30 research outputs found

    Recruitment Kinetics of Tropomyosin Tpm3.1 to Actin Filament Bundles in the Cytoskeleton Is Independent of Actin Filament Kinetics

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    <div><p>The actin cytoskeleton is a dynamic network of filaments that is involved in virtually every cellular process. Most actin filaments in metazoa exist as a co-polymer of actin and tropomyosin (Tpm) and the function of an actin filament is primarily defined by the specific Tpm isoform associated with it. However, there is little information on the interdependence of these co-polymers during filament assembly and disassembly. We addressed this by investigating the recovery kinetics of fluorescently tagged isoform Tpm3.1 into actin filament bundles using FRAP analysis in cell culture and <i>in vivo</i> in rats using intracellular intravital microscopy, in the presence or absence of the actin-targeting drug jasplakinolide. The mobile fraction of Tpm3.1 is between 50% and 70% depending on whether the tag is at the C- or N-terminus and whether the analysis is <i>in vivo</i> or in cultured cells. We find that the continuous dynamic exchange of Tpm3.1 is not significantly impacted by jasplakinolide, unlike tagged actin. We conclude that tagged Tpm3.1 may be able to undergo exchange in actin filament bundles largely independent of the assembly and turnover of actin.</p></div

    Titration model parameters.

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    <p>These parameters are obtained from fitting the theoretical titration model to the dataset. The error on the concentration measured at [<i>Ca<sup>2+</sup></i>]<i> = 1 µM</i> is shown in the last column on the right.</p

    Fluorescence decay parameters obtained from the fitting of ΔC11CFP and mTFP1.

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    <p><τ> is the average lifetime calculated as described by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049200#pone.0049200.e007" target="_blank">equation (7)</a>. χ<sup>2</sup> is the fit quality criterion calculated by the fitting software (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049200#pone.0049200.e006" target="_blank">equation (6)</a>). Errors are the 67% confidence intervals returned by the TRFA analysis software. FP: fluorescent protein.</p

    N- and C-terminal tagged Tpm3.1 constructs have similar mobile fractions but dissimilar recovery rates.

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    <p>(A,B) Representative images of FRAP assay in MEFs transfected with either N- or C-Tpm3.1. FRAP zones (white arrows) were bleached and cells imaged at 1 fps for 2 min. (inset A,B). Enlarged images of FRAP zones over time (s). (C,D) FRAP curves of N- or C-Tpm3.1 transfected MEFs. (E) Half-times of N- and C-Tpm3.1 recovery (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s003" target="_blank">S1 Table</a>). Data obtained from 6 experiments, 3–15 cells per experiment. Error bars are +/- <i>SEM</i>. Scale bars = 10 μm.</p

    Tpm3.1 maintains constant and rapid cycling on stress fibers in the presence of jasplakinolide.

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    <p>(A) Representative image and FRAP sequence of MEFs transfected with C-Tpm3.1. FRAP zone indicated by white arrow. Top panel: FRAP sequence of untreated control cells. Bottom panel: FRAP sequence after treatment with 7 μM jasplakinolide. (B) FRAP curves of C-Tpm3.1 in control and drug-treated conditions. (C) Mobile fraction of control and drug-treated condition (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168203#pone.0168203.s007" target="_blank">S5 Table</a>). (D,E) Curve fits for C-Tpm3.1 in control (D) and drug-treated condition (E). Data obtained from 3 separate experiments, 3–8 cells per experiment. Error bars are +/- <i>SEM</i>. Scale bars = 10 μm.</p

    Temperature dependence of the average fluorescence lifetime of ΔC11CFP and mTFP1.

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    <p>A triple exponential model was used to describe ΔC11CFP fluorescence decays and a double exponential model for mTFP1. The average lifetimes were calculated using <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049200#pone.0049200.e007" target="_blank">equation (7)</a>. Errors are the 67% confidence intervals returned by the TRFA analysis software. The straight lines represent the linear regression of the corresponding dataset with a slope of −0.048 ns/°C for ΔC11CFP and −0.019 ns/°C for mTFP1.</p

    Fluorescence Lifetime Readouts of Troponin-C-Based Calcium FRET Sensors: A Quantitative Comparison of CFP and mTFP1 as Donor Fluorophores

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    <div><p>We have compared the performance of two Troponin-C-based calcium FRET sensors using fluorescence lifetime read-outs. The first sensor, TN-L15, consists of a Troponin-C fragment inserted between CFP and Citrine while the second sensor, called mTFP-TnC-Cit, was realized by replacing CFP in TN-L15 with monomeric Teal Fluorescent Protein (mTFP1). Using cytosol preparations of transiently transfected mammalian cells, we have measured the fluorescence decay profiles of these sensors at controlled concentrations of calcium using time-correlated single photon counting. These data were fitted to discrete exponential decay models using global analysis to determine the FRET efficiency, fraction of donor molecules undergoing FRET and calcium affinity of these sensors. We have also studied the decay profiles of the donor fluorescent proteins alone and determined the sensitivity of the donor lifetime to temperature and emission wavelength. Live-cell fluorescence lifetime imaging (FLIM) of HEK293T cells expressing each of these sensors was also undertaken. We confirmed that donor fluorescence of mTFP-TnC-Cit fits well to a two-component decay model, while the TN-L15 lifetime data was best fitted to a constrained four-component model, which was supported by phasor analysis of the measured lifetime data. If the constrained global fitting is employed, the TN-L15 sensor can provide a larger dynamic range of lifetime readout than the mTFP-TnC-Cit sensor but the CFP donor is significantly more sensitive to changes in temperature and emission wavelength compared to mTFP and, while the mTFP-TnC-Cit solution phase data broadly agreed with measurements in live cells, this was not the case for the TN-L15 sensor. Our titration experiment also indicates that a similar precision in determination of calcium concentration can be achieved with both FRET biosensors when fitting a single exponential donor fluorescence decay model to the fluorescence decay profiles. We therefore suggest that mTFP-based probes are more suitable for FLIM experiments than CFP-based probes.</p> </div

    Fluorescence decays of eGFP at 21°C.

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    <p>(A) eGFP from cytosol preparation of HEK293T cells expressing eGFP. (B) purified eGFP. IRF: Instrument Response Function. Res. : residuals.</p

    Fluorescence decays of TN-L15.

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    <p><b>(A) and mTFP-TnC-Cit (D) in 0 µM and 40 µM of free calcium.</b> The resulting fit quality criteria (χ<sup>2</sup>) from the global fits are 1.128 and 1.316 for the TN-L15 and mTFP-TnC-Cit datasets respectively. (B) and (C) show the corresponding fit residuals at 0 µM and 40 µM for TN-L15. (E) and (F) show the fit residuals for mTFP-TnC-Cit at 0 µM and 40 µM. Res. : residuals.</p
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