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.

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 Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability

2012 ◽  
Vol 198 (2) ◽  
pp. 251-263 ◽  
Author(s):  
Enora Moutin ◽  
Fabrice Raynaud ◽  
Jonathan Roger ◽  
Emilie Pellegrino ◽  
Vincent Homburger ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Receptors ◽  
Physical Interaction ◽  
Scaffolding Proteins ◽  
Cellular Functions ◽  
Electrophysiological Recordings ◽  
Living Neurons ◽  
Activity Dependent

Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor–mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.

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 Tension Sensor Based on Fluorescence Resonance Energy Transfer Reveals Fiber Diameter-Dependent Mechanical Factors During Myelination

2021 ◽  
Vol 15 ◽  
Author(s):  
Takeshi Shimizu ◽  
Hideji Murakoshi ◽  
Hidetoshi Matsumoto ◽  
Kota Ichino ◽  
Atsunori Hattori ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fiber Diameter ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Physical Factors ◽  
Axon Diameter ◽  
Myelin Formation ◽  
Tension Sensor ◽  
Fluorescence Resonance

Oligodendrocytes (OLs) form a myelin sheath around neuronal axons to increase conduction velocity of action potential. Although both large and small diameter axons are intermingled in the central nervous system (CNS), the number of myelin wrapping is related to the axon diameter, such that the ratio of the diameter of the axon to that of the entire myelinated-axon unit is optimal for each axon, which is required for exerting higher brain functions. This indicates there are unknown axon diameter-dependent factors that control myelination. We tried to investigate physical factors to clarify the mechanisms underlying axon diameter-dependent myelination. To visualize OL-generating forces during myelination, a tension sensor based on fluorescence resonance energy transfer (FRET) was used. Polystyrene nanofibers with varying diameters similar to neuronal axons were prepared to investigate biophysical factors regulating the OL-axon interactions. We found that higher tension was generated at OL processes contacting larger diameter fibers compared with smaller diameter fibers. Additionally, OLs formed longer focal adhesions (FAs) on larger diameter axons and shorter FAs on smaller diameter axons. These results suggest that OLs respond to the fiber diameter and activate mechanotransduction initiated at FAs, which controls their cytoskeletal organization and myelin formation. This study leads to the novel and interesting idea that physical factors are involved in myelin formation in response to axon diameter.

scholarly journals Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement

2021 ◽  
Vol 22 (21) ◽  
pp. 11727
Author(s):  
Maria J. Sarmento ◽  
Luís Borges-Araújo ◽  
Sandra N. Pinto ◽  
Nuno Bernardes ◽  
Joana C. Ricardo ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cellular Functions ◽  
Cytoskeleton Organization

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells.

scholarly journals Development of a High-Throughput Cul3-Keap1 Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for Identifying Nrf2 Activators

2018 ◽  
Vol 24 (2) ◽  
pp. 175-189 ◽  
Author(s):  
Derek D. Poore ◽  
Glenn Hofmann ◽  
Lawrence A. Wolfe ◽  
Hongwei Qi ◽  
Ming Jiang ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adaptor Protein ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Full Diversity ◽  
Ubiquitin Ligase Complex ◽  
Fluorescence Resonance

Nrf2, a master regulator of the phase II gene response to stress, is kept at low concentrations in the cell through binding to Keap1, an adaptor protein for the Cul3 ubiquitin ligase complex. To identify Nrf2 activators, two separate time-resolved fluorescence resonance energy transfer (TR-FRET) assays were developed to monitor the binding of Nrf2-Keap1 and Cul3-Keap1, respectively. The triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) and its analogs, exhibited approximately 100-fold better potency in the Cul3-Keap1 assay than in the Nrf2-Keap1 assay, and this difference was more profound at 37 °C than at room temperature in the Nrf2-Keap1 assay, but this phenomenon was not observed in the Cul3-Keap1 assay. A full diversity screen of approximately 2,200,000 GSK compounds was run with the Cul3-Keap1 TR-FRET assay and multiple chemical series were identified and characterized.

scholarly journals R-Ras Regulates Exocytosis by Rgl2/Rlf-mediated Activation of RalA on Endosomes

2007 ◽  
Vol 18 (5) ◽  
pp. 1850-1860 ◽  
Author(s):  
Akiyuki Takaya ◽  
Takahiro Kamio ◽  
Michitaka Masuda ◽  
Naoki Mochizuki ◽  
Hirofumi Sawa ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Small Gtpase ◽  
Nucleotide Exchange ◽  
Cellular Functions ◽  
Recycling Endosomes ◽  
Nucleotide Exchange Factor ◽  
Wide Range ◽  
Ras Family

R-Ras is a Ras-family small GTPase that regulates various cellular functions such as apoptosis and cell adhesion. Here, we demonstrate a role of R-Ras in exocytosis. By the use of specific anti-R-Ras antibody, we found that R-Ras was enriched on both early and recycling endosomes in a wide range of cell lines. Using a fluorescence resonance energy transfer-based probe for R-Ras activity, R-Ras activity was found to be higher on endosomes than on the plasma membrane. This high R-Ras activity on the endosomes correlated with the accumulation of an R-Ras effector, the Rgl2/Rlf guanine nucleotide exchange factor for RalA, and also with high RalA activity. The essential role played by R-Ras in inducing high levels of RalA activity on the endosomes was evidenced by the short hairpin RNA (shRNA)-mediated suppression of R-Ras and by the expression of R-Ras GAP. In agreement with the reported role of RalA in exocytosis, the shRNA of either R-Ras or RalA was found to suppress calcium-triggered exocytosis in PC12 pheochromocytoma cells. These data revealed that R-Ras activates RalA on endosomes and that it thereby positively regulates exocytosis.

Single-Molecule FRET of Intrinsically Disordered Proteins

2020 ◽  
Vol 71 (1) ◽  
pp. 391-414 ◽  
Author(s):  
Lauren Ann Metskas ◽  
Elizabeth Rhoades
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Intrinsically Disordered Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Disordered Proteins ◽  
Protein Protein Interactions ◽  
Cellular Functions ◽  
Intrinsically Disordered ◽  
Single Molecule Fret

Intrinsically disordered proteins (IDPs) are now widely recognized as playing critical roles in a broad range of cellular functions as well as being implicated in diverse diseases. Their lack of stable secondary structure and tertiary interactions, coupled with their sensitivity to measurement conditions, stymies many traditional structural biology approaches. Single-molecule Förster resonance energy transfer (smFRET) is now widely used to characterize the physicochemical properties of these proteins in isolation and is being increasingly applied to more complex assemblies and experimental environments. This review provides an overview of confocal diffusion-based smFRET as an experimental tool, including descriptions of instrumentation, data analysis, and protein labeling. Recent papers are discussed that illustrate the unique capability of smFRET to provide insight into aggregation-prone IDPs, protein–protein interactions involving IDPs, and IDPs in complex experimental milieus.

scholarly journals A Generic, Scalable, and Rapid Time-Resolved Förster Resonance Energy Transfer-Based Assay for Antigen Detection—SARS-CoV-2 as a Proof of Concept

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Juuso Rusanen ◽  
Lauri Kareinen ◽  
Leonora Szirovicza ◽  
Hasan Uğurlu ◽  
Lev Levanov ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Virus Isolation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Antigen Detection ◽  
Förster Resonance Energy Transfer ◽  
Proof Of Concept ◽  
Rt Pcr ◽  
Time Resolved ◽  
Förster Resonance

ABSTRACT The ongoing coronavirus disease 2019 (COVID-19) pandemic has seen an unprecedented increase in the demand for rapid and reliable diagnostic tools, leaving many laboratories scrambling for resources. We present a fast and simple assay principle for antigen detection and demonstrate its functionality by detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigens in nasopharyngeal swabs. The method is based on the detection of SARS-CoV-2 nucleoprotein (NP) and S protein (SP) via time-resolved Förster resonance energy transfer (TR-FRET) with donor- and acceptor-labeled polyclonal anti-NP and -SP antibodies. Using recombinant proteins and cell culture-grown SARS-CoV-2, the limits of detection were established as 25 pg of NP or 20 infectious units (IU) and 875 pg of SP or 625 IU. Testing reverse transcription-PCR (RT-PCR)-positive (n = 48, with cycle threshold [CT] values from 11 to 30) or -negative (n = 96) nasopharyngeal swabs demonstrated that the assay yielded positive results for all samples with CT values of <25 and for a single RT-PCR-negative sample. Virus isolation from the RT-PCR-positive nasopharyngeal swabs showed a strong association between the presence of infectious virus and a positive antigen test result. The NP-based assay showed 97.4% (37/38) sensitivity and 100% (10/10) specificity in comparison with virus isolation and 77.1% (37/48) sensitivity and 99.0% (95/96) specificity in comparison with SARS-CoV-2 RT-PCR. The assay is performed in a buffer that neutralizes SARS-CoV-2 infectivity, and the assay is relatively simple to set up as an “in-house” test. Here, SARS-CoV-2 served as the model pathogen, but the assay principle is applicable to other viral infections, and the test format could easily be adapted to high-throughput testing. IMPORTANCE PCR is currently the gold standard for the diagnosis of many acute infections. While PCR and its variants are highly sensitive and specific, the time from sampling to results is measured in hours at best. Antigen tests directly detect parts of the infectious agent, which may enable faster diagnosis but often at lower sensitivity and specificity. Here, we describe a technique for rapid antigen detection and demonstrate the test format’s potential using SARS-CoV-2 as the model pathogen. The 10-min test, performed in a buffer that readily inactivates SARS-CoV-2, from nasopharyngeal samples identified 97.4% (37/38) of the samples from which we could isolate the virus. This suggests that the test performs well in identifying patients potentially shedding the virus. Although SARS-CoV-2 served as the model pathogen to demonstrate proof of concept, the test principle itself would be applicable to a wide variety of infectious and perhaps also noninfectious diseases.

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