Highly specific and non-invasive imaging of Piezo1-dependent activity across scales using GenEPi

Mechanosensing is a ubiquitous process to translate external mechanical stimuli into biological responses.Piezo1 ion channels are directly gated by mechanical forces and play an essential role in cellular mechanotransduction.However, readouts of Piezo1 activity are mainly examined by invasive or indirect techniques, such as electrophysiological analyses and cytosolic calcium imaging.Here, we introduce GenEPi, a genetically-encoded fluorescent reporter for non-invasive optical monitoring of Piezo1-dependent activity.We demonstrate that GenEPi has high spatiotemporal resolution for Piezo1dependent stimuli from the single-cell level to that of the entire organism.GenEPi reveals transient, local mechanical stimuli in the plasma membrane of single cells, resolves repetitive contraction-triggered stimulation of beating cardiomyocytes within microtissues, and allows for robust and reliable monitoring of Piezo1-dependent activity in vivo.GenEPi will enable non-invasive optical monitoring of Piezo1 activity in mechanochemical feedback loops during development, homeostatic regulation, and disease.Throughout an organism's lifetime, cell mechanosensation (i.e., the ability to perceive and respond to mechanical stimuli in the form of shear stress, tension, or compression) is essential in a myriad of developmental, physiological, and pathophysiological processes, including embryogenesis, homeostasis, metastasis, and wound healing 1 .How these processes incorporate active feedback via force sensing at the cellular level is an area of active study, and a wide range of tools have been developed to interrogate cell mechanics 2,3 .Stretch-activated ion channels, including the Piezo proteins, can respond to various external mechanical stimuli 4,5 .Most vertebrates have two Piezo genes, Piezo1 and Piezo2 4 , and functional homologs have been identified both in plants 6 and invertebrates 7 .While Piezo2 function is mainly restricted to the peripheral nervous system, Piezo1 is expressed in a wide range of tissues and has been shown to contribute to mechanotransduction in various organs 5 .Mutations in human Piezo1 have been implicated in diseases, such as dehydrated hereditary