PROJECT SUMMARY/ABSTRACT Disruptions in mechanosensation are commonly responsible for symptoms in diseases of gut-brain interaction (DGBI), like irritable bowel syndrome (IBS), which affect ~15% of the US population. Therefore, my laboratory’s long-term goal is to elucidate the cellular and molecular mechanisms of gastrointestinal (GI) mechanosensitivity in health and in DGBIs. There are several mechanosensory pathways in the GI tract. One such important mechanosensory pathway that is involved in DGBIs is initiated through the specialized sensory epithelial enteroendocrine cells (EECs). Upon stimulation, EECs release diverse signaling molecules that have a range of physiologic effects, and disruption in this signaling is involved in DGBIs. We discovered an EECs sub-population which is mechanosensitive. These EECs express Piezo2, a mechano-gated ion channel that connects forces to release of EEC signaling molecules and endows the intestine with an ability to sense small luminal forces to adjust GI motility and secretion. The objective of this proposal, based on strong rationale and extensive preliminary data, is to test the hypothesis that rectal Piezo2+ mechanosensitive EECs co-express select GPCRs sensitive to digestive metabolites, which modulate Piezo2 mechanosensitivity. These rectal mechanosensitive EECs connect chemo- and mechano- signaling in rectal EECs, and thereby regulate regional and proximal intestinal motility. We have established novel transgenic mouse models that allow us to lineage track, stimulate, and interrogate specific EEC sub- populations, and we will use these mouse models and validated EEC lines with a range of innovative and established gold-standard approaches from single cells to in vivo to study mechanosensitive EECs and their roles in GI physiology. Aim 1 will determine the cellular mechanisms of chemo- and mechano-signaling in EECs. Aim 2 will determine how the mechanosensitive EECs stimulated by forces and luminal digestive metabolite to modulate GI motility. The proposed experiments are foundationally linked to our previous work, but they represent a new and exciting direction and can be completed in the defined award period. The results from this study are important because they will allow us to deeply understand mechanosensitive EEC cellular physiology and their roles in GI motility, which will enable us to examine alterations in disease, and then potentially target these pharmacologically as novel and specific therapies for DGBIs.