our team’s expertise in quantitative proteomics, high-resolution microscopy, and biophysics uniquely positions us to elucidate crucial insights into FUS dysregulation and its role in causing ALS. Our work will generate an invaluable resource—a comprehensive list of FUS interactors in healthy and disease-associated conditions—that will shed light on molecular pathways contributing to FUS-mediated ALS pathogenesis. Furthermore, our findings and approaches are applicable to several other ALS-associated RNA-binding proteins that share similar properties as FUS, providing a primary model for future studies. Together, the novel regulators we identify will illuminate novel avenues for therapeutic design that could make ALS a treatable disease.