The Piconewton Force Awakens: Quantifying Mechanics in Cells

The development of calibrated Forster resonance energy transfer (FRET)-based tension sensors has allowed the first analyses of mechanical processes with piconewton (pN) sensitivity in cells. Here, we introduce the working principle of this emerging microscopy method and discuss how it has been utilized to obtain quantitative insights into the mechanisms of intracellular force transduction in cell-matrix adhesions, cell-cell junctions, and at the cell cortex. These examples demonstrate that genetically encoded tension sensors are powerful tools to unravel force transduction mechanisms, but also indicate current limitations. We propose that further technical improvements are needed to develop a truly molecular understanding of mechanobiological processes in cells and tissues

Investigating piconewton forces in cells by FRET-based molecular force microscopy

The ability of cells to sense and respond to mechanical forces is crucial for a wide range of developmental and pathophysiological processes. The molecular mechanisms underlying cellular mechanotransduction, however, are largely unknown because suitable techniques to measure mechanical forces across individual molecules in cells have been missing. In this article, we highlight advances in the development of molecular force sensing techniques and discuss our recently expanded set of FRET-based tension sensors that allows the analysis of mechanical forces with piconewton sensitivity in cells. In addition, we provide a theoretical framework for the design of additional tension sensor modules with adjusted force sensitivity. (C) 2016 Elsevier Inc. All rights reserved

Generation and Analysis of Biosensors to Measure Mechanical Forces Within Cells

The inability to measure mechanical forces within cells has been limiting our understanding of how mechanical information is processed on the molecular level. In this chapter, we describe a method that allows the analysis of force propagation across distinct proteins within living cells using Förster resonance energy transfer (FRET)-based biosensors

Multiplexing molecular tension sensors reveals piconewton force gradient across talin-1

Förster resonance energy transfer (FRET)-based tension sensor modules (TSMs) are available for investigating how distinct proteins bear mechanical forces in cells. Yet, forces in the single piconewton (pN) regime remain difficult to resolve, and tools for multiplexed tension sensing are lacking. Here, we report the generation and calibration of a genetically encoded, FRET-based biosensor called FL-TSM, which is characterized by a near-digital force response and increased sensitivity at 3–5 pN. In addition, we present a method allowing the simultaneous evaluation of coexpressed tension sensor constructs using two-color fluorescence lifetime microscopy. Finally, we introduce a procedure to calculate the fraction of mechanically engaged molecules within cells. Application of these techniques to new talin biosensors reveals an intramolecular tension gradient across talin-1 that is established upon integrin-mediated cell adhesion. The tension gradient is actomyosin- and vinculin-dependent and sensitive to the rigidity of the extracellular environment

Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap

New tools for applying force to animals, tissues, and cells are critically needed in order to advance the field of mechanobiology, as few existing tools enable simultaneous imaging of tissue and cell deformation as well as cellular activity in live animals. Here, we introduce a novel microfluidic device that enables high-resolution optical imaging of cellular deformations and activity while applying precise mechanical stimuli to the surface of the worm\u27s cuticle with a pneumatic pressure reservoir. To evaluate device performance, we compared analytical and numerical simulations conducted during the design process to empirical measurements made with fabricated devices. Leveraging the well-characterized touch receptor neurons (TRNs) with an optogenetic calcium indicator as a model mechanoreceptor neuron, we established that individual neurons can be stimulated and that the device can effectively deliver steps as well as more complex stimulus patterns. This microfluidic device is therefore a valuable platform for investigating the mechanobiology of living animals and their mechanosensitive neurons