PROJECT SUMMARY / ABSTRACT This R35 program aims to delineate a previously unappreciated role of Piezo1 mechanosensitive ion channels in regulating cellular activity under inflammatory conditions using articular chondrocytes (ACs) as a model system. Mechanotransduction is a highly dynamic and ubiquitous process, and virtually all mammalian cells exhibit rapid and robust ion influx upon mechanical stimulus. In disease states, cells present abnormal mechanotransduction revealed by altered mechanosensitivity and biochemical transduction. Piezo1 is a novel mechanically-activated (MA) Ca2+-permeating channel abundantly expressed in cardiovascular, musculo- skeletal, and neural tissues. Augmented functional expression of Piezo1 in ACs is associated with cartilage disorders. Interestingly, ACs present a morphological differentiation from spherical ACs to cells with point-like processes in osteoarthritis. The actin filament (F-actin) networks have been focused on understanding morpho- logical differentiation and mechanical stability of AC, yet our preliminary data suggest a potential role of Piezo1 in F-actin remodeling in process formation under IL1α-mediated inflammatory conditions. Here, we propose a paradigm-shifting concept of Piezo1-mediated morphological changes and pro- cess formation in inflammatory conditions. Both mechanotransduction and inflammation influence or are re- sponsible for a wide array of physiological processes and abnormalities. A knowledge gap exists in the mecha- notransduction-inflammation coupling, morphology-dependent cellular mechanosensitivity, Piezo1-mediated morphological changes, Piezo1 localization on the membrane cortex of round-shape ACs, and Piezo1-recruit- ment in processes of fibroblast-like shaped ACs. This proposed program will identify: (i) if activated Piezo1 al- ters local membrane curvature and triggers to form cellular protrusions and reorganize cytoskeleton meshwork, (ii) specific inflammatory cytokines triggering morphological changes and augmenting Piezo1 channels in pro- cesses, which in turn altering the gating properties of Piezo1, and (iii) if Piezo1-mediated inflammatory re- sponse increases mechanical fatigue and death. The whole-cell patch-clamp method allows to record ion channel activities, yet ACs present technical challenges on sealing the membrane primarily due to their round morphology and small size (~10 μm in diameter). Thus, we have custom-built a novel microscopy, named ‘Mechano-microscopy’, by combining an atomic force microscope (AFM), a high-speed ratiometric Ca2+ im- aging microscope, and a confocal microscope to simultaneously record cellular morphology and biochemical signals in response to mechanical cues under inflammatory conditions. In addition, we will utilize the super- resolution STED microscope and Piezo1-TdTomato reporter mice to interrogate dynamics of membrane curva- ture and Piezo1 localization in ACs after treating specific inflammatory cytokines. Our program will c...