Using FRET to analyse signals controlling cell adhesion and migration

Kemp-O'Brien, Karl; Parsons, Maddy

doi:10.1111/j.1365-2818.2012.03686.x

Cited by 7 publications

(4 citation statements)

References 47 publications

(55 reference statements)

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“…The translation of this approach into cellular applications is of significant interest, and we expect that this concept of using optically active molecules localized to distinct areas of the cell membrane can ultimately inform the design of molecularly encoded sensors to monitor membrane dynamics in cellular systems. To date, molecularly encoded and optically active molecules that have been used to measure forces in cellular systems have been predominantly limited to cytoskeletal components and focal adhesion complexes (52)(53)(54)(55). With the growing awareness of the important role of membrane tension in cellular regulation, molecularly encoded dyes that localize to various structural components of the membrane would undoubtedly help elucidate the role tension in a variety of cellular behaviors.…”

Section: Discussionmentioning

confidence: 99%

Visualizing Tension and Growth in Model Membranes Using Optical Dyes

Boyd

Kamat

2018

Biophysical Journal

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Cells dynamically regulate their membrane surface area during a variety of processes critical to their survival. Recent studies with model membranes have pointed to a general mechanism for surface area regulation under tension in which cell membranes unfold or take up lipid to accommodate membrane strain. Yet we lack robust methods to simultaneously measure membrane tension and surface area changes in real time. Using lipid vesicles that contain two dyes isolated to spatially distinct parts of the membrane, we introduce, to our knowledge, a new method to monitor the processes of membrane stretching and lipid uptake in model membranes. Laurdan, located within the bilayer membrane, and Förster resonance energy transfer dyes, localized to the membrane exterior, act in concert to report changes in membrane tension and lipid uptake during osmotic stress. We use these dyes to show that membranes under tension take up lipid more quickly and in greater amounts compared to their nontensed counterparts. Finally, we show that this technique is compatible with microscopy, enabling real-time analysis of membrane dynamics on a single vesicle level. Ultimately, the combinatorial use of these probes offers a more complete picture of changing membrane morphology. Our optical method allows us to remotely track changes in membrane tension and surface area with model membranes, offering new opportunities to track morphological changes in artificial and biological membranes and providing new opportunities in fields ranging from mechanobiology to drug delivery.

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Section: Discussionmentioning

confidence: 99%

Visualizing Tension and Growth in Model Membranes Using Optical Dyes

Boyd

Kamat

2018

Biophysical Journal

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“…A similar design has been used to make biosensors for other signaling molecules, such as kinases and membrane-bound proteins, shedding light on SH2-domain-mediated scaffolding, and rapid signal transduction by FAK (94) and Src (95). The use of these activity biosensors in live cells has 12 led to many novel insights into adhesion-dependent signaling mechanisms (96,97), including the direct visualization of force-induced Src activation (95).…”

Section: Spatiotemporal Regulation Of the Cytoskeleton And Adhesion-dmentioning

confidence: 98%

Molecular-Scale Tools for Studying Mechanotransduction

LaCroix

Rothenberg

Hoffman

2015

Annu. Rev. Biomed. Eng.

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Mechanical stimuli are known to be potent regulators of the form and function of cells and organisms. Although biological regulation has classically been understood in terms of principles from solution biochemistry, advancements in many fields have led to the development of a suite of techniques that are able to reveal the interplay between mechanical loading and changes in the biochemical properties of proteins in systems ranging from single molecules to living organisms. Here, we review these techniques and highlight the emergence of a new molecular-scale understanding of the mechanisms mediating the detection and response of cells to mechanical stimuli, a process termed mechanotransduction. Specifically, we focus on the role of subcellular adhesion structures in sensing the stiffness of the surrounding environment because this process is pertinent to applications in tissue engineering as well the onset of several mechanosensitive disease states, including cancer.

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“…Upon excitation, the electrons of the donor fluorophore jump from their ground state to a higher energy level. As they return to the ground state, a photon is emitted [232]. FRET efficiency depends on the inverse sixth power of the distance between donor and acceptor and the angles between the dipole moments.…”

Section: Fret-based Force Sensors To Estimate Force In Live Cellsmentioning

confidence: 99%

Mechanical dynamics in live cells and fluorescence-based force/tension sensors

Yang

Zhang

Guo

et al. 2015

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research

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Three signaling systems play the fundamental roles in modulating cell activities: chemical, electrical, and mechanical. While the former two are well studied, the mechanical signaling system is still elusive because of the lack of methods to measure structural forces in real time at cellular and subcellular levels. Indeed, almost all biological processes are responsive to modulation by mechanical forces that trigger dispersive downstream electrical and biochemical pathways. Communication among the three systems is essential to make cells and tissues receptive to environmental changes. Cells have evolved many sophisticated mechanisms for the generation, perception and transduction of mechanical forces, including motor proteins and mechanosensors. In this review, we introduce some background information about mechanical dynamics in live cells, including the ubiquitous mechanical activity, various types of mechanical stimuli exerted on cells and the different mechanosensors. We also summarize recent results obtained using genetically encoded FRET (fluorescence resonance energy transfer)-based force/tension sensors; a new technique used to measure mechanical forces in structural proteins. The sensors have been incorporated into many specific structural proteins and have measured the force gradients in real time within live cells, tissues, and animals.

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Using FRET to analyse signals controlling cell adhesion and migration

Cited by 7 publications

References 47 publications

Visualizing Tension and Growth in Model Membranes Using Optical Dyes

Visualizing Tension and Growth in Model Membranes Using Optical Dyes

Molecular-Scale Tools for Studying Mechanotransduction

Mechanical dynamics in live cells and fluorescence-based force/tension sensors

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