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

Freikamp, Andrea; Mehlich, Alexander; Klingner, C.; Grashoff, Carsten

doi:10.1016/j.jsb.2016.03.011

Cited by 52 publications

(35 citation statements)

References 63 publications

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“…One of the applications is the use of FRET-based sensors for determination and quantification of biomolecular crowding in live cells as an indicator of the physicochemical state of the cytoplasm [131]. Mechanical tension FRET sensors can also be used to study extra- and intracellular single-molecule force measurements [132]. DNA FRET sensors combined with TIRF imaging have been used to monitor DNA synthesis in real time where a simple setup utilizes a DNA primer labelled with a fluorophore acceptor and annealed to the DNA template which is labelled with the donor fluorophore.…”

Section: Main Techniques and Applications Of Single-molecule Fluorescmentioning

confidence: 99%

Single-molecule fluorescence microscopy review: shedding new light on old problems

2017

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Fluorescence microscopy is an invaluable tool in the biosciences, a genuine workhorse technique offering exceptional contrast in conjunction with high specificity of labelling with relatively minimal perturbation to biological samples compared with many competing biophysical techniques. Improvements in detector and dye technologies coupled to advances in image analysis methods have fuelled recent development towards single-molecule fluorescence microscopy, which can utilize light microscopy tools to enable the faithful detection and analysis of single fluorescent molecules used as reporter tags in biological samples. For example, the discovery of GFP, initiating the so-called ‘green revolution’, has pushed experimental tools in the biosciences to a completely new level of functional imaging of living samples, culminating in single fluorescent protein molecule detection. Today, fluorescence microscopy is an indispensable tool in single-molecule investigations, providing a high signal-to-noise ratio for visualization while still retaining the key features in the physiological context of native biological systems. In this review, we discuss some of the recent discoveries in the life sciences which have been enabled using single-molecule fluorescence microscopy, paying particular attention to the so-called ‘super-resolution’ fluorescence microscopy techniques in live cells, which are at the cutting-edge of these methods. In particular, how these tools can reveal new insights into long-standing puzzles in biology: old problems, which have been impossible to tackle using other more traditional tools until the emergence of new single-molecule fluorescence microscopy techniques.

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Section: Main Techniques and Applications Of Single-molecule Fluorescmentioning

confidence: 99%

Single-molecule fluorescence microscopy review: shedding new light on old problems

2017

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“…Novel engineering approaches are already helping to overcome the difficulty of controlling mechanical forces, which cannot be manipulated as simply as molecules, and to address the problem of surveying or stimulating the membrane through the thick pectocellulosic plant cell wall. For example, genetically encoded biosensors developed for ions, pH, and small molecules have revolutionized the study of plant cell biology and signal transduction [95–97], and one way to maintain endogenous conditions during mechanobiology experimentation may be the future development of fluorescent biosensors that quantitatively report on physical parameters such as turgor or membrane tension [51, 98]. …”

Section: Plant Mechanoperception: the Molecular Scalementioning

confidence: 99%

Life behind the wall: sensing mechanical cues in plants

2017

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There is increasing evidence that all cells sense mechanical forces in order to perform their functions. In animals, mechanotransduction has been studied during the establishment of cell polarity, fate, and division in single cells, and increasingly is studied in the context of a multicellular tissue. What about plant systems? Our goal in this review is to summarize what is known about the perception of mechanical cues in plants, and to provide a brief comparison with animals.

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“…This is different from traction force microscopy, which is used to determine forces at the cellular level using specialized gels or fabricated micropillars (Polacheck & Chen, ). To analyze forces across individual molecules in vitro mostly atomic force microscopy or optical and magnetic tweezers are employed (Freikamp, Mehlich, Klingner, & Grashoff, ). Forces across cell surface receptors are accessible with chemically modified surfaces using polyethylene glycol‐, protein‐, or DNA‐based force sensors (Liu, Galior, Ma, & Salaita, ; Morimatsu, Mekhdjian, Adhikari, & Dunn, ; Zhang, Ge, Zhu, & Salaita, ).…”

Section: Commentarymentioning

confidence: 99%

“…Current Protocols in Cell Biology (Freikamp, Mehlich, Klingner, & Grashoff, 2017). Forces across cell surface receptors are accessible with chemically modified surfaces using polyethylene glycol-, protein-, or DNA-based force sensors (Liu, Galior, Ma, & Salaita, 2017;Morimatsu, Mekhdjian, Adhikari, & Dunn, 2013;Zhang, Ge, Zhu, & Salaita, 2014).…”

Section: Of 29mentioning

confidence: 99%

Genetically Encoded FRET‐Based Tension Sensors

2019

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Genetically encoded Förster resonance energy transfer (FRET)-based tension sensors measure piconewton-scale forces across individual molecules in living cells or whole organisms. These biosensors show comparably high FRET efficiencies in the absence of tension, but FRET quickly decreases when forces are applied. In this article, we describe how such biosensors can be generated for a specific protein of interest, and we discuss controls to confirm that the observed differences in FRET efficiency reflect changes in molecular tension. These FRET efficiency changes can be related to mechanical forces as the FRET-force relationship of the employed tension sensor modules are calibrated. We provide information on construct generation, expression in cells, and image acquisition using live-cell fluorescence lifetime imaging microscopy (FLIM). Moreover, we describe how to analyze, statistically evaluate, and interpret the resulting data sets. Together, these protocols should enable the reader to plan, execute, and interpret FRET-based tension sensor experiments. C 2019 by John Wiley & Sons, Inc.Keywords: biosensor r FLIM r FRET r mechanotransduction r tension sensor

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

Cited by 52 publications

References 63 publications

Single-molecule fluorescence microscopy review: shedding new light on old problems

Single-molecule fluorescence microscopy review: shedding new light on old problems

Life behind the wall: sensing mechanical cues in plants

Genetically Encoded FRET‐Based Tension Sensors

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