Molecular Force Sensors for Biological Application

Abstract

The mechanical forces exerted by cells on their surrounding microenvironment are known as cellular traction forces. These forces play crucial roles in various biological processes, such as tissue development, wound healing and cell functions. However, it is hard for traditional techniques to measure cellular traction forces accurately because their magnitude (from pN to nN) and the length scales over which they occur (from nm to μm) are extremely small. In order to fully understand mechanotransduction, highly sensitive tools for measuring cellular forces are needed. Current powerful techniques for measuring traction forces include traction force microscopy (TFM) and fluorescent molecular force sensors (FMFS). In this review, we elucidate the force imaging principles of TFM and FMFS. Then we highlight the application of FMFS in a variety of biological processes and offer our perspectives and insights into the potential applications of FMFS.

Keywords: cellular traction forces; fluorescent molecular force sensors; mechanotransduction; traction force microscopy.

Figure 2

Overview of force-to-fluorescence conversion based on different principles: (a) distance-based FRET or fluorescence quenching, adapted from Refs. [102,103,104,105]; (b) orientation-based FRET, adapted from Ref. [95]; (c) distance-based emission spectrum, adapted from Refs. [106,107,108]; (d) fluorescence loss, adapted from Ref. [109].

Figure 3

Examples of FMFS for measuring intracellular mechanical forces. (a) General schematic of genetically encoded FMFS. (b) FRET cassette (stFRET), which can be inserted into host structural proteins within cells to observe in situ strain [100]. Copyright 2008, reproduced with permission from John Wiley and Sons. (c) Spectrin stFRET (sstFRET), utilized for reporting mechanical forces on the cellular α-actinin protein, adapted from Ref. [134]. (d) PriSSM, designed for myosin–actin interaction, adapted from Refs. [106,107]. (e) TSMod for measuring mechanical forces on vinculin [122]. Copyright 2010, reproduced with permission from Springer Nature. (f) FMFS, designed for measuring mechanical forces on talin. Scale bars: 20 μm [136]. Copyright 2015, reproduced with permission from Springer Nature. (g) TSMod-based E-cadherin tension sensor (EcadTSMod), adapted from Ref. [137].

Figure 4

Examples of FMFS for measuring cell–ligand mechanical forces. (a) FMFS for measuring the mechanical force on EGFR during the early stages of endocytosis [102]. Copyright 2011, reproduced with permission from Springer Nature. (b) FMFS for measuring the mechanical force during the process of host cell uptake of viral particles [138]. Copyright 2020, reproduced with permission from Springer Nature. (c–g) FMFS used for measuring the mechanical forces during the cell adhesion process: (c) FMFS based on peptides [101], copyright 2016, reproduced with permission from American Chemical Society; (d) FMFS based on PEG [103], copyright 2013, reproduced with permission from American Chemical Society; (e) FMFS based on I27. The mechanical forces transmitted by integrins extended the I27 protein, leading to an increase in the intensity of the dye (stars). [127], copyright 2016, reproduced with permission from American Chemical Society; (f) FMFS based on dsDNA [142], copyright 2017, reproduced with permission from Elsevier Publisher; (g) FMFS based on ssDNA [104], copyright 2021, reproduced with permission from Springer Nature.

Figure 1

Overview of TFM used to quantify cell traction forces (F) in different dimensions. F are spatial vectors that can cause substrate deformation both perpendicular to and within the viewing plane. (a) 2D TFM embedded with fluorescent microbeads; (b) substrate surface equipped with micropillar arrays; (c) 3D TFM embedded with fluorescent microbeads.

Figure 5

Examples of FMFS for monitoring and regulating cellular mechanical functions. (a) FMFS used for monitoring the maturation of cardiomyocytes [91]. Copyright 2022, reproduced with permission from American Chemical Society. (b) FMFS employed for monitoring receptor-mediated rigidity sensing [146]. Copyright 2023, reproduced with permission from Springer Nature. (c) FMFS designed for monitoring the activation of Notch [147]. Copyright 2016, reproduced with permission from American Chemical Society. (d) FMFS designed for monitoring the activation of T cells, adapted from Ref. [148]. (e) FMFS used for regulating cell morphology and motility [149]. Copyright 2021, reproduced with permission from Wiley-VCH GmbH. (f) FMFS developed for monitoring single-molecule loading rate during cell adhesion, adapted from Ref. [150]. (g) FMFS utilized for sorting cell mixtures [151]. Copyright 2020, reproduced with permission from Wiley-VCH GmbH.

Figure 6

Examples of FMFS applications in high-throughput screening platforms. (a) Schematic showing the mechano-Cas12a assisted tension sensor (MCATS) [159]. Copyright 2023, reproduced with permission from Springer Nature. (b) Overview of mechanotriggered hybridization chain reaction (mechano-HCR) based on TGT [160]. Copyright 2021, reproduced with permission from Wiley-VCH GmbH. (c) Principle of tension-activated cell tagging (TaCT). Stars represent Atto674N [161]. Copyright 2023, reproduced with permission from Springer Nature. (d) Schematic showing TGT-based rupture and delivery tension gauge system (RAD-TGT) [162]. Copyright 2023, reproduced with permission from Springer Nature. (e) Combination of DNA tensioners and microfluidic-based cell arrays. The DNA tensioner consists of three DNA sequences: F, Q, and H. Sequences H is divided into three parts: a (hybridizes with the cholesterol-labeled sequence F), b (assembled into a “hairpin” structure by the complementation of sequences b1 and b3), and c (hybridized with the quencher-labeled sequence Q) [163]. Copyright 2022, reproduced with permission from Wiley-VCH GmbH.

Figure 7

Examples of FMFS applications in cell–cell interactions. The arrows represent the direction of tensile forces generated by cells. (a) Schematic showing a membrane DNA tension probe (MDTP). Scale bars: 20 μm [165]. Copyright 2017, reproduced with permission from American Chemical Society. (b) Overview of a DNA-based ratiometric fluorescence probe (DNAMeter) [166]. Copyright 2020, reproduced with permission from the Royal Society of Chemistry. (c) Combination of DNA tension probe and fluorescence lifetime imaging microscopy (FLIM), named FLIM-MDTP [167]. Copyright 2021, reproduced with permission from Wiley-VCH GmbH. (d) Principle of a molecular tension fluorescence microscopy (MTFM) based on a “spring-like” DNA beacon. Scale bars: 25 μm [168]. Copyright 2020, reproduced with permission from American Chemical Society. (e) Intercellular forces and energy costs in confined microchannels [169]. Copyright 2023, reproduced with permission from Elsevier.