The Piconewton Force Awakens: Quantifying Mechanics in Cells

Freikamp, Andrea; Cost, Anna-Lena; Grashoff, Carsten

doi:10.1016/j.tcb.2016.07.005

Cited by 68 publications

(66 citation statements)

References 60 publications

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“…To some extent, this is being realized with genetically encoded force sensors that have been expressed within force-bearing cytoskeletal components of cultured cells (114). These force sensors utilize two fluorescent proteins that are able to exchange energy through non-radiative transfer (FRET), separated with a short unstructured or structured peptide.…”

Section: Testable Predictions and The Techniques To Examine Themmentioning

confidence: 99%

The Work of Titin Protein Folding as a Major Driver in Muscle Contraction

Eckels

Tapia‐Rojo

Rivas‐Pardo

et al. 2018

Annu. Rev. Physiol.

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Single molecule atomic force microscopy and magnetic tweezers experiments have demonstrated that titin Ig domains are capable of folding against a pulling force, generating mechanical work which exceeds that produced by a myosin motor. We hypothesize that upon muscle activation, formation of actomyosin crossbridges reduces the force on titin causing entropic recoil of the titin polymer and triggering the folding of the titin Ig domains. In the physiological force range of 4–15 pN under which titin operates in muscle, the folding contraction of a single Ig domain can generate 200% of the work of entropic recoil, and occurs at forces which exceed the maximum stalling force of single myosin motors. Thus titin operates like a mechanical battery storing elastic energy efficiently by unfolding Ig domains, and delivering the charge back by folding when the motors are activated during a contraction. We advance the hypothesis that titin folding and myosin activation act as inextricable partners during muscle contraction.

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Section: Testable Predictions and The Techniques To Examine Themmentioning

confidence: 99%

The Work of Titin Protein Folding as a Major Driver in Muscle Contraction

Eckels

Tapia‐Rojo

Rivas‐Pardo

et al. 2018

Annu. Rev. Physiol.

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“…To date, a range of proteins has been investigated with FRET‐based tension sensors to quantify molecular tension in subcellular structures like cell adhesion complexes or the actin cortex (reviewed in Freikamp, Cost, & Grashoff, ; Gayrard & Borghi, ; Jurchenko & Salaita, ). If conducted properly, such experiments can provide unprecedented insights into the molecular mechanisms of cellular force transduction.…”

Section: Introductionmentioning

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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“…Many of the molecular components within the mitotic spindle have been identified and functionally characterized (Sauer, Korner, Hanisch, Ries, Nigg, & Sillje, 2005), but our understanding of how these components act in concert to produce the forces that give rise to spindle morphology and power chromosome movements during cell division lags behind. To address this gap in knowledge and provide a tool to measure and map MT sliding forces, we designed a fluorescence resonance energy transfer (FRET)-based probe consisting of a tension sensor module (Cost, Ringer, Chrostek-Grashoff, & Grashoff, 2015;Freikamp, Cost, & Grashoff, 2016;Freikamp, Mehlich, Klingner, & Grashoff, 2017;Grashoff et al, 2010;LaCroix, Rothenberg, Berginski, Urs, & Hoffman, 2015) flanked at its N and C termini with the mTMBD. This double-handed tension sensor (DH-TSmod) is shown schematically in Fig.…”

Section: Design Of a Double-handed Mtmbd-fp Crosslinkermentioning

confidence: 99%

Tau‐based fluorescent protein fusions to visualize microtubules

et al. 2017

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The ability to visualize cytoskeletal proteins and their dynamics in living cells has been critically important in advancing our understanding of numerous cellular processes, including actin- and microtubule-dependent phenomena such as cell motility, cell division, and mitosis. Here we describe a novel set of fluorescent protein fusions designed specifically to visualize microtubules in living systems using fluorescence microscopy. Each fusion contains a fluorescent protein module linked in frame to a modified phospho-deficient version of the microtubule-binding domain of Tau (mTMBD). We found that expressed and purified constructs containing a single mTMBD decorated Xenopus egg extract spindles more homogenously than similar constructs containing the microtubule-binding domain of Ensconsin, suggesting that the binding affinity of mTMBD is minimally affected by localized signaling gradients generated during mitosis. Furthermore, microtubule dynamics were not grossly perturbed by the presence of Tau-based fluorescent protein fusions. Interestingly, the addition of a second mTMBD to the opposite terminus of our construct caused dramatic changes to the spatial localization of probes within spindles. These results support the use of Tau-based fluorescent protein fusions as minimally perturbing tools to accurately visualize microtubules in living systems.

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The Piconewton Force Awakens: Quantifying Mechanics in Cells

Cited by 68 publications

References 60 publications

The Work of Titin Protein Folding as a Major Driver in Muscle Contraction

The Work of Titin Protein Folding as a Major Driver in Muscle Contraction

Genetically Encoded FRET‐Based Tension Sensors

Tau‐based fluorescent protein fusions to visualize microtubules

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