Visualization of Ca²⁺ influx spots across individual SA channels activated by tension in stress fibers

Abstract

In response to mechanical stretch in the vessel, endothelial cells show a variety of reactions such as secretion of vasoactive substances and morphological changes, which are triggered by Ca²⁺-influx across stretch-activated cation channel (SACatC).Two crucial questions, however, remain on SACatC activation: how are forces conveyed to SACatCs? and :where are SACatCs in the cell? We suspected that membrane-connected cytoskeleton (actually stress fiber, SF) may act as an intracellular force transmitter and that SACtaCs exist in the vicinity of focal adhesions (FA) at which SF is terminated. To prove this hypothesis we developed a method by which we can apply localized mechanical stimuli onto FAs via SFs by dragging SFs with laser tweezers while measuring Ca²⁺ influx across individual SACatCs by high speed near-field microscopy (NFM). It was found that SACatCs were activated by small forces as low as 1pN and located near FAs as close as 300-500nm.