scholarly journals Myosin II Tension Sensors Visualize Force Generation within the Actin Cytoskeleton in Living Cells

2019 ◽  
Author(s):  
Ryan G. Hart ◽  
Divya Kota ◽  
Fangjia Li ◽  
Diego Ramallo ◽  
Andrew J. Price ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging ◽  
Cellular Functions ◽  
Initial Report ◽  
Spatial And Temporal Heterogeneity ◽  
Tension Sensor ◽  
Myosin Motors ◽  
Type Ii Myosin

AbstractType II myosin motors generate cytoskeletal forces that are central to cell division, embryogenesis, muscle contraction, and many other cellular functions. However, at present there is no method that can directly measure the forces generated by myosins in living cells. Here we describe a Förster resonance energy transfer (FRET)-based tension sensor that can measure forces generated by Nonmuscle Myosin IIB (NMIIB) in living cells with piconewton (pN) sensitivity. Fluorescence lifetime imaging microscopy (FLIM)-FRET measurements indicate that the forces generated by NMIIB exhibit significant spatial and temporal heterogeneity, with inferred tensions that vary widely in different regions of the cell. This initial report highlights the potential utility of myosin-based tension sensors in elucidating the roles of cytoskeletal contractility in a wide variety of contexts.

scholarly journals Looking at the Functional State of Proteins Inside Cells

1999 ◽  
Vol 7 (8) ◽  
pp. 3-4
Author(s):  
Stephen W. Carmichael
Keyword(s):  
Functional State ◽  
Spatial Data ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Cellular Functions ◽  
Lifetime Imaging ◽  
Intracellular Proteins

Many intracellular proteins are catalysts that regulate cellular functions. These catalysts can be assayed to determine their functional state, but untii now it was not possible to simultaneously obtain a functional analysis and spatial data. Tony Ng, Anthony Squire, and others, working in the laboratories of Phillippe Bastiaens and Peter Parker, have combined Fluorescence Lifetime Imaging Microscopy (FLIM) with Fluorescence Resonance Energy Transfer (FRET) to spatially resolve the activation of a protein catalyst within living cells. Their technique was also applied to fixed cells.

scholarly journals The in vivo mechanics of the magnetotactic backbone as revealed by correlative FLIM-FRET and STED microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Erika Günther ◽  
André Klauß ◽  
Mauricio Toro-Nahuelpan ◽  
Dirk Schüler ◽  
Carsten Hille ◽  
...  
Keyword(s):  
Magnetic Particles ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Stimulated Emission ◽  
Fluorescence Lifetime Imaging ◽  
Sted Microscopy

AbstractProtein interaction and protein imaging strongly benefit from the advancements in time-resolved and superresolution fluorescence microscopic techniques. However, the techniques were typically applied separately and ex vivo because of technical challenges and the absence of suitable fluorescent protein pairs. Here, we show correlative in vivo fluorescence lifetime imaging microscopy Förster resonance energy transfer (FLIM-FRET) and stimulated emission depletion (STED) microscopy to unravel protein mechanics and structure in living cells. We use magnetotactic bacteria as a model system where two proteins, MamJ and MamK, are used to assemble magnetic particles called magnetosomes. The filament polymerizes out of MamK and the magnetosomes are connected via the linker MamJ. Our system reveals that bacterial filamentous structures are more fragile than the connection of biomineralized particles to this filament. More importantly, we anticipate the technique to find wide applicability for the study and quantification of biological processes in living cells and at high resolution.

scholarly journals Single-Cell Biochemical Multiplexing by Multidimensional Phasor Demixing and Spectral Fluorescence Lifetime Imaging Microscopy

2021 ◽  
Vol 9 ◽  
Author(s):  
Kalina T. Haas ◽  
Maximilian W. Fries ◽  
Ashok R. Venkitaraman ◽  
Alessandro Esposito
Keyword(s):  
Fluorescence Lifetime ◽  
Cell Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Fluorescence Lifetime Imaging ◽  
Biochemical Reactions ◽  
Lifetime Imaging ◽  
Spectrally Resolved ◽  
Single Living Cells

Revealing mechanisms underpinning cell function requires understanding the relationship between different biochemical reactions in living cells. However, our capabilities to monitor more than two biochemical reactions in living cells are limited. Therefore, the development of methods for real-time biochemical multiplexing is of fundamental importance. Here, we show that data acquired with multicolor (mcFLIM) or spectrally resolved (sFLIM) fluorescence lifetime imaging can be conveniently described with multidimensional phasor transforms. We demonstrate a computational framework capable of demixing three Forster resonance energy transfer (FRET) probes and quantifying multiplexed biochemical activities in single living cells. We provide a comparison between mcFLIM and sFLIM suggesting that sFLIM might be advantageous for the future development of heavily multiplexed assays. However, mcFLIM—more readily available with commercial systems—can be applied for the concomitant monitoring of three enzymes in living cells without significant losses.

scholarly journals STReTCh: a strategy for facile detection of mechanical forces across proteins in cells

2022 ◽  
Author(s):  
Brian L Zhong ◽  
Vipul T Vachharajani ◽  
Alexander R Dunn
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adaptor Protein ◽  
Magnetic Tweezers ◽  
Cellular Adhesion ◽  
Mechanical Forces ◽  
Proof Of Concept ◽  
Cellular Functions ◽  
Practical Challenge ◽  
Tension Sensor

Numerous proteins experience and respond to mechanical forces as an integral part of their cellular functions, but measuring these forces remains a practical challenge. Here, we present a compact, 11 kDa molecular tension sensor termed STReTCh (Sensing Tension by Reactive Tag Characterization). Unlike existing genetically encoded tension sensors, STReTCh does not rely on experimentally demanding Förster resonance energy transfer (FRET)-based measurements and is compatible with typical fix-and-stain protocols. Using a magnetic tweezers assay, we calibrate the STReTCh module and show that it responds to physiologically relevant, piconewton forces. As proof-of-concept, we use an extracellular STReTCh-based sensor to visualize cell-generated forces at integrin-based adhesion complexes. In addition, we incorporate STReTCh into vinculin, a cytoskeletal adaptor protein, and show that STReTCh reports on forces transmitted between the cytoskeleton and cellular adhesion complexes. These data illustrate the utility of STReTCh as a broadly applicable tool for the measurement molecular-scale forces in biological systems.

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