BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Abstract
Understanding the gating mechanism of the mechanosensitive ion channel PIEZO2 is crucial because of its involvement in key physiological and pathological processes. The use of a bead-functionalized cantilever by atomic force microscopy (AFM) provides a well-defined contact area, enabling accurate and reproducible force delivery to the cell surface. This approach enables the application of a spatiotemporally controlled mechanical stimulation to single PIEZO2-transfected HEK-293 cell at short (0.5s) and long (10s) stimulus duration, while monitoring the channel activity in single stimulated cell and neighboring cells by calcium signal. Two distinct dyes were used: Rhod-3AM restricted to the cytoplasm and Rhod-2AM permeable to whole cell including organelles. We observed that in a single cell, a 50nN local force elicited stronger responses with short stimuli, particularly with the organelle-permeable dye. Moreover, upon mechanical stimulation of a single PIEZO2-overexpressing cell, calcium transients were also detected in neighboring, non-stimulated cells, particularly with a dye permeable to organelles, thus suggesting a mechanosensitive intercellular communication pathway. These results confirmed that PIEZO2 channels are highly sensitive, efficiently respond to short-duration stimuli, and are involved in a mechanosensitive cell-cell communication.
