Quantitative mechanical stimulation of GPR68 using a novel 96 well flow plugin.

Abstract

Mechanosensitive proteins play a crucial role in a range of physiological processes, including hearing, tactile sensation and regulating blood flow. While previous work has demonstrated the mechanosensitivity of several proteins, the ability to apply precisely defined mechanical forces to cells in a consistent, replicable manner remains a significant challenge. In this work we present a novel 96-well plate-compatible plugin device for generating highly-controlled flow-based mechanical simulation of cells, which enables quantitative assessment of mechanosensitive protein function. The device is used to mechanically stimulate HEK 293T cells expressing the mechanosensitive protein GPR68, a G protein-coupled receptor. By assaying intracellular calcium levels during flow-based cell stimulation, we determine that GPR68 signalling is a function of the applied shear-force. As this approach is compatible with conventional cell culture plates and allows for simultaneous readout in a conventional fluorescence plate reader, this represents a valuable new tool to investigate mechanotransduction.