SUMMARY Tools capable of imaging and perturbing mechanical signaling pathways with fine spatiotemporal resolution have been elusive despite their importance in diverse cellular processes. The challenge in developing a mechanogenetic toolkit (i.e. selective and quantitative activation of genetically encoded mechanoreceptors) stems from the fact that many mechanically-activated processes are localized in space and time, yet additionally require mechanical loading to become activated. To address this challenge, we synthesized magnetoplasmonic nanoparticles that can image, localize, and mechanically load targeted proteins with high spatiotemporal resolution. We demonstrate their utility by investigating the cell surface activation of two mechanoreceptors: Notch and E-cadherin. By measuring cellular responses to a spectrum of spatial, chemical, temporal, and mechanical inputs at the single molecule and single cell level, we reveal how spatial segregation and mechanical force cooperate to direct receptor activation dynamics. This generalizable technique can be used to control and understand diverse mechanosensitive processes in cell signaling.
Spatiotemporal interrogation of signal transduction at the single cell level is necessary to answer a host of important biological questions. This protocol describes 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. The key components of this tool are a magnetoplasmonic nanoparticle (MPN) actuator that delivers defined spatial and mechanical cues to receptors through target-specific one-to-one engagement, and a micromagnetic tweezers (µMT) that remotely controls the magnitude of force exerted on a single MPN. This protocol consists of three major parts: 1) Preparation of small (40-50 nm), monovalent, and modular MPN probes, including chemical synthesis of MPN nanoparticles, conjugation with DNA, and purification of monovalent MPNs, 2) µMT setup and single particle force calibration as a function of µMT-to-MPN distance, and 3) control of spatial and mechanical properties of targeted mechanosensitive receptors in live cells by adjusting the µMT-to-MPN COMPETING FINANCIAL INTEREST The authors declare no competing financial interests.Supplementary Information is available in the online version of the paper. HHS Public Access
The ability to sense and manipulate the state of biological systems has been extensively advanced during the past decade with the help of recent developments in physical tools. Unlike standard genetic and pharmacological perturbation techniques-knockdown, overexpression, small molecule inhibition-that provide a basic on/off switching capability, these physical tools provide the capacity to control the spatial, temporal, and mechanical properties of the biological targets. Among the various physical cues, magnetism offers distinct advantages over light or electricity. Magnetic fields freely penetrate biological tissues and are already used for clinical applications. As one of the unique features, magnetic fields can be transformed into mechanical stimuli which can serve as a cue in regulating biological processes. However, their biological applications have been limited due to a lack of high-performance magnetism-to-mechanical force transducers with advanced spatiotemporal capabilities. In this Account, we present recent developments in magnetic nanotweezers (MNTs) as a useful tool for interrogating the spatiotemporal control of cells in living tissue. MNTs are composed of force-generating magnetic nanoparticles and field generators. Through proper design and the integration of individual components, MNTs deliver controlled mechanical stimulation to targeted biomolecules at any desired space and time. We first discuss about MNT configuration with different force-stimulation modes. By modulating geometry of the magnetic field generator, MNTs exert pulling, dipole-dipole attraction, and rotational forces to the target specifically and quantitatively. We discuss the key physical parameters determining force magnitude, which include magnetic field strength, magnetic field gradient, magnetic moment of the magnetic particle, as well as distance between the field generator and the particle. MNTs also can be used over a wide range of biological time scales. By simply adjusting the amplitude and phase of the applied current, MNTs based on electromagnets allow for dynamic control of the magnetic field from microseconds to hours. Chemical design and the nanoscale effects of magnetic particles are also essential for optimizing MNT performance. We discuss key strategies to develop magnetic nanoparticles with improved force-generation capabilities with a particular focus on the effects of size, shape, and composition of the nanoparticles. We then introduce various strategies and design considerations for target-specific biomechanical stimulations with MNTs. One-to-one particle-receptor engagement for delivering a defined force to the targeted receptor and the small size of the nanoparticles are important. Finally, we demonstrate the utility of MNTs for manipulating biological functions and activities with various spatial (single molecule/cell to organisms) and temporal resolution (microseconds to days). MNTs have the potential to be utilized in many exciting applications across diverse biological systems spanning from fundament...
SUMMARYTools capable of imaging and perturbing mechanical signaling pathways with fine spatiotemporal resolution have been elusive despite their importance in diverse cellular processes. The challenge in developing a mechanogenetic toolkit (i.e. selective and quantitative activation of genetically encoded mechanoreceptors) stems from the fact that many mechanically-activated processes are localized in space and time, yet additionally require mechanical loading to become activated. To Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. HHS Public Access
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