Fluorescence Resonance Energy Transfer Biosensor Research Articles

Diminished nuclear-localized β-adrenoceptor signalling activates YAP to promote kidney fibrosis in diabetic nephropathy.

Diabetic nephropathy (DN) is a leading cause of chronic kidney disease (CKD), which is characterized by mesangial matrix expansion that involves dysfunctional mesangial cells (MCs). However, the underlying mechanisms remain unclear. This study aims to delineate the spatiotemporal contribution of adrenergic signalling in diabetic kidney fibrosis to reveal potential therapeutic targets. A model of diabetic nephropathy was induced by in db/db mice. Gene expression in kidneys was profiled by RNA-seq analyses, western blot and immunostaining. Subcellular-localized fluorescence resonance energy transfer (FRET) biosensors determined adrenergic signalling microdomains in MCs. Effects of oral rolipram, a phosphodiesterase 4 (PDE4) inhibitor, on the model were measured. Our model exhibited impaired kidney function with elevated expression of adrenergic and fibrotic genes, including Adrb1, PDEs, Acta2 and Tgfβ. RNA-seq analysis revealed that MCs with dysregulated YAP pathway were crucial to the extracellular matrix secretion in kidneys from diabetic nephropathy patients. In cultured MCs, TGF-β promoted profibrotic gene transcription, which was regulated by nuclear-localized β-adrenoceptor signalling. Mechanistically, TGF-β treatment diminished nuclear-specific cAMP signalling in MCs and reduced PKA-dependent phosphorylation of YAP, leading to its activation. In parallel, db/db mouse kidneys showed increased expressions of PDE4B and PDE4D. Treatment with oral rolipram alleviated kidney fibrosis in db/db mice. Diabetic nephropathy impaired nuclear-localized β1-adrenoceptor-cAMP signalling microdomain through upregulating PDE4 expression, promoting fibrosis in MCs via PKA dephosphorylation-dependent YAP activation. Our results suggest PDE4 inhibition as a promising strategy for alleviating kidney fibrosis in diabetic nephropathy.

Development of mKate3/HaloTag7 (JFX650) and CFP/YFP Dual-Fluorescence (or Förster) Resonance Energy Transfer Pairs for Visualizing Dual-Molecular Activity.

Although several imaging strategies for dual fluorescence (or Förster) resonance energy transfer (FRET) biosensors have been reported, their implementation is challenging because of the limited performance of fluorescent proteins and the spectral overlap of FRET biosensors. These processes often require additional data calibration to eliminate artifacts. Many CFP/YFP FRET biosensors have been developed. In this study, we introduced the mKate3/HT7(JFX650) FRET pair, which effectively formed two pairs of FRET pairs for dual-FRET imaging when combined with the CFP/YFP FRET pair. The FRET donor mKate3 exhibited higher brightness than its predecessor mKate. The FRET acceptor, HT7(JFX650), is a HaloTag7 protein covalently conjugated with a far-red JFX650-THL ligand. The pair comprising mKate3 and HT7(JFX650) represents an excellent FRET dyad, exhibiting a high FRET efficiency ratio. To use the FRET pair for dual FRET biosensor imaging, we constructed PKA and K+ biosensors based on the mKate3/HT7(JFX650) FRET pair. These biosensors can be used along with CFP/YFP biosensors to simultaneously detect the responses of intracellular PKA/Src, PKA/Ca2+, and K+/Ca2+ under different stimuli. The findings revealed that dual FRET biosensors, which are based on the combination of CFP/YFP and mKate3/HT7 (JFX650), exhibit adequate compatibility and can be used to visualize multiple molecular activities in a live cell.

A Reliable System for Quantitative G-Protein Activation Imaging in Cancer Cells.

Fluorescence resonance energy transfer (FRET) biosensors have proven to be an indispensable tool in cell biology and, more specifically, in the study of G-protein signalling. The best method of measuring the activation status or FRET state of a biosensor is often fluorescence lifetime imaging microscopy (FLIM), as it does away with many disadvantages inherent to fluorescence intensity-based methods and is easily quantitated. Despite the significant potential, there is a lack of reliable FLIM-FRET biosensors, and the data processing and analysis workflows reported previously face reproducibility challenges. Here, we established a system in live primary mouse pancreatic ductal adenocarcinoma cells, where we can detect the activation of an mNeonGreen-Gαi3-mCherry-Gγ2 biosensor through the lysophosphatidic acid receptor (LPAR) with 2-photon time-correlated single-photon counting (TCSPC) FLIM. This combination gave a superior signal to the commonly used mTurquoise2-mVenus G-protein biosensor. This system has potential as a platform for drug screening, or to answer basic cell biology questions in the field of G-protein signalling.

Development of FRET Biosensor to Characterize CSK Subcellular Regulation.

C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs' activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg107 site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones.

Highly sensitive and specific graphene oxide-based FRET aptasensor for quantitative detection of human soluble growth stimulating gene protein 2

Soluble growth stimulation expressed gene 2 (sST2) is a new generation biomarker in the diagnosis and prognosis of heart failure (HF). Here, the sST2-specific aptamers were selected from a random ssDNA library with the full length of 88 nucleotides (nt) via target-immobilized magnetic beads (MB)-based systematic evolution of ligands by exponential enrichment (SELEX) technology. After eight rounds of selection, six aptamers with the most enrichment were selected. Among, the aptamer L1 showed the high-affinity binding to sST2 with the lowest Kd value (77.3 ± 0.05 nM), which was chosen as the optimal aptamer for further molecular docking. Then, the aptamer L1 was used to construct a graphene oxide (GO) - based fluorescence resonance energy transfer (FRET) biosensor for sST2, which exhibits a linear detection range of 0.1–100 μg/ml and a detection limit of 3.7 ng/ml. The aptasensor was applied to detect sST2 in real samples, with a good correlation and agreement with the traditional enzyme-linked immunosorbent assay (ELISA) when quantitative analyzing the sST2 concentration in serum samples from HF patients. The results show that not only an efficient strategy for screening the practicable aptamer, but also a rapid and sensitive detection platform for sST2 were established.

Genetically Encoded Fluorescence Resonance Energy Transfer Biosensor for Live-Cell Visualization of Lamin A Phosphorylation at Serine 22.

Extensive phosphorylation at serine 22 (pSer22) on lamin A is the hallmark of cell mitosis, which contributes to the breakdown of nuclear envelope. In the interphase, pSer22 lamin A exists in low abundance and is involved in mechanotransduction, virus infection, and gene expression. Numerous evidences emerge to support lamin A regulation on cell function and fate by phosphorylation. However, live-cell imaging tools for visualizing the dynamics of pSer22 lamin A are yet to be established. Herein, we developed a novel lamin A phosphorylation sensor (LAPS) based on fluorescence resonance energy transfer (FRET) with high sensitivity and specificity. We observed the dynamic lamin A phosphorylation during the cell cycle progression in single living cells: the increase of pSer22 modification when cells entered the mitosis and recovered upon the mitosis exit. Our biosensor also showed the gradual reduction of pSer22 modification during cell adhesion and in response to hypotonic environment. By applying LAPS, we captured the propagation of pSer22 modification from inside to outside of the inner nuclear membrane, which further led to the breakdown of nuclear envelope. Meanwhile, we found the synchronous phosphorylation of pSer22 lamin A and H3S10ph at mitosis entry. Inhibition of Aurora B, the responsible kinase for H3S10ph, could shorten the mitotic period without obvious effect on the pSer22 modification level of lamin A. Thus, LAPS allows the spatiotemporal visualization of the lamin A pSer22, which will be useful for elucidating the molecular mechanisms underlying cell mitosis and mechanoresponsive processes.

Advances in FRET‐based biosensors from donor‐acceptor design to applications

AbstractFluorogenic biosensors are essential tools widely used in biomedicine, chemical biology, environmental protection and food safety. Fluorescence resonance energy transfer (FRET) is a crucial technique for developing fluorogenic biosensors that provide mechanistic insight into bioprocesses through time‐spatial bioimaging in living cells and organisms. Although extensive FRET‐based sensors have been developed for detecting or imaging analytes of interest over the past decade, few comprehensive reviews have summarized the recent studies from the fundamental chemical angle about the design and application. In this work, the recent advance in the discovery of FRET biosensors using donor‐acceptor dye combinations is described and they are classified based on different types of analytes, such as mall molecules, proteins, enzymes, nucleic acids and metal ions. This review provides molecular‐level inspiration for the design of FRET‐based biosensors, aiding in their application in biosensing and bioimaging.

Development of a high-throughput fluorescence resonance energy transfer assay for biotin in food

A new fluorescence resonance energy transfer (FRET) biosensor to evaluate the presence of biotin has been developed and optimized. The biosensor is based on the direct evaluation of BirA enzyme from Escherichia coli homodimerization as a result of biotin binding. BirA monomer was genetically fused either to fluorescent protein mTurquoise2 (mTur) or to cp173Venus (Venus). In the presence of biotin, BirA dimerization brings mTur and Venus molecules close enough for an energy transfer. The analytical performances of the biosensor in the presence biotin using the purified fusion proteins which expressed in Escherichia coli have been evaluated and optimized. The biotin assay can be performed in 96-well microplate format with a 10 min incubation. The FRET signal increased with the biotin concentration in the range of 0.4 nmol/L - 4 nmol/L, and the linear equation was Y = 0.235 + 0.333X (R2 = 0.991), which was slightly affected by other water-soluble vitamins and trace metal elements. The FRET biosensor was used to detect biotin in infant formula samples, and the results were in good agreement with those obtained from the microbial method, indicating that the developed FRET biosensor was applicable to the biotin detection in food.

Plasmon‐enhanced FRET biosensor based on Tm3+/Er3+ co‐doped core‐shell upconversion nanoparticles for ultrasensitive virus detection

AbstractOutbreaks of infectious viruses offer a formidable challenge to public healthcare systems and early detection of viruses is essential for preventing virus propagation. In this work, an ultrasensitive plasmon‐enhanced fluorescence resonance energy transfer (FRET) biosensor based on core‐shell upconversion nanoparticle (csUCNP) and gold nanoparticle (AuNP) for accurate detection of SARS‐CoV‐2 viral RNA is presented. In this biodetection assay, the Tm3+/Er3+ co‐doped csUCNP NaGdF4:Yb/Tm@NaYF4:Yb/Er acts as an energy donor and AuNP serves as an energy acceptor. The upconversion emission of Tm3+ and the design of the core‐shell structure led to a simultaneous surface plasmon effect of AuNP. The localized surface plasmon resonance (LSPR) arising from collective oscillations of free electrons significantly enhanced FRET efficiency between Er3+ and AuNP. The as‐prepared biosensor obtained a limit of detection (LOD) as low as 750 am, indicating that the integration of FRET and surface plasmon into one biodetection assay significantly boosted the sensitivity of the biosensor. In addition, samples extracted from clinical samples are also utilized to validate the effectiveness of the biosensor. Therefore, this innovative plasmon‐enhanced FRET biosensor based on Tm3+/Er3+ co‐doped csUCNP may pave the way for rapid and accurate biodetection applications.

Attomolar-level detection of respiratory virus long-chain oligonucleotides based on FRET biosensor with upconversion nanoparticles and Au–Au dimer

Upconversion nanoparticles (UCNPs) are promising nanoprobes in DNA/RNA detection, such as respiratory viral RNAs, and siRNA in cancer. However, recent studies have indicated that the sensitivity of UCNP-based biosensors is restricted, ranging from picomolar to femtomolar level. Moreover, most of existing UCNP-based probes are only able to detect short-chain oligonucleotides, which are not suitable for detection of long-chain oligonucleotides in many real applications. In this work, we introduced a new UCNP-based fluorescence resonance energy transfer (FRET) nanoprobe design composed of NaGdF4:Yb3+, Er3+@NaGdF4 core-shell UCNPs (csUCNPs) linking with Au–Au dimer to detect long-chain oligonucleotides of SARS-CoV-2 N-gene. Compared with typical single gold nanoparticles (AuNPs) in FRET biosensors, our theoretical investigation shows that a stronger electromagnetic field is generated in the Au–Au dimer where the plasmon resonance can enhance FRET efficiency and increase the working distance. Thus, the synergetic effect of plasmonic resonance and FRET enables a greater quenching efficiency (QE) of Au–Au dimer to UCNPs, which leads to more remarkable upconversion luminescence (UCL) recovery for each target gene recognition. Importantly, our design significantly improved the limit of detection (LOD) to attomolar level, with a linear response ranging from 2 aM to 2 fM. Moreover, the clinical detection with inactivated SARS-CoV-2 samples was successfully performed with excellent specificity within 30 min using the developed UCNPs biosensors incorporated with Au–Au dimer. This UCNP biosensor based on Au–Au dimer strategy with ultra-sensitivity and good selectivity opens a new path for clinical diagnosis without target amplification and plays an instructive role in other virus diagnosis.

Zafirlukast Is a Promising Scaffold for Selectively Inhibiting TNFR1 Signaling.

Tumor necrosis factor (TNF) plays an important role in the pathogenesis of inflammatory and autoimmune diseases such as rheumatoid arthritis and Crohn's disease. The biological effects of TNF are mediated by binding to TNF receptors, TNF receptor 1 (TNFR1), or TNF receptor 2 (TNFR2), and this coupling makes TNFR1-specific inhibition by small-molecule therapies essential to avoid deleterious side effects. Recently, we engineered a time-resolved fluorescence resonance energy transfer biosensor for high-throughput screening of small molecules that modulate TNFR1 conformational states and identified zafirlukast as a compound that inhibits receptor activation, albeit at low potency. Here, we synthesized 16 analogues of zafirlukast and tested their potency and specificity for TNFR1 signaling. Using cell-based functional assays, we identified three analogues with significantly improved efficacy and potency, each of which induces a conformational change in the receptor (as measured by fluorescence resonance energy transfer (FRET) in cells). The best analogue decreased NF-κB activation by 2.2-fold, IκBα efficiency by 3.3-fold, and relative potency by two orders of magnitude. Importantly, we showed that the analogues do not block TNF binding to TNFR1 and that binding to the receptor's extracellular domain is strongly cooperative. Despite these improvements, the best candidate's maximum inhibition of NF-κB is only 63%, leaving room for further improvements to the zafirlukast scaffold to achieve full inhibition and prove its potential as a therapeutic lead. Interestingly, while we find that the analogues also bind to TNFR2 in vitro, they do not inhibit TNFR2 function in cells or cause any conformational changes upon binding. Thus, these lead compounds should also be used as reagents to study conformational-dependent activation of TNF receptors.

Enhancing interaction of actin and actin-binding domain 1 of dystrophin with modulators: Toward improved gene therapy for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full- length dystrophin. Thus increasing the actin-binding affinity of micro-dystrophin, using small molecules, could be a beneficial therapeutic approach. Here we have developed and validated a novel high-throughput screening (HTS) assay to discover small molecules that increase the binding affinity of dystrophin's actin-binding domain1 (ABD1). We engineered a novel fluorescence resonance energy transfer (FRET) biosensor, consisting of the mClover3, fluorescent protein (donor) attached to the C- terminus of dystrophin ABD1, and Alexa Fluor 568 (acceptor) attached to the C-terminal cysteine of actin. We used this biosensor in small-molecule screening, using a unique high-precision, high-throughput screening (HTS) fluorescence lifetime assay, identifying several compounds from a FDA-approved library that significantly increase the binding between actin and ABD1. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the actin-dystrophin interaction, with the ultimate goal of developing therapies for muscular dystrophy.

Astrocyte-secreted glypican-4 drives APOE4-dependent tau hyperphosphorylation

Tau protein aggregates are a major driver of neurodegeneration and behavioral impairments in tauopathies, including in Alzheimer's disease (AD). Apolipoprotein E4 (APOE4), the highest genetic risk factor for late-onset AD, has been shown to exacerbate tau hyperphosphorylation in mouse models. However, the exact mechanisms through which APOE4 induces tau hyperphosphorylation remains unknown. Here, we report that the astrocyte-secreted protein glypican-4 (GPC-4), which we identify as a binding partner of APOE4, drives tau hyperphosphorylation. We discovered that first, GPC-4 preferentially interacts with APOE4 in comparison to APOE2, considered to be a protective allele to AD, and second, that postmortem APOE4-carrying AD brains highly express GPC-4 in neurotoxic astrocytes. Furthermore, the astrocyte-secreted GPC-4 induced both tau accumulation and propagation invitro. CRISPR/dCas9-mediated activation of GPC-4 in a tauopathy mouse model robustly induced tau hyperphosphorylation. In the absence of GPC4, APOE4-induced tau hyperphosphorylation was largely diminished using invitro tau fluorescence resonance energy transfer-biosensor cells, in human-induced pluripotent stem cell-derived astrocytes and in an invivo mouse model. We further show that APOE4-mediated surface trafficking of APOE receptor low-density lipoprotein receptor-related protein 1 through GPC-4 can be a gateway to tau spreading. Collectively, these data support that APOE4-induced tau hyperphosphorylation is directly mediated by GPC-4.

A Higher Affinity Melamine Binding Aptamer Mutant for More Sensitive Detection

AbstractMelamine is an illegal additive in milk products. Mel‐34 is an aptamer specific for melamine. In this paper, we studied the base‐pair function in the stem regions of Mel‐34 using label‐free thiazole orange (TO) staining assay. Then, we used isothermal titration calorimetry (ITC) to verify the binding activity, and the fluorescence resonance energy transfer (FRET) biosensor based molecule‐beacon was established to detect melamine in yogurt sample. We found one of the mutants (mut6) has four folds higher affinity than Mel‐34, which indicated its critical binding domain was G18‐C25 base pair. We detected melamine in 1 % yogurt sample using mut6 as recognition molecule, the linear range was below 20 μM and the limit of detection was 4.09 μM which is below the melamine threshold (1.0 mg/kg) in milk products. This work calls for carefully study the critical binding domain to discover higher affinity aptamer specific for small molecule using label‐free method.

Live Cell Imaging of Cyclic Nucleotides in Human Cardiomyocytes.

The ubiquitous second messengers' 3',5'-cyclic adenosine monophosphate (cAMP ) and 3',5'-cyclic guanosine monophosphate (cGMP) are crucial in regulating cardiomyocyte function, as well as pathological processes, by acting in distinct subcellular microdomains and thus controlling excitation-contraction coupling. Spatio-temporal intracellular dynamics of cyclic nucleotides can be measured in living cells using fluorescence resonance energy transfer (FRET ) by transducing isolated cells with genetically encoded biosensors. While FRET experiments have been regularly performed in cardiomyocytes from different animal models, human-based translational experiments are very challenging due to the difficulty to culture and transduce adult human cardiomyocytes. Here, we describe a technique for obtaining human atrial and ventricular myocytes which allows to keep them alive in culture long enough to transduce them and visualize cAMP and cGMP in physiological and pathological human settings.

Development of a single quantum dot-mediated FRET biosensor for amplification-free detection of ten-eleven translocation 2

Ten-eleven translocation (TET) family proteins play key roles in multiple cellular processes by mediating the oxidation of 5-methylcytosine to directly participate in DNA demethylation, and often aberrantly expressed in various diseases. In this research, we develop a single-quantum-dot (QD)-mediated fluorescence resonance energy transfer (FRET) biosensor for amplification-free measurement of ten-eleven translocation 2 (TET2). When TET2 is present, it catalyzes the oxidation of 5-vinylcytosine in dsDNA to 5-formylmethylcytosine, and the subsequent labeling of dsDNA with Cy5 generates a biotinylated Cy5-dsDNA complex. Biotinylated Cy5-dsDNA complexes are conjugated to the streptavidin-coated 605QDs to obtain a Cy5-dsDNA-605QD nanostructures, inducing FRET from 605QD to Cy5. FRET signal can be simply measured by single-molecule counting. This biosensor enables homogeneous detection of TET2 with a limit of detection (LOD) of 0.042 ng/μL, and it can accurately measure cellular TET2 down to 1 cell. Moreover, this biosensor can be used to screen TET2 inhibitors, offering a new platform for TET2-related medical research and clinical diagnostics.

Tracking the Dynamic Histone Methylation of H3K27 in Live Cancer Cells.

Histone methylations play a crucial role in chromatin remodeling and genome regulations. However, there is a lack of tools to visualize these histone modifications with high spatiotemporal resolutions in live cells. We have developed a biosensor based on fluorescence resonance energy transfer (FRET) and incorporated it into nucleosomes, capable of monitoring the trimethylation of H3K27 (H3K27me3) in live cells. We also revealed that the performance of the FRET biosensor can be significantly improved by adjusting the linkers within the biosensor. An improved biosensor enables the live-cell imaging of different histone methylation status, induced by the suppressive H3.3K27M or existing in breast cancer cells with varying genetic backgrounds. We have further applied the biosensor to reveal the dynamic coupling between H3K27me3 changes and caspase activity representing the initiation of apoptosis in cancer cells by imaging both H3K27me3 and caspase activity simultaneously in the same live cells. Thus, this new FRET biosensor can provide a powerful tool to visualize the epigenetic regulation in live cells with high spatial temporal resolutions.

Cover Feature: A DNA‐Encoded FRET Biosensor for Visualizing the Tension across Paxillin in Living Cells upon Shear Stress (Anal. Sens. 1/2022)

The cover shows a DNA-encoded biosensor based on a Fluorescence Resonance Energy Transfer (FRET) technique named PaxTS. The biosensor consists of an LD12 domain, an LIM1234 domain, a nano-spring and a pair of fluorescent proteins to convert the tension across paxillin to FRET efficiency in living cells, indicating that there exists a force transmission pathway described as ‘membrane-cytoskeleton-FAs’ and involves paxillin upon mechanical force. More information can be found in the Full Paper by Bo Liu and co-workers.

Optogenetic control of receptors reveals distinct roles for actin- and Cdc42-dependent negative signals in chemotactic signal processing

During chemotaxis, neutrophils use cell surface G Protein Coupled Receptors to detect chemoattractant gradients. The downstream signaling system is wired with multiple feedback loops that amplify weak inputs and promote spatial separation of cell front and rear activities. Positive feedback could promote rapid signal spreading, yet information from the receptors is transmitted with high spatial fidelity, enabling detection of small differences in chemoattractant concentration across the cell. How the signal transduction network achieves signal amplification while preserving spatial information remains unclear. The GTPase Cdc42 is a cell-front polarity coordinator that is predictive of cell turning, suggesting an important role in spatial processing. Here we directly measure information flow from receptors to Cdc42 by pairing zebrafish parapinopsina, an optogenetic G Protein Coupled Receptor with reversible ON/OFF control, with a spectrally compatible red/far red Cdc42 Fluorescence Resonance Energy Transfer biosensor. Using this toolkit, we show that positive and negative signals downstream of G proteins shape a rapid, dose-dependent Cdc42 response. Furthermore, F-actin and Cdc42 itself provide two distinct negative signals that limit the duration and spatial spread of Cdc42 activation, maintaining output signals local to the originating receptors.

A DNA‐Encoded FRET Biosensor for Visualizing the Tension across Paxillin in Living Cells upon Shear Stress

AbstractPaxillin is a potential participant in direct intracellular force transmission, which is considered as the foundation of cells sensing and responding to the extracellular environment. However, the detection of tension across paxillin has not been achieved due to lacking microsized tools. Herein, a paxillin tension sensor (PaxTs) based on the Fluorescence Resonance Energy Transfer (FRET) technique was constructed. PaxTs can be expressed and assembled to FA sites spontaneously to visualize the tension across paxillin with FRET efficiency of ∼62.4 % in living cells. The tension across paxillin was found to decrease upon shear stress, in which the membrane fluidity and contractility of actin acted as cushions. It is observed that paxillin participates in the pathway of cell membrane‐cytoskeleton‐FAs for force transmission upon mechanical force in real time visualization, which provides a promising new method to investigate the direct intracellular force transmission in biology and technology.