Magnetic nano-tweezer for interrogating mechanosensitive signaling proteins in space and time.

ABSTRACT

Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to understand how extracellular cues are converted into biochemical signals and differentially regulate cellular responses. Using single-cell perturbation tools such as optogenetics, specific biochemical cues can be delivered to selective molecules or cells at any desired location and time. By measuring cellular responses to provided perturbations, investigators have decoded and deconstructed the working mechanisms of a variety of neuroelectric and biochemical signaling processes. However, analogous methods for deciphering the working mechanisms of mechanosensitive signaling by regulating mechanical inputs to cell receptors have remained elusive. To address this unmet need, we have recently developed a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. This tool combines a magnetofluorescent nanoparticle (MFN) actuator, which provides precise spatial and mechanical signals to receptors via target-specific one-to-one interaction, with a micromagnetic tweezers that remotely controls the force exerted on a single nanoparticle. This chapter provides comprehensive experimental protocols of mechanogenetics consisting of four stages (i) chemical synthesis of MFNs, (ii) bio-conjugation and purification of monovalent MFNs, (iii) establishment of cells with genetically encoded mechanosensitive proteins, and (iv) modular targeting and control of cell-surface receptors in live cells. The entire procedure takes up to 1 week. This mechanogenetic tool can be generalized to study many outstanding questions related to the dynamics of cell signaling and transcriptional control, including the mechanism of mechanically activated receptor.