8 research outputs found

    The Spectroscopic Basis of Fluorescence Triple Correlation Spectroscopy

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    We have developed fluorescence triple correlation spectroscopy (F3CS) as an extension of the widely used fluorescence microscopy technique fluorescence correlation spectroscopy. F3CS correlates three signals at once and provides additional capabilities for the study of systems with complex stoichiometry, kinetic processes, and irreversible reactions. A general theory of F3CS was developed to describe the interplay of molecular dynamics and microscope optics, leading to an analytical function to predict experimental triple correlations of molecules that freely diffuse through the tight focus of the microscope. Experimental correlations were calculated from raw fluorescence data using triple correlation integrals that extend multiple-tau correlation theory to delay times in two dimensions. The quality of experimental data was improved by tuning specific spectroscopic parameters and employing multiple independent detectors to minimize optoelectronic artifacts. Experiments with the reversible system of freely diffusing 16S rRNA revealed that triple correlation functions contain symmetries predicted from time-reversal arguments. Irreversible systems are shown to break these symmetries, and correlation strategies were developed to detect time-reversal asymmetries in a comprehensive way with respect to two delay times, each spanning many orders of magnitude in time. The correlation strategies, experimental approaches, and theory developed here enable studies of the composition and dynamics of complex systems using F3CS

    <i>In vivo</i> RNA binding by Rev mutants.

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    <p>(A) Schematic representation of the pSLIIB/CAT reporter used to analyze Rev:RRE RNA binding <i>in vivo</i>. (B) <i>In vivo</i> RRE RNA-binding phenotypes of various <i>trans</i>-activators are shown. HeLa cells were transiently cotransfected with the Rev reporter construct pSLIIB/CAT, pBC12/CMV/╬▓-Gal (internal control) and the indicated Tat-Rev fusion constructs. At 48 h post-transfection, CAT and ╬▓-Gal expression was analysed by ELISA. CAT values were adjusted for transfection efficiency to the ╬▓-Gal level in each culture. Data are expressed as a percentage of wildtype Rev activity (set to 100%).</p

    Nucleocytoplasmic shuttling of Rev.

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    <p>Standard interspecies heterokaryon fusion assay for detection of protein nuclear export. HeLa cells were transiently transfected with the indicated Rev expression plasmids and fused at 24 h post-transfection with untransfected NIH3T3 cells (indicated by an asterisk). CRM1-mediated protein shuttling was blocked by treatment of the cultures with leptomycin B (LMB). Localization and nuclear export of Rev proteins was visualized by indirect immunofluorescence microscopy using specific anti-Rev antibody (red label; panel b, e, h, k, n, q, t). Nuclei were stained with Hoechst 33258 (blue label; panel a, d, g, j, m, p, s) and merged with the fluorescence and bright light pictures (panel c, f, i, l, o, r, u).</p

    FRET measurement of Rev oligomer formation.

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    <p>(A) Expression level and <i>trans</i>-activation capacity of Rev-CFP and Rev-YFP fusion proteins. COS cells were transiently cotransfected with the Rev reporter plasmid pGPV-RRE and the indicated <i>trans</i>-activator constructs. At 24 h post-transfection Rev-dependent expression of HIV-1 structural proteins p55<sup>Gag</sup> and p24<sup>Gag</sup>, the respective Rev <i>trans</i>-activators and actin (gel loading control) was detected by Western blot analysis using specific antibodies. (B) Representative FACS plots illustrating the percentage of FRET positive cells. COS cells were transiently transfected with expression vectors for control CFP and RevWT-YFP, or the indicated combinations of Rev donor and acceptor constructs. To provide RRE RNA the plasmid pGPV-RRE was included in each transfection. At 24 h post-transfection cells were harvested and analyzed by flow cytometry for Rev:Rev interaction, represented as FRET positive cells. (C) Mean percentage of FRET-positive cells (adjusted to the background) determined in FRET-FACS experiments.</p

    Analysis of Rev-mediated nuclear RNA export.

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    <p>(A) Schematic representation of the nuclease S1 protection assay. The Rev reporter, the end-labelled input probe and the protected sequences, recognizing unspliced and spliced RNA are depicted. (B) Nucleocytoplasmic distribution of Rev-regulated unspliced and spliced RNA determined by nuclease S1 assay. COS cells were transiently cotransfected with Rev reporter DNA pDM128/CMV and the indicated Rev expression vectors. Nuclear and cytoplasmic RNA was isolated at 48 h post-transfection. Unspliced and spliced reporter-derived RNA was detected by autoradiography using the <sup>32</sup>P-labeled input probe. (N, nuclear RNA; C, cytoplasmic RNA). (C) Quantification of Rev-regulated cytoplasmic and nuclear viral RNA. HeLa cells were transiently cotransfected with pHXB2Δ<i>rev</i> and the indicated Rev constructs. At 48 h post-transfection nuclear and cytoplasmic RNAs were isolated and the accumulation of Rev-regulated unspliced <i>gag</i> RNA was quantified by real-time PCR. Total viral copy numbers were adjusted to the endogenous <i>gapdh</i> mRNA level in each sample.</p

    Regulated homodimerization of HIV-1 Rev.

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    <p>(A) Expression of FKBP-Rev fusion constructs. HeLa cells were transiently transfected with plasmids expressing the indicated Rev fusion proteins. Levels of protein expression were determined by Western blot analysis at 48 h post-transfection using specific anti-Rev and anti-actin (gel loading control) antibodies. (B) <i>Trans</i>-activation capacity of Rev fusion proteins. A provirus rescue assay was performed by transient cotransfection of HeLa cells with pHXB2Δ<i>rev</i> DNA, the internal control plasmid pBC12/CMV/SEAP and the indicated Rev expression vectors in the absence or presence of the chemical homodimerizer AP20187. Culture supernatants were collected at 48 h post-transfection and the accumulation of HIV-1 particles was quantified by p24<sup>Gag</sup> antigen ELISA. Values of p24<sup>Gag</sup> antigen were adjusted for transfection efficiency to SEAP activity in each culture supernatant.</p
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