Quantitative real-time imaging of intracellular FRET biosensor dynamics using rapid multi-beam confocal FLIM

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

A Comparison of Donor-Acceptor Pairs for Genetically Encoded FRET Sensors: Application to the Epac cAMP Sensor as an Example

We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used in an attempt to improve the FRET span of the Epac-based cAMP sensor. The results show significant albeit not perfect correlation between performance in the spacer construct and in the Epac sensor. Finally, this strategy enabled us to identify improved sensors both for detection by sensitized emission and by fluorescent lifetime imaging. The present overview should be helpful in guiding development of future FRET sensors

Zygotic vinculin is not essential for embryonic development in zebrafish

<div><p>The formation of multicellular tissues during development is governed by mechanical forces that drive cell shape and tissue architecture. Protein complexes at sites of adhesion to the extracellular matrix (ECM) and cell neighbors, not only transmit these mechanical forces, but also allow cells to respond to changes in force by inducing biochemical feedback pathways. Such force-induced signaling processes are termed mechanotransduction. Vinculin is a central protein in mechanotransduction that in both integrin-mediated cell-ECM and cadherin-mediated cell-cell adhesions mediates force-induced cytoskeletal remodeling and adhesion strengthening. Vinculin was found to be important for the integrity and remodeling of epithelial tissues in cell culture models and could therefore be expected to be of broad importance in epithelial morphogenesis <i>in vivo</i>. Besides a function in mouse heart development, however, the importance of vinculin in morphogenesis of other vertebrate tissues has remained unclear. To investigate this further, we knocked out vinculin functioning in zebrafish, which contain two fully functional isoforms designated as vinculin A and vinculin B that both show high sequence conservation with higher vertebrates. Using TALEN and CRISPR-Cas gene editing technology we generated vinculin-deficient zebrafish. While single vinculin A mutants are viable and able to reproduce, additional loss of zygotic vinculin B was lethal after embryonic stages. Remarkably, vinculin-deficient embryos do not show major developmental defects, apart from mild pericardial edemas. These results lead to the conclusion that vinculin is not of broad importance for the development and morphogenesis of zebrafish tissues.</p></div

Cardiac and skeletal muscle phenotypes of vinculin-null mutants.

<p>(A) Western blot of lysates from the posterior half of WT, <i>vcla</i>^-/- and <i>vcla</i>^-/-<i>vclb</i>^<i>-/-</i> embryos at 5 dpf, probed for vinculin and β-actin. (B) Some vinculin mutants show cardiac edema of which representative mild and severe cases are depicted. Wild type and <i>vcla</i>^-/-<i>vclb</i>^<i>-/-</i> mutants at 3 dpf (left) and 5 dpf (right). For corresponding images of the other genotypes described in (C), see supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182278#pone.0182278.s006" target="_blank">S6 Fig</a>. (C) Quantification of the presence of cardiac edemas in offspring from a <i>vcla</i>^-/-<i>vclb</i>^<i>+/-</i> incross. Classification as depicted in (B). Data was obtained from three independent experiments. WT embryos from an independent WT strain were analyzed as control from two independent experiments. Data is represented as mean ± s.e.m. A two-tailed paired student t-test was performed to compare the incidence of severe edemas between 3 dpf and 5 dpf within each genotype (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182278#pone.0182278.s007" target="_blank">S7 Fig</a>). (D) Immunostaining of actin in skeletal muscle of 5 dpf embryos. Images at the bottom are zoomed in parts of the upper images as indicated by the yellow squares (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182278#pone.0182278.s008" target="_blank">S8 Fig</a> for additional images and quantifications).</p

UV-induced photochromism.

<p>Change in ratio of YFP to CFP emission in CFP^nd-linker-YFP^nd (squares) and CFP^nd-linker-Venus^d (triangles) following exposure to UV light for the indicated times. CFP^nd-linker-YFP^nd as well as free YFP (data not shown) display a dose-dependent increase in emission that maximizes at about 10%, whereas Venus and cp¹⁷³Venus (not shown) are insensitive to UV exposure. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001916#s3" target="_blank">Methods</a> for further detail.</p

Generation of vinculin B-deficient zebrafish using CRISPR-Cas.

<p>(A) Schematic representation of the endogenous <i>vclb</i> locus showing the CRISPR guide RNA target site in exon 1. (B) Sequence chromatograms of genomic DNA from non-injected control (top) and <i>vclb</i> CRISPR-injected embryos. The arrowhead denotes the expected CRISPR cleavage site (3 nucleotides upstream of the PAM site). (C) Summary of mutations found in the F1 offspring of <i>vclb</i> CRISPR-injected fish of either a wild-type (top) or <i>vcla</i> mutant (bottom) background. Arrows denote the mutation of the <i>vclb</i> mutant used in further experiments. (D) Amino acid sequence of <i>vclb</i> in wild-type (top) and <i>vclb</i> mutant (bottom). Asterisk denotes the premature stop codon found in the vinculin B coding sequence of <i>vclb</i> mutants.</p

Generation of vinculin A-deficient zebrafish mutants using TALENs.

<p>(A) Schematic representation of the endogenous <i>vcla</i> locus targeted by TALEN gene editing technology at the exon4-intron4 boundary. The TALEN arms flank a BclI restriction enzyme recognition site (highlighted in red) used for screening mutant alleles through restriction fragment length polymorphism (RFLP) analysis. (B) RFLP analysis of embryos injected with increasing dosages of <i>vcla</i> TALEN mRNA. Uncleaved bands represent efficient TALEN activity. (C) Summary of the range of mutations found at the vcla TALEN target locus in the F1 offspring of <i>vcla</i> TALEN-injected fish. Arrow denotes the mutation of the <i>vcla</i> mutant used in further experiments. (D) Sequence chromatograms from cDNA of wild-type vinculin (top) and of the <i>vcla Δ8B</i> mutant allele (bottom).</p

FRET in donor-linker-acceptor constructs as detected by frequency-domain FLIM.

<p>The indicated constructs were expressed in HEK293 cells and FRET efficiency E was determined as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001916#s3" target="_blank">Material and Methods</a>. Shown are mean (bars), standard deviation (SD) and standard error of the mean (SEM) of 20–400 cells. For further detail, see text.</p

Slow green-to-red maturation of tdTomato and its effects on FRET.

<p>(A) Cells expressing CFP^nd-EPAC-tdTomato for 24 hr display a spectrum of colors when viewed by eye using an Omega X154 triple-color (CFP-YFP-RFP) cube. For reproduction reasons, the confocal picture shows a mix of green (470–530 nm) and red (570–670) emission to closely match the image visible by eye. In contrast, CFP^nd-EPAC-mRFP and CFP^nd-EPAC-mCherry show a more homogeneous red color. (B) Cell-to-cell variability in maturation of CFP^nd-linker-tdTomato causes significant deviations in the fluorescence decay times detected in the CFP channel, as measured by frequency-domain FLIM. Scale bar, 12 µm.</p

Schematic overview of the constructs used in this study.

<p>Donor and acceptor fluorophore are connected by a peptide stretch (Linker A: SGLRSRYPFASEL) or by the Epac1(ΔDEP, CD) domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001916#pone.0001916-Ponsioen1" target="_blank">[1]</a>. Within this stretch, the amino acids PF were replaced by the Epac domain itself, leaving linkers B: SGLRSRY and C: ASEL. For truncated donor constructs (CFPΔ and GFPΔ) GITLGMDELYK was deleted from the donor FPs and SGLRS from the linker. In tandem acceptor constructs the acceptors were separated by a supplementary linker (Linker D: PNFVFLIGAAGILFVSGEL) except for tdHcRed and tdTomato which have distinctive linkers, namely NG(GA)₆PVAT) and (GHGTGSTGSGSSGTASSEDNNMA), respectively.</p