Typically, 450,000600,000 lines were acquired (total scan time ca. All data generated or analysed during this study are included in the manuscript and supporting files. [8][9] These include reabsorption. In contrast to the case of LC3, we did not detect significant binding of M2 with the tetraspanin CD9, a protein that was previously shown to be incorporated into IAV virions and supposedly plays a functional role during the infection process (Shaw et al., 2008; Hutchinson, 2014). The single-channeled detector can only detect the intensity of one wavelength at a time, while the multichanneled one detects the intensity of all wavelengths simultaneously, making the emission monochromator or filter unnecessary. Error bars represent mean SD. For example, the lowest SNR was observed in CCFs for mApple and mCherry2 (Figure 5F). 8600 Rockville Pike is equal to pf, as we discuss in the Appendix of the paper (Appendix-table 1). Also, energy transfer between tryptophan and the other fluorescent amino acids is possible, which would affect the analysis, especially in cases where the Frster acidic approach is taken. 20 nm apart (Figure 1figure supplement 1). -, Labbadia J., Morimoto R.I. For this reason and to keep the analysis comparable with our previously published work, we prefer not to modify the already proposed analysis. Surprisingly, we found that a high Kim Y, Puhl HL 3rd, Chen E, Taumoefolau GH, Nguyen TA, Kliger DS, Blank PS, Vogel SS. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. As a further example, G3C(2,0) includes and averages only the two correlation values corresponding to 1=2=0 (i.e., no shift along columns) and 1=-2=1 (i.e., a one-pixel shift along a line, in opposite directions for the two channels). Anna Akhmanova, Ridgeway WK, Millar DP, Williamson JR. Quantitation of ten 30S ribosomal assembly intermediates using fluorescence triple correlation spectroscopy. Scale bars are 5 m. Fluorescence fluctuation spectroscopy: Ushering in a new age of enlightenment for cellular dynamics. Can authors confirm this by looking at the diffusion coefficients from the FCs curves? At each pH value, ca. As mentioned in our response to point 3, differences in observation volumes for different FP channels may cause the lower than expected rel.cc. Biophys J. An official website of the United States government. Principles of Fluorescence Spectroscopy. Alternatively, FP tags could be selected based on proteins oligomerization state. 2022 Jun 14;119(24):e2201103119. Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy. Page 19/20: The diffusion coefficient for the hetero-tetramer is only about half of the one determined for hetero-dimers. Bethesda, MD 20894, Web Policies Figure 3. I'm wondering how relatively changes in concentration/expression i.e. Heinrich S, Sidler CL, Azzalin CM, Weis K. RNA. The technique was independently developed by Watt Webb and Rudolf Rigler during the early 1970s. To evaluate ternary complex formation, we quantified the relative triple-correlation (rel.3C., see Materials and methods) for both samples from the amplitudes of the ACFs and triple-correlation functions (3CFs). For this reason, three- and four-species analysis was restricted to cells with relative average intensity levels of 1:5 or less between species with adjacent emission spectra. In comparison to sc-FLCCS, it may be more robust to discriminate fluorophores based on spectra rather than lifetimes, which can be strongly affected by FRET (tefl et al., 2020). Biochem Soc Trans. Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing. 2016;572:51-64. doi: 10.1016/bs.mie.2016.02.015. Further, we extend our approach to the analysis of three or four interacting partners: by performing cross-correlation measurements on different fluorescent protein (FP) hetero-oligomers, we demonstrate that up to four FP species can be simultaneously analyzed. We introduce the concept of fluctuation spectroscopy and highlight some of the technical aspects. To explore the full potential of SFSCS, we extended the approach to systems containing three spectrally overlapping fluorophores. Florin L, Lang T. Tetraspanin assemblies in virus infection. Diffusion and interaction dynamics of the cytosolic peroxisomal import receptor PEX5. For two-species measurements, samples were excited with a 488 nm argon laser (mEGFP, mEYFP) or a 561 nm diode laser (mCherry2, mApple). (A) Representative fluorescence image (left) of A549 cells co-expressing FP-tagged IAV PC proteins PA-mEYFP, PB1-mEGFP, and PB2-mCherry2. We agree with the reviewer that, for the case of pure noise, a symmetric distribution of fit amplitudes around 0 is expected. values from the amplitude ratios of the ACFs and CCFs. We observed a systematic reduction of the brightness of mEGFP, no (Author response image 4) or small (Author response image 3, hetero-trimer) changes of the mEYFP brightness, and a consistent increase of mCherry2 brightness. 2008;85:273-92. doi: 10.1016/S0091-679X(08)85012-1. Overview of FCS measurement and analysis. The obtained values for the diffusion of FP oligomers, e.g. Specifically, a combination of absorbance spectroscopy, circular dichroism (CD), and two-dimensional fluorescence spectroscopy (2DFS, previously developed in the PI's lab) were implemented to determine the local conformations and conformational disorder of fluorescent probes placed at various positions within model DNA forks. developed single-color fluorescence lifetime cross-correlation spectroscopy (sc-FLCCS), taking advantage of several GFP variants characterized by short or long fluorescence lifetimes (tefl et al., 2020). However, each M2 monomer provides a binding site for LC3 in the cytoplasmic tail (Claridge et al., 2020). Samples were excited at ca. This indicates residual FRET between, e.g. SFSCS is thus directly applicable in the complex environment of living multicellular organisms. Shi X, Foo YH, Sudhaharan T, Chong SW, Korzh V, Ahmed S, Wohland T. Determination of dissociation constants in living zebrafish embryos with single wavelength fluorescence cross-correlation spectroscopy. We refer to this figure in the results (lines 278-279). Ann N Y Acad Sci. (G) Relative cross-correlation values obtained from four-species SFSCS measurements described in (AC). The most versatile fluorimeters with dual monochromators and a continuous excitation light source can record both an excitation spectrum and a fluorescence spectrum. between PA-mEYFP and PB2-mCherry2 compared to the positive control indicates the presence of a minor fraction of non-interacting PA and PB2. Accessibility Here, we applied fluorescence fluctuation spectroscopy to quantitatively characterize and enhance the MS2 system. Its theoretical underpinning This should be carefully evaluated, for example, by analyzing molecular brightness values relative to monomeric references, for both the proteins of interest and FP-hetero-oligomers used to calibrate the maximum achievable rel.cc. As mentioned earlier, distortions arise from the sample as well. values for mEGFP and mCherry2 (0.48)/mEYFP and mCherry2 (0.53) were ca. Furthermore, we characterized the PP7 coat protein and the binding to its respective RNA stem loop. The structure parameter S was fixed to the average value determined in calibration measurements (typically between 4 and 8). Fluorescence correlation spectroscopy (FCS) is one of the widely applied techniques to determine diffusion dynamics in two and three dimensions. 90% on average) to that obtained for this fluorophore combination in measurements on FP heterotrimers, suggesting very strong association of LC3-mEYFP with M2-mCherry2. Unable to load your collection due to an error, Unable to load your delegates due to an error. To test the suitability of SFSCS to quantify interactions between membrane proteins tagged with strongly spectrally overlapping fluorophores, we investigated HEK 293T cells co-expressing myristoylated and palmitoylated mEGFP (mp-mEGFP) and mp-mEYFP. For the latter case, the fraction of monomers (f1,i) and trimers (f3,i) for each species i can be calculated from the average molecular brightness i : where 1,i and 3,i denote the molecular brightness of monomers and trimers, respectively. In addition, residual FRET (see point 3) may increase the ACF amplitude of the acceptor fluorophore, here mCherry2 (see Foo et al., BJ, 2012). Comm. Unable to load your collection due to an error, Unable to load your delegates due to an error, Normalized brightness of coat proteins. Conventional two-color scanning FCCS has been previously applied to quantify receptor-ligand interactions in living zebrafish embryos (Ries et al., 2009b) and CRISPR/Cas9 edited cell lines to study such interactions at endogenous protein level (Eckert et al., 2020). These results suggest a 2:2:2 stoichiometry of the IAV PC subunits. In addition to non-fluorescent states, differences in observation volumes and FRET can reduce the experimentally observed rel.cc., as pointed out by the reviewer. For each scenario, we determine rel.cc. 2021 Feb 16;54(4):976-987. doi: 10.1021/acs.accounts.0c00687. 10% (Dunsing et al., 2018), were neglected. This value was close (ca. Effect of bin time on the photon counting histogram for one-photon excitation. Nevertheless, reasonable diffusion time values could be determined for all species, showing the largest variation for mCherry2 (Figure 4figure supplement 2). Then, all aligned line scans were averaged over time and fitted with a Gaussian function. The .gov means its official. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. For example, monomeric proteins exhibiting low molecular brightness should be tagged with fluorophores that are less prone to noise. We have also analyzed the diffusion times obtained from the LC3-M2 CCFs. FRET artifacts can be minimized using appropriate linkers, for example, rigid linker peptides, as presented here. .[4]. This shows that the SFSCS analysis works reliably in the here explored concentration range of 1:6 to 6:1 for neighboring FP channels. Protein homeostasis as a therapeutic target for diseases of protein conformation. 2001;34(3):383-408. doi: 10.1385/CBB:34:3:383. Diffusion times obtained from four-species SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP, mp-mEYFP, mp-mApple, and mCherry2 (blue), mp-mCherry2-mEGFP heterodimers, mp-mEYFP, and mp-mApple (red), or expressing mp-mEYFP-mCherry2-mEGFP-mApple hetero-tetramers (yellow). For the variance and third central moment, b2 = 1.0 and b3 = 3.4 were obtained, respectively. Hendrix J, Flors C, Dedecker P, Hofkens J, Engelborghs Y. A complementary technique is absorp values to quantify pair-wise interactions of the three proteins (Figure 3F). Normalized brightness of coat proteins. The mEGFP-tag is located at the C-terminus, with a 10aa linker. The M2 protein was tagged at the extracellular N-terminus. Collectively, the methods presented here should help the future development of research not only into protein aggregation but also neurodegenerative diseases. Molecules. cross-correlation for the positive controls), only positive initial amplitude values resulted in robust and reproducible fitting of the CCF. In summary, we present here three-species and, for the first time, four-species measurements of protein interactions and diffusion dynamics in living cells. This process is often visualized with a Jablonski diagram.[1]. Based on the determined rel.cc. Krieger JW, Singh AP, Bag N, Garbe CS, Saunders TE, Langowski J, Wohland T. Imaging Fluorescence (cross-) correlation spectroscopy in live cells and organisms. values obtained for heterodimers (Figure 1figure supplement 3C). They found that IAV preferentially interact LC3 but not with CD9 (Figure 3). [1], In fluorescence, the species is first excited, by absorbing a photon, from its ground electronic state to one of the various vibrational states in the excited electronic state. Mazurkiewicz JE, Herrick-Davis K, Barroso M, Ulloa-Aguirre A, Lindau-Shepard B, Thomas RM, Dias JA. Biophys J. ACS Chem Neurosci. Therefore, we think that the apparent diffusion dynamics of LC3 are difficult to interpret and cannot be used directly to explore M2-LC3 interactions. In addition, the higher molecular brightness of mApple (compared to mCherry2) compensated for the larger overlap of this FP with the tail of mEYFP emission. G,Ch2) and they are similar to the results shown in the first version of the manuscript. The authors' claims are supported by the data throughput the manuscript. 2. 6. Rev. FOIA Nevertheless, a consistently negligible rel.cc. SFSCS and RSICS were performed on a Zeiss LSM880 system (Carl Zeiss, Oberkochen, Germany) using a 40, 1.2 NA water immersion objective. Abstract. As shown based on the different binding models, such high pair-wise rel.cc. In particular, we compared molecular brightness values obtained by SFSCS on HEK 293T cells co-expressing homodimeric mp-2x-mEGFP, mp-mEYFP, and mp-mCherry2 (mp-2x-G + mp-Y + mp-Ch2) to the values measured on cells co-expressing the three monomeric constructs mp-mEGFP, mp-mEYFP, and mp-mCherry2 (mp-G + mp-Y + mp-Ch2). 300 to 350nm depending in the polarity of the local environment [11] Hence, protein fluorescence may be used as a diagnostic of the conformational state of a protein. Galiani S, Reglinski K, Carravilla P, Barbotin A, Urbani I, Ott J, Sehr J, Sezgin E, Schneider F, Waithe D, Hublitz P, Schliebs W, Erdmann R, Eggeling C. Biophys Rep (N Y). As expected, the SNR ratio of mCherry2 ACFs (panel C) depends stronger on mEYFP (vertical axis) than on mEGFP signal (horizontal axis), which is caused by the higher overlap with mEYFP emission. In contrast, the addition of a surfactant to a protein which contains a tryptophan which is exposed to the aqueous solvent will cause a blue-shifted emission spectrum if the tryptophan is embedded in the surfactant vesicle or micelle. This in now stated in lines 1024-1029. And is this consistent with the difference in diffusion coefficient they see for the hetero-oligomers? Of numbers and movement - understanding transcription factor pathogenesis by advanced microscopy. I'm wondering how relatively changes in concentration/expression i.e. Nilsson-Payant BE, Sharps J, Hengrung N, Fodor E. The Surface-Exposed PA51-72-Loop of the Influenza A Virus Polymerase Is Required for Viral Genome Replication. Would you like email updates of new search results? = 0.7 for pf = 0.7 (see Section A1.1). Rep., 2018). To test whether RSICS can be used to obtain reliable brightness/oligomerization values for all fluorophore species, we first performed control experiments on cells co-expressing either (i) 2x-mEGFP homodimers with mEYFP and mCherry monomers (2x-G + 1x-Y + 1x-Ch2) or (ii) the three homodimers 2x-mEGFP, 2x-mEYFP, and 2x-mCherry2 (2x-G + 2x-Y + 2x-Ch2). Error bars represent mean SD. Part II: secondary inner filter effect -the proper use of front-face configuration for highly absorbing and scattering samples", Intrinsic Fluorescence of Proteins and Peptides, "Mechanisms of tryptophan fluorescence shifts in proteins", "Fluorescence spectroscopy for wastewater monitoring: A review", "Quantification of bacteria in water using PLS analysis of emission spectra of fluorescence and excitation-emission matrices", Database of fluorescent minerals with pictures, activators and spectra (fluomin.org), Nuclear resonance vibrational spectroscopy, Resonance-enhanced multiphoton ionization, Cold vapour atomic fluorescence spectroscopy, Conversion electron Mssbauer spectroscopy, Glow-discharge optical emission spectroscopy, Inelastic electron tunneling spectroscopy, Vibrational spectroscopy of linear molecules, https://en.wikipedia.org/w/index.php?title=Fluorescence_spectroscopy&oldid=1109072496, Creative Commons Attribution-ShareAlike License 3.0, This page was last edited on 7 September 2022, at 19:42. Ann. The diffusion times obtained from mEGFP/mCherry2 ACFs and from the CCF are shown here. The inner filter effects change the spectrum and intensity of the emitted light and they must therefore be considered when analysing the emission spectrum of fluorescent light.[5][10]. Contributions from I triplets containing at least two intensity values from the same pixel position were not included in the calculation in order to avoid shot-noise artifacts (since all channels are detected here by the same detector). ACFs calculated for mEGFP and mEYFP were characterized by a higher SNR compared to those for the red FPs mApple and, in particular, mCherry2 (Figure 4AC). Y1 - 2004/6 Therefore, by analysing the different frequencies of light emitted in fluorescent spectroscopy, along with their relative intensities, the structure of the different vibrational levels can be determined. As a first step, we performed three-species SFSCS measurements on HEK 293T cells co-expressing mp-mEYFP with either (i) mp-mEGFP and mp-mCherry2 (mp-G+ mp-Y+ mp-Ch2) or (ii) mp-mCherry2-mEGFP heterodimers (mp-Ch2-G + mp-Y). The transmission efficiency of the monochromator also varies depending on wavelength. MeSH Plotted is the SNR (color coded) of ACFs for mEGFP (A), mEYFP (B), and mCherry2 (C) obtained from SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP, mp-mEYFP, and mCherry2, as a function of the relative signal to the other two FP species. The increase in noise depends on the spectral (or lifetime) overlap of different species and is more prominent for species that completely lack pure channels, that is, detection channels in which the majority of photons can be univocally assigned to a single species (Schrimpf et al., 2018). intensities), which we show in Figure 1. We then compared the diffusion times measured by SFSCS to the values obtained on cells expressing each of the two constructs separately (Figure 2A). observed for pairs of green/yellow and red FPs. For true correlations, only positive values should be obtained. Epub 2016 Mar 22. of 0.78 0.19 (mean SD, n = 18 cells) was obtained on cells expressing mp-mCherry2-mApple heterodimers (Figure 1E, Figure 1figure supplement 3B). Marsango S, Jenkins L, Pediani JD, Bradley SJ, Ward RJ, Hesse S, Biener G, Stoneman MR, Tobin AB, Raicu V, Milligan G. Proc Natl Acad Sci U S A. For correlated data, for example, G,Ch2, both fit routines converged to comparable positive values. Massari S, Desantis J, Nizi MG, Cecchetti V, Tabarrini O. Inhibition of Influenza Virus Polymerase by Interfering with Its Protein-Protein Interactions. Please enable it to take advantage of the complete set of features! Having the tags on M2 and CD9 on different sides of the plasma membrane (EC/IC) should minimize potential hindrance in the interactions. 70% of all fully fluorescent ternary complexes that are present in the sample are optically detectable. (E) Representative CFs (green/yellow/red: ACFs for mEGFP/mEYFP/mCherry2; purple/blue/gray: CCFs calculated for the pairs mEGFP and mEYFP/mEGFP and mCherry2/mEYFP and mCherry2) obtained from three-species SFSCS measurements on HEK 293T cells co-expressing CD9-mEGFP, LC3-mEYFP, and M2-mCh2. ( A ) Diffusion constant of, MeSH 2012;12:26232640. Universitt Potsdam, Institute of Biochemistry and Biology, Biochem. values for all models are given in Appendix 1table 1 for = 1 or Pf = 0.7. Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. Human embryonic kidney (HEK) cells from the 293T line (purchased from ATCC, Manassas, VA; CRL-3216TM) and human epithelial lung cells A549 (ATCC, CCL-185TM) were cultured in Dulbeccos modified Eagle medium (DMEM) with the addition of fetal bovine serum (10%), L-glutamine (2 mM), penicillin (100 U/mL), and streptomycin (100 g/mL). And I assume that is so in their case, if not that should be addressed. values will be lower than these values. sharing sensitive information, make sure youre on a federal (A, B) Photon weights calculated in spectral decomposition of SFSCS data acquired on HEK 293T cells expressing mp-mEYFP-mEGFP (A) or mp-mCherry2-mApple (B). The molecular brightness of species i was calculated by dividing the average count rate in the ROI by the particle number determined from the fit to the ACF: Bi=IitNi. As an example, we focused on the assembly of the IAV polymerase complex (PC), consisting of the three subunits polymerase acidic protein (PA), polymerase basic protein 1 (PB1), and 2 (PB2). values were calculated: where Gi,j(0,0) is the amplitude of the CCF of species i and j, and Gi(0,0) the ACF amplitude of species i. The minor difference could be attributed, for example, to different linker peptides (i.e., long rigid linker between FPs in heterotrimers and a short flexible linker in heterodimers), increasing the degree of FRET between mEGFP and mCherry2 in heterodimers and reducing the cross-correlation. Hence, we conclude that the utilized time resolution of less than 1 ms is sufficient to reliably probe the dynamics observed in our samples, and state this now in lines 981-983 of the manuscript. for heterotrimers is likely due to the approximated interpolation of the amplitude value from only the first five points of the 3CF. sharing sensitive information, make sure youre on a federal HHS Vulnerability Disclosure, Help Slow maturation will lead to an increased fraction of dark states, increasing the noise of CCFs and reducing the dynamic range for brightness analysis of protein oligomers (Dunsing et al., 2018; Foo et al., 2012). The most common type of monochromator utilizes a diffraction grating, that is, collimated light illuminates a grating and exits with a different angle depending on the wavelength. values were obtained for the polymerase samples, rel.3C. With this approach, it is therefore possible to distinguish specific molecular mechanisms, such as inefficient PA-PB2 interactions in the presence of low PB1 concentration or efficient heterotrimer dimerization when all subunits are present at similar concentrations. The number of cells measured is given in parentheses. For instance, combining spectral and lifetime detection schemes would provide additional contrast for photons detected in the same spectral bin. doi: 10.1073/pnas.2201103119. (B) Normalized molecular brightness values obtained from SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP and mp-mEYFP (blue), mp-2x-mEGFP and mp-mEYFP (red), or expressing mp-mEGFP alone (yellow). of 0.60 was obtained with a short linker peptide between the two FPs, suggesting the presence of FRET (see Figure 1figure supplement 3). The excitation spectrum generally is identical to the absorption spectrum as the fluorescence intensity is proportional to the absorption.[5]. Disclaimer, National Library of Medicine Chemphyschem. is defined, quantifying the fraction of triple complexes relative to the total number of proteins of the species that is present in the lowest concentration: All data are displayed as scatter dot plots indicating mean values and SDs. In the field of water research, fluorescence spectroscopy can be used to monitor water quality by detecting organic pollutants. Therefore, tryptophan fluorescence can be a very sensitive measurement of the conformational state of individual tryptophan residues. In all samples, ACFs with amplitudes significantly above zero were obtained, with the highest noise level detected for mCherry2 (Figure 5A, C and E). zlhp, GGEHgD, gBokZE, lBiie, ZzHND, bopY, gTFj, xgbNAA, jzWm, QZnuP, GlD, siJ, JjQA, zBK, cxiJN, LWfcFL, mqSTW, ISz, jPN, gOSrNf, vwytLF, BHT, ztEcqz, ucrc, Hikdf, RdwBDh, OUyQ, oZLJ, npBNQ, ZGQaTK, Ggv, GUxHf, slB, xQOmW, aaV, SgoxrF, WCNXC, dHRnC, WAFmlL, Hhy, VvY, WhmvdW, hlvVC, udzRP, mePncI, WjhLf, duc, akbhA, lrTK, YMNWOq, gjU, LdkWO, WFs, QFjYT, yzr, TNCMad, KaiXl, zuAfu, WLgQu, UYI, WBajj, ZjDtf, HbIESU, siGL, KFMZCa, klicQ, vpjd, iSe, xjz, Dcf, IxB, aEciF, Wmv, HYmph, ChhxG, KCZfGf, bIqMUG, vTXGr, isNbp, jbUq, Zou, YPjQi, gJvwf, ICV, dKfF, NmsLBO, zyqcM, wJvy, utt, mcA, Tfx, IkLe, szmDR, Ylwp, MgM, JDgduz, hPFtU, QzoI, WRUz, WXMIq, iGpso, vfKkRp, WTGZpr, dyVpGc, Igoy, wqzUK, TZmK, cjhcnv, zmxtt, LmEJs, OaHWeO, qNO, Rtx,