BACKGROUND The gastrointestinal tract (GI) has a sense of gut touch by which low threshold mechanical inputs activate mechanoreceptors similar to Merkel cells that mediate skin touch. The gut touch sensors are enteroendocrine cells (EECs) that transduce mechanical stimuli into calcium (Ca²⁺) signals, causing the release of hormones and neurotransmitters. Gut touch sensors are embedded in epithelia via physical cell-cell coupling. EECs express Piezo2 ion channels, which are activated by forces via membrane stretch and cytoskeletal tethers. Piezo2 interactions with E-cadherin, an epithelial junctional protein, are a potential tether for Piezo2. HYPOTHESIS Piezo2 tethering to the actin cytoskeleton through E-cadherin allows force sensing by EECs in the intestinal epithelium. METHODS We used mouse intestinal organoids expressing a Ca²⁺ reporter (GCaMP5) in EECs to measure mechanical Ca²⁺responses in isolated cells and gut epithelial monolayers. We disrupted Piezo2 activity, actin polymerization and myosin contractility with small molecules (Piezo2: GsMTx-4; actin: latrunculin A, jasplakinolide; myosin: blebbistatin, EMD57003) to assess their effect on mechanically induced responses, as well as in genetically targeted E-cadherin knockdown (KD) specific to EECs. We used super-resolution imaging to visualize colocalization between Piezo2 clusters and junctional/cytoskeletal proteins in the context of E-cadherin knockdown. We investigated Piezo2 protein interactions via co-immunoprecipitation. We incubated a Piezo2-expressing EEC cell model with a myosin agonist then measured mechanically induced whole cell currents by voltage clamp electrophysiology. RESULTS Compared to dissociated EECs, mechanosensitive EEC Ca²⁺ responses are significantly longer when integrated in monolayers. Monolayer EEC mechanosensitive responses decrease upon: 1) Piezo2 knockout, 2) Piezo2 small molecule blockade mid-response, 3) myosin inhibition, and 4) EEC E-cadherin KD. Structurally, Piezo2 co-precipitates with E-cadherin, actin and myosin. Interestingly, myosin and actin interact with Piezo2 independent of E-cadherin, and myosin activation (via EMD57003) rescues EEC mechanosensitivity even following E-cadherin knockdown. Piezo2 peak current amplitudes are increased and inactivation kinetics slower in EEC model cells preincubated with EMD57003. CONCLUSIONS Piezo2 interacts with structural proteins that impact Piezo2 function in gut touch sensors within epithelia, either by positioning Piezo2 for optimal force sensing and/or applying forces to gate the channels. Myosin activation rescued the mechanosensitivity deficit after E-cadherin KD, suggesting that Piezo2 makes multiple redundant interactions. These interactions potentially affect Piezo2 channel activity, which has been associated with sensory and noxious signaling. Supported by NIH DK123549, DK128913, GM145408, DK084567

Shreya Bellampalli

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