The physiological process of maintaining cellular solute and water content is termed osmotic homeostasis, or osmoregulation, and is essential for all forms of cellular life. In humans, osmotic homeostasis plays vital roles in several contexts, including regulation of the kidney's urinary concentrating mechanism, control of blood pressure, and activation of immune responses. Osmotic dyshomeostasis is associated with several age- related diseases, including chronic kidney disease, renal failure, hypertension, and peripheral neuropathy. Despite the obvious importance of osmoregulation in both physiological and pathophysiological disease states, little is known about the mechanisms by which animal cells sense and respond to osmotic stress. A better understanding of these mechanisms may allow earlier detection and intervention in age-related diseases. Most studies of osmoregulation have been carried out using cultured cells, which fail to mimic the complex environments in which most cells are found. These studies have led to many hypotheses to explain the mechanism(s) of cellular osmosensing, such as mechanical 'stretching' of the membrane and/or cytoskeleton, macromolecular crowding, and alterations in cytoplasmic ionic content, to name a few. However, there is little data supporting any of these models. To gain an in vivo perspective on mechanisms of cellular osmosensing in animals, we are studying this process in the model organism C. elegans, in which complex cell-cell and cell- extracellular matrix (ECM) interactions are preserved. Using unbiased forward and reverse genetic approaches, we discovered critical roles for the extracellular matrix (Rohlfing et al, PLoS Genetics, 2011) and protein misfolding (Moronetti Mazzeo et al, PNAS, 2012) in the regulation of cellular osmosensing in C. elegans. Based on these findings we hypothesize that animal cells use both mechanotransduction and protein damage detection mechanisms to sense osmotic disturbances and activate osmosensitive gene expression. In Aim 1, we will determine if the C. elegans cuticular ECM acts as a structural 'osmosensor' to transduce information via interactions between the mucin-like protein OSM-8 and a transmembrane protein PTR-23. In Aim 2, we will define the native proteins susceptible to stress-induced protein aggregation and determine how aging and aging regulators influence osmotic stress induced protein damage and osmosensitive gene expression. In Aim 3, we will examine how ECM and protein damage detection pathways interact with each other to control osmoregulatory physiology. Our studies take maximal advantage of the C. elegans system to fill an important gap in our knowledge of metazoan cell physiology. These findings will provide transformative insights into the conserved process of osmoregulation that will allow us to better understand, detect, and manage age-related diseases of osmotic dyshomeostasis.