Simultaneous readout of multiple FRET pairs using photochromism.
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ABSTRACT:
SUBMITTER: Roebroek T
PROVIDER: S-EPMC8012603 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:We describe a strategy for experimentally-constraining computational simulations of intrinsically disordered proteins (IDPs), using ?-synuclein, an IDP with a central role in Parkinson's disease pathology, as an example. Previously, data from single-molecule Förster Resonance Energy Transfer (FRET) experiments have been effectively utilized to generate experimentally constrained computational models of IDPs. However, the fluorophores required for single-molecule FRET experiments are not amenable to the study of short-range (<30 Å) interactions. Using ensemble FRET measurements allows one to acquire data from probes with multiple distance ranges, which can be used to constrain Monte Carlo simulations in PyRosetta. To appropriately employ ensemble FRET data as constraints, we optimized the shape and weight of constraining potentials to afford ensembles of structures that are consistent with experimental data. We also used this approach to examine the structure of ?-synuclein in the presence of the compacting osmolyte trimethylamine-N-oxide. Despite significant compaction imparted by 2 M trimethylamine-N-oxide, the underlying ensemble of ?-synuclein remains largely disordered and capable of aggregation, also in agreement with experimental data. These proof-of-concept experiments demonstrate that our modeling protocol enables one to efficiently generate experimentally constrained models of IDPs that incorporate atomic-scale detail, allowing one to study an IDP under a variety of conditions.
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Project description:Genetically encoded FRET-based sensor proteins have significantly contributed to our current understanding of the intracellular functions of Zn2+. However, the external excitation required for these fluorescent sensors can give rise to photobleaching and phototoxicity during long-term imaging, limits applications that suffer from autofluorescence and light scattering, and is not compatible with light-sensitive cells. For these applications, sensor proteins based on Bioluminescence Resonance Energy Transfer (BRET) would provide an attractive alternative. In this work, we used the bright and stable luciferase NanoLuc to create the first genetically encoded BRET sensors for measuring intracellular Zn2+. Using a new sensor approach, the NanoLuc domain was fused to the Cerulean donor domain of two previously developed FRET sensors, eCALWY and eZinCh-2. In addition to preserving the excellent Zn2+ affinity and specificity of their predecessors, these newly developed sensors enable both BRET- and FRET-based detection. While the dynamic range of the BRET signal for the eCALWY-based BLCALWY-1 sensor was limited by the presence of two competing BRET pathways, BRET/FRET sensors based on the eZinCh-2 scaffold (BLZinCh-1 and -2) yielded robust 25-30% changes in BRET ratio. In addition, introduction of a chromophore-silencing mutation resulted in a BRET-only sensor (BLZinCh-3) with increased BRET response (50%) and an unexpected 10-fold increase in Zn2+ affinity. The combination of robust ratiometric response, physiologically relevant Zn2+ affinities, and stable and bright luminescence signal offered by the BLZinCh sensors allowed monitoring of intracellular Zn2+ in plate-based assays as well as intracellular BRET-based imaging in single living cells in real time.
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Project description:PurposeTo develop and evaluate a simultaneous multislice (SMS) reconstruction technique that provides noise reduction and leakage blocking for highly accelerated cardiac MRI.MethodsReadOut Concatenated k-space SPIRiT (ROCK-SPIRiT) uses the concept of readout concatenation in image domain to represent SMS encoding, and performs coil self-consistency as in SPIRiT-type reconstruction in an extended k-space, while allowing regularization for further denoising. The proposed method is implemented with and without regularization, and validated on retrospectively SMS-accelerated cine imaging with three-fold SMS and two-fold in-plane acceleration. ROCK-SPIRiT is compared with two leakage-blocking SMS reconstruction methods: readout-SENSE-GRAPPA and split slice-GRAPPA. Further evaluation and comparisons are performed using prospectively SMS-accelerated cine imaging.ResultsResults on retrospectively three-fold SMS and two-fold in-plane accelerated cine imaging show that ROCK-SPIRiT without regularization significantly improves on existing methods in terms of PSNR (readout-SENSE-GRAPPA: 33.5 ± 3.2, split slice-GRAPPA: 34.1 ± 3.8, ROCK-SPIRiT: 35.0 ± 3.3) and SSIM (readout-SENSE-GRAPPA: 84.4 ± 8.9, split slice-GRAPPA: 85.0 ± 8.9, ROCK-SPIRiT: 88.2 ± 6.6 [in percentage]). Regularized ROCK-SPIRiT significantly outperforms all methods, as characterized by these quantitative metrics (PSNR: 37.6 ± 3.8, SSIM: 94.2 ± 4.1 [in percentage]). The prospectively five-fold SMS and two-fold in-plane accelerated data show that ROCK-SPIRiT and regularized ROCK-SPIRiT have visually improved image quality compared with existing methods.ConclusionThe proposed ROCK-SPIRiT technique reduces noise and interslice leakage in accelerated SMS cardiac cine MRI, improving on existing methods both quantitatively and qualitatively.
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Project description:Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn²⁺based on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn²⁺ is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn²⁺ levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn²⁺. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn²⁺ uptake simultaneously in two compartments, revealing that nuclear Zn²⁺ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn²⁺ more slowly and with a delay of 600-700 sec. Lastly, these studies provide the first glimpse of nuclear Zn²⁺ and reveal that nuclear Zn²⁺ is buffered at a higher level than cytosolic Zn²⁺.
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Project description:Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca(2+) and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.
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Project description:Many dichotomous traits for complex diseases are often involved more than one locus and/or associated with quantitative biomarkers or environmental factors. Incorporating these quantitative variables into linkage analysis as well as localizing two linked disease loci simultaneously could therefore improve the efficiency in mapping genes. We extended the robust multipoint Identity-by-Descent (IBD) approach with incorporation of covariates developed previously to simultaneously estimate two linked loci using different types of affected relative pairs (ARPs).We showed that the efficiency was enhanced by incorporating a quantitative covariate parametrically or non-parametrically while localizing two disease loci using ARPs. In addition to its help in identifying factors associated with the disease and in improving the efficiency in estimating disease loci, this extension also allows investigators to account for heterogeneity in risk-ratios for different ARPs. Data released from the collaborative study on the genetics of alcoholism (COGA) for Genetic Analysis Workshop 14 (GAW 14) were used to illustrate the application of this extended method.The simulation studies and example illustrated that the efficiency in estimating disease loci was demonstratively enhanced by incorporating a quantitative covariate and by using all relative pairs while mapping two linked loci simultaneously.
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Project description:We present a highly sensitive bioanalytical microarray assay that enables the analysis of small genomic sample material. By combining an optimized cDNA purification step with single molecule cDNA detection on the microarray, the platform has improved sensitivity compared to conventional systems, allowing amplification-free determination of expression profiles with as little as 600ng total RNA. Total RNA from cells was reverse transcribed into fluorescently labeled cDNA and purified employing a precipitation method that minimizes loss of cDNA material. The microarray was scanned on a fluorescence chip-reader with single molecule sensitivity. Using the newly developed platform we were able to analyze the RNA expression profile of a subpopulation of rare multiple myeloma CD138 negative progenitor (MM CD138(neg)) cells. The high-sensitivity microarray approach led to the identification of a set of 20 genes differentially expressed in MM CD138(neg) cells. Our work demonstrates the applicability of a straight-forward single-molecule DNA array technology to current topics of molecular and cellular cancer research, which are otherwise difficult to address due to the limited amount of sample material.
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Project description:PurposeReadout-segmented echo-planar imaging (rs-EPI) can provide high quality diffusion data because it is less prone to distortion and blurring artifacts than single-shot echo-planar imaging (ss-EPI), particularly at higher resolution and higher field. Readout segmentation allows shorter echo-spacing and echo train duration, resulting in reduced image distortion and blurring, respectively, in the phase-encoding direction. However, these benefits come at the expense of longer scan times because the segments are acquired in multiple repetitions times (TRs). This study shortened rs-EPI scan times by reducing the TR duration with simultaneous multislice acceleration.MethodsThe blipped-CAIPI method for slice acceleration with reduced g-factor SNR loss was incorporated into the diffusion-weighted rs-EPI sequence. The rs- and ss-EPI sequences were compared at a range of resolutions at both 3 and 7 Tesla in terms of image fidelity and diffusion postprocessing results.ResultsSlice-accelerated clinically useful trace-weighted images and tractography results are presented. Tractography analysis showed that the reduced artifacts in rs-EPI allowed better discrimination of tracts than ss-EPI.ConclusionSlice acceleration reduces rs-EPI scan times providing a practical alternative to diffusion-weighted ss-EPI with reduced distortion and high resolution. Magn Reson Med 74:136-149, 2015. © 2014 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine.
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Project description:BACKGROUND:To dissect the intricate workings of neural circuits, it is essential to gain precise control over subsets of neurons while retaining the ability to monitor larger-scale circuit dynamics. This requires the ability to both evoke and record neural activity simultaneously with high spatial and temporal resolution. NEW METHOD:In this paper we present approaches that address this need by combining micro-electrocorticography (?ECoG) with optogenetics in ways that avoid photovoltaic artifacts. RESULTS:We demonstrate that variations of this approach are broadly applicable across three commonly studied mammalian species - mouse, rat, and macaque monkey - and that the recorded ?ECoG signal shows complex spectral and spatio-temporal patterns in response to optical stimulation. COMPARISON WITH EXISTING METHODS:While optogenetics provides the ability to excite or inhibit neural subpopulations in a targeted fashion, large-scale recording of resulting neural activity remains challenging. Recent advances in optical physiology, such as genetically encoded Ca(2+) indicators, are promising but currently do not allow simultaneous recordings from extended cortical areas due to limitations in optical imaging hardware. CONCLUSIONS:We demonstrate techniques for the large-scale simultaneous interrogation of cortical circuits in three commonly used mammalian species.
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