Imaging real-time HIV-1 virion fusion with FRET-based biosensors.
Ontology highlight
ABSTRACT:
SUBMITTER: Jones DM
PROVIDER: S-EPMC4547133 | biostudies-literature | 2015 Aug
REPOSITORIES: biostudies-literature
Publications
Imaging real-time HIV-1 virion fusion with FRET-based biosensors.
Jones Daniel M DM Padilla-Parra Sergi S
Scientific reports 20150824
We have produced a novel, simple and rapid method utilising genetically encodable FRET-based biosensors to permit the detection of HIV-1 virion fusion in living cells. These biosensors show high sensitivity both spatially and temporally, and allow the real-time recovery of HIV-1 fusion kinetics in both single cells and cell populations simultaneously. ...[more]
Similar Datasets
Project description:Mechanical stress is an unmapped source of free energy in cells. Mapping the stress fields in a heterogeneous time-dependent environment like that found in cells requires probes that are specific for different proteins and respond to biologically relevant forces with minimal disturbance to the host system. To meet these goals, we have designed a genetically encoded stress sensor with minimal volume and high sensitivity and dynamic range. The new FRET-based sensor, called cpstFRET, is designed to be modulated by the angles between the donor and acceptor rather than the distance between them. Relative to other probes, it is physically smaller and exhibits a greater dynamic range and sensitivity and expresses well. For in vivo testing, we measured stress gradients in time and space in non-erythroid spectrin in several different cell types and found that spectrin is under constitutive stress in some cells but not in others. Stresses appear to be generated by both F-actin and tubulin. The probe revealed, for the first time, that spectrin undergoes time-dependent force modulation during cell migration. cpstFRET can be employed in vitro, in vivo and in situ, and when incorporated into biologically expressed extracellular polymers such as collagen, it can report multidimensional stress fields.
| S-EPMC3367834 | biostudies-literature
Project description:HIV-1 infectivity is achieved through virion maturation. Virus particles undergo structural changes via cleavage of the Gag polyprotein mediated by the viral protease, causing the transition from an uninfectious to an infectious status. The majority of proviruses in people living with HIV-1 treated with combination antiretroviral therapy are defective with large internal deletions. Defective proviral DNA frequently preserves intact sequences capable of expressing viral structural proteins to form virus-like particles whose maturation status is an important factor for chronic antigen-mediated immune stimulation and inflammation. Thus, novel methods to study the maturation capability of defective virus particles are needed to characterize their immunogenicity. To build a quantitative tool to study virion maturation in vitro, we developed a novel single virion visualization technique based on fluorescence resonance energy transfer (FRET). We inserted an optimized intramolecular CFP-YPF FRET donor-acceptor pair bridged with an HIV-1 protease cleavage sequence between the Gag MA-CA domains. This system allowed us to microscopically distinguish mature and immature virions via their FRET signal when the FRET donor and acceptor proteins were separated by the viral protease during maturation. We found that approximately 80% of the FRET labeled virus particles were mature with equivalent infectivity to wild type. The proportion of immature virions was increased by treatment of virus producer cells with a protease inhibitor in a dose-dependent manner, which corresponded to a relative decrease in infectivity. Potential areas of application for this tool are assessing maturation efficiency in different cell type settings of intact or deficient proviral DNA integrated cells. We believe that this FRET-based single-virion imaging platform will facilitate estimating the impact on the immune system of both extracellular intact and defective viruses by quantifying the Gag maturation status.
| S-EPMC7985248 | biostudies-literature
Project description:Understanding the formation process of nanoparticles is of the utmost importance to improve their design and production. This especially holds true for self-assembled nanoparticles whose formation processes have been largely overlooked. Herein, we present a new technology that integrates a microfluidic-based nanoparticle synthesis method and Förster resonance energy transfer (FRET) microscopy imaging to visualize nanoparticle self-assembly in real time. Applied to different nanoparticle systems, for example, nanoemulsions, drug-loaded block-copolymer micelles, and nanocrystal-core reconstituted high-density lipoproteins, we have shown the approach's unique ability to investigate key parameters affecting nanoparticle formation.
| S-EPMC5589959 | biostudies-literature
Project description: Not available
| S-EPMC10509129 | biostudies-literature
Project description:Multiplexed imaging of Förster Resonance Energy Transfer (FRET)-based biosensors potentially presents a powerful approach to monitoring the spatio-temporal correlation of signalling pathways within a single live cell. Here, we discuss the potential of homo-FRET based biosensors to facilitate multiplexed imaging. We demonstrate that the homo-FRET between pleckstrin homology domains of Akt (Akt-PH) labelled with mCherry may be used to monitor 3'-phosphoinositide accumulation in live cells and show how global analysis of time resolved fluorescence anisotropy measurements can be used to quantify this accumulation. We further present multiplexed imaging readouts of calcium concentration, using fluorescence lifetime measurements of TN-L15-a CFP/YFP based hetero-FRET calcium biosensor-with 3'-phosphoinositide accumulation.
| S-EPMC4519867 | biostudies-literature
Project description:Fluorescent protein (FP) biosensors based on Förster resonance energy transfer (FRET) are commonly used to study molecular processes in living cells. There are FP-FRET biosensors for many cellular molecules, but it remains difficult to perform simultaneous measurements of multiple biosensors. The overlapping emission spectra of the commonly used FPs, including CFP/YFP and GFP/RFP make dual FRET measurements challenging. In addition, a snapshot imaging modality is required for simultaneous imaging. The Image Mapping Spectrometer (IMS) is a snapshot hyperspectral imaging system that collects high resolution spectral data and can be used to overcome these challenges. We have previously demonstrated the IMS's capabilities for simultaneously imaging GFP and CFP/YFP-based biosensors in pancreatic ?-cells. Here, we demonstrate a further capability of the IMS to image simultaneously two FRET biosensors with a single excitation band, one for cAMP and the other for Caspase-3. We use these measurements to measure simultaneously cAMP signaling and Caspase-3 activation in pancreatic ?-cells during oxidative stress and hyperglycemia, which are essential components in the pathology of diabetes.
| S-EPMC5718502 | biostudies-literature
Project description:Fluorescence lifetime imaging (FLIM) is a quantitative, intensity-independent microscopical method for measurement of diverse biochemical and physical properties in cell biology. It is a highly effective method for measurements of Förster resonance energy transfer (FRET), and for quantification of protein-protein interactions in cells. Time-domain FLIM-FRET measurements of these dynamic interactions are particularly challenging, since the technique requires excellent photon statistics to derive experimental parameters from the complex decay kinetics often observed from fluorophores in living cells. Here we present a new time-domain multi-confocal FLIM instrument with an array of 64 visible beamlets to achieve parallelised excitation and detection with average excitation powers of ~ 1-2 μW per beamlet. We exemplify this instrument with up to 0.5 frames per second time-lapse FLIM measurements of cAMP levels using an Epac-based fluorescent biosensor in live HeLa cells with nanometer spatial and picosecond temporal resolution. We demonstrate the use of time-dependent phasor plots to determine parameterisation for multi-exponential decay fitting to monitor the fractional contribution of the activated conformation of the biosensor. Our parallelised confocal approach avoids having to compromise on speed, noise, accuracy in lifetime measurements and provides powerful means to quantify biochemical dynamics in living cells.
| S-EPMC7083966 | biostudies-literature
Project description:We have developed a high-throughput drug discovery platform, measuring fluorescence resonance energy transfer (FRET) with fluorescent alpha-synuclein (αSN) biosensors, to detect spontaneous pre-fibrillar oligomers in living cells. Our two αSN FRET biosensors provide complementary insight into αSN oligomerization and conformation in order to improve the success of drug discovery campaigns for the treatment of Parkinson's disease. We measure FRET by fluorescence lifetime, rather than traditional fluorescence intensity, providing a structural readout with greater resolution and precision. This facilitates identification of compounds that cause subtle but significant conformational changes in the ensemble of oligomeric states that are easily missed using intensity-based FRET. We screened a 1280-compound small-molecule library and identified 21 compounds that changed the lifetime by >5 SD. Two of these compounds have nanomolar potency in protecting SH-SY5Y cells from αSN-induced death, providing a nearly tenfold improvement over known inhibitors. We tested the efficacy of several compounds in a primary mouse neuron assay of αSN pathology (phosphorylation of mouse αSN pre-formed fibrils) and show rescue of pathology for two of them. These hits were further characterized with biophysical and biochemical assays to explore potential mechanisms of action. In vitro αSN oligomerization, single-molecule FRET, and protein-observed fluorine NMR experiments demonstrate that these compounds modulate αSN oligomers but not monomers. Subsequent aggregation assays further show that these compounds also deter or block αSN fibril assembly.
| S-EPMC8238948 | biostudies-literature
Project description:A molecular force sensing cassette (stFRET) was incorporated into actinin, filamin, and spectrin in vascular endothelial cells (BAECs) and into collagen-19 in Caenorhabditis elegans. To estimate the stress sensitivity of stFRET in solution, we used DNA springs. A 60-mer loop of single stranded DNA was covalently linked to the external cysteines of the donor and acceptor. When the complementary DNA was added it formed double stranded DNA with higher persistence length, stretching the linker and substantially reducing FRET efficiency. The probe stFRET detected constitutive stress in all cytoskeletal proteins tested, and in migrating cells the stress was greater at the leading edge than the trailing edge. The stress in actinin, filamin and spectrin could be reduced by releasing focal attachments from the substrate with trypsin. Inhibitors of actin polymerization produced a modest increase in stress on the three proteins suggesting they are mechanically in parallel. Local shear stress applied to the cell with a perfusion pipette showed gradients of stress leading from the site of perfusion. Transgenic C. elegans labeled in collagen-19 produced a behaviorally and anatomically normal animal with constitutive stress in the cuticle. Stretching the worm visibly stretched the probe in collagen showing that we can trace the distribution of mean tissue stress in specific molecules. stFRET is a general purpose dynamic sensor of mechanical stress that can be expressed intracellularly and extracellularly in isolated proteins, cells, tissues, organs and animals.
| S-EPMC3101475 | biostudies-literature
Project description:With the technical development of ultrasonography (US), electromagnetic tracking-based fusion imaging of real-time US and computed tomography/magnetic resonance (CT/MR) images has been used for percutaneous hepatic intervention such as biopsy and radiofrequency ablation (RFA). Because of the fusion imaging technique, the fused CT or MR images show the same plane and move synchronously while performing real-time US. With this information, fusion imaging can enhance lesion detectability and reduce the false positive detection of focal hepatic lesions with poor sonographic conspicuity. Three-dimensional US can also be fused with realtime US for the percutaneous RFA of liver tumors requiring overlapping ablation. When fusion imaging is not sufficient for identifying small focal hepatic lesions, contrast-enhanced US can be added to fusion imaging.
| S-EPMC4176112 | biostudies-literature