For non-correlated data (e.g. For the presented three- and four-species SFSCS and RSICS experiments, relative signals were limited to 1:5 (i.e., range of 1:5 to 5:1). 2. Shoup D, Roth A, Thapa R, Puchalla J, Rye HS. 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). (solely based on pf, i.e. This value was comparable to that measured for HA-mEGFP expressed separately (36 8 ms, mean SD, n = 18 cells). 2016;85:14. 2.54 min). They found that IAV preferentially interact LC3 but not with CD9 (Figure 3). FOIA Solid thick lines show fits of a two-dimensional diffusion model to the CFs. We then calculated four ACFs, six CCFs, and rel.cc. In addition to diffusion analysis, we also analyzed the cross-correlation of HA-mEGFP and mp-mEYFP signal for two-species measurements, resulting in negligible rel.cc. Handbook of spectroscopy. Biochem. Methods. 1. Hendrix J, Flors C, Dedecker P, Hofkens J, Engelborghs Y. In future studies, the approach presented here may be used to further elucidate the complex interaction network of viral proteins, for example, matrix protein 1 (M1) (Hilsch et al., 2014), M2, HA, and neuraminidase, cellular host factors, and PM lipids (Bobone et al., 2017) during the assembly process of IAV at the PM of living cells (Rossman and Lamb, 2011). Schwille P, Meyer-Almes FJ, Rigler R. Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution. 2001;34(3):383-408. doi: 10.1385/CBB:34:3:383. Federal government websites often end in .gov or .mil. The formation of ternary PC complexes in these samples could be extrapolated from the observed high rel.cc. We think that this comment might arise from a misunderstanding. 8942980. The set of FPs may be optimized for specific applications. Unable to load your collection due to an error, Unable to load your delegates due to an error. We excited mEGFP, mEYFP, and mCherry2 with 488 nm and 561 nm lines simultaneously and detected their fluorescence in 23 spectral bins in the range of 491695 nm. - ! For FPs with a probability pf to be fluorescent, the expected rel.cc. Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E. Measuring fast dynamics in solutions and cells with a laser scanning microscope. This suggests that not all potential binding sites in the cytoplasmic tail of M2 may be available to fluorescently tagged LC3, either due to binding of endogenous LC3, other cellular host factors, or steric hindrance. Hwang LC, Gsch M, Lasser T, Wohland T. Simultaneous multicolor fluorescence cross-correlation spectroscopy to detect higher order molecular interactions using single wavelength laser excitation. -, Labbadia J., Morimoto R.I. 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. To minimize FRET artifacts, FPs in hetero-trimers and tetramers were linked by a rigid linker. Methods Cell Biol. To assess whether this time resolution is sufficient to detect the diffusion dynamics observed in the experiments, we re-analyzed a three-species SFSCS measurement acquired on cells co-expressing membrane-anchored FPs (mp-1x-G + mp-1x-Y + mp-1x-Ch2) using a variable time binning (i.e., effective time resolution varying here from ca. Cross-correlation analysis may be further affected by FRET between different FP species, potentially reducing experimental rel.cc. All measurements were performed at room temperature. Some of this fluorescent light passes through a second filter or monochromator and reaches a detector, which is usually placed at 90 to the incident light beam to minimize the risk of transmitted or reflected incident light reaching the detector. Overlapping fluorescence emission from different species detected in the same channels provides unwanted background signal and thus reduces the signal-to-noise ratio (SNR) of the CFs (Schrimpf et al., 2018). It is worth noting that these values, albeit consistently negligible, appear to depend on the specific fitting procedure (see Figure 3figure supplement 2 and Materials and methods for details). We use the TTM to demonstrate live-cell super-resolved fluorescence lifetime image scanning microscopy and fluorescence lifetime fluctuation spectroscopy. Comm. We compared the molecular brightness values for mEGFP and mEYFP in samples co-expressing monomeric FP constructs mp-mEGFP and mp-mEYFP with the values obtained for cells co-expressing mp-2x-mEGFP homodimers and mp-mEYFP (Figure 2B). If this is the case, some or all of the photons emitted by the fluorophore may be absorbed again. There remain mainly technical questions and some points that should be discussed to ensure consistency of the results. Influenza virus assembly and budding. 2021 Jun 1;120(11):2192-2204. doi: 10.1016/j.bpj.2021.03.035. Stefl et al. Pathways of cellular proteostasis in aging and disease. In particular, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), combining SFCS and FSCS. Normalized brightness values were calculated by dividing molecular brightness values detected in each SFSCS measurement by the average brightness obtained for mEGFP and mEYFP in cells co-expressing mp-mEGFP and mp-mEYFP. 7. Notably, applying this empirical correction to the auto- and cross-correlation amplitudes confirmed the previously introduced correction formula (see Appendix 1figure 1), Gcorr,=FF-1G, (Hendrix et al., 2016). (C) Relative cross-correlation values obtained from SFSCS measurements on HEK 293T cells expressing mp-mEYFP-mEGFP (rigid linker between the two FPs, see Supplementary file 1a) or mp-mEGFP-mEYFP (short linker between the two FPs, Supplementary file 1a) heterodimers. Ries J, Chiantia S, Schwille P. Accurate determination of membrane dynamics with line-scan FCS. We agree with the reviewer that, for the case of pure noise, a symmetric distribution of fit amplitudes around 0 is expected. We have added an additional comment on this issue to the new limitations section (lines 833-841). cross-correlation for the positive controls), only positive initial amplitude values resulted in robust and reproducible fitting of the CCF. Cell Biochem Biophys. Cross-correlation RICS analysis was performed in the arbitrary region RICS framework (Hendrix et al., 2016). ( a ) Jablonski diagram showing, MeSH ( A ) Diffusion constant of, MeSH Schrimpf et al. LC3 docks with the LIR motif via hydrophobic pockets located in the middle region of its sequence (Hamacher-Brady and Brady, Cell. The expected experimental rel.3C. The average laser power used in two (G, H) Relative cross-correlation values (G) and diffusion coefficients (H) obtained from four-species RSICS measurements described in (AF). Filtering based on unreasonable cross-correlation diffusion time may remove some of the residual positive rel.cc. Kim Y, Puhl HL 3rd, Chen E, Taumoefolau GH, Nguyen TA, Kliger DS, Blank PS, Vogel SS. On the contrary, mCherry2 (3-fold change of SNR, panel C) and mEGFP (2-fold change of SNR, panel A) are only moderately affected. obtained on proteins of interest. Overview of FCS measurement and analysis. between mEGFP and mEYFP for the polymerase subunits indicates higher order interactions, that is, higher stoichiometry than 1:1 (Kaliszewski et al., 2018). Careers. This confirms that reliable brightness values were obtained and that dimeric and monomeric species can be correctly identified. In the case of interactions, the fit converges to positive values. Overview of fluorescence correlation spectroscopy, Overview of fluorescence correlation spectroscopy (FCS). FFS studies are conventionally limited to the analysis of two spectrally distinguished species due to (i) broad emission spectra of fluorophores with consequent cross-talk artifacts and (ii) limited overlap of detection/excitation geometries for labels with large spectral separation. The number of cells measured is given in parentheses. 3. HHS Vulnerability Disclosure, Help This indicates residual FRET between, e.g. doi: 10.1146/annurev-biochem-060614-033955. Accessibility Recently, three-species implementations of RICCS and FCCS were successfully demonstrated for the first time in living cells. The use of intrinsic fluorescence for the study of protein conformation is in practice limited to cases with few (or perhaps only one) tryptophan residues, since each experiences a different local environment, which gives rise to different emission spectra. Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy. LC3 is recruited to the plasma membrane (PM) in cells showing higher expression of M2 (top cell) relative to M2, but remains in the cytosol in cells expressing only low levels of M2 compared to LC3 (bottom cell). This could also be compared with the actual correlation times measured. We demonstrate that SFSCS enables cross-talk-free cross-correlation, diffusion, and oligomerization analysis of up to four protein species labeled with strongly overlapping fluorophores. rel.cc.=0.72 for mEYFP-mEGFP hetero-dimers in 2-species SFSCS, Figure 1). Jorba N, Area E, Ortn J. Oligomerization of the influenza virus polymerase complex in vivo. Reviewer #3 (Recommendations for the authors): This manuscript is a carefully conducted study of multi-color fluorescence fluctuation spectroscopy as applied to plasma membranes. Ibilir A, Serfling R, Mller J, Thomas R, De Faveri C, Zabel U, Scarselli M, Beck-Sickinger AG, Bock A, Coin I, Lohse MJ, Annibale P. Determination of G-protein-coupled receptor oligomerization by molecular brightness analyses in single cells. Therein, we stress the point that for two-species measurements, the fact that only one laser line is needed provides a clear advantage of the spectral approach, resulting in higher rel.cc. We therefore performed an approximate calculation of the expected rel.3C. To clone mp-mCherry2-(L)-mApple, mp-mCherry2-(L)_pcDNA3.1+ plasmid was first generated by amplifying mp-mCherry2 (using a primer encoding a long rigid linker sequence) and inserting it into pcDNA3.1+ using NheI and KpnI restriction. 2015;84:435464. 4. The https:// ensures that you are connecting to the N461919. Aggregation distributions on cells determined by photobleaching image correlation spectroscopy. Clipboard, Search History, and several other advanced features are temporarily unavailable. Statistical significance was determined using Welchs corrected two-tailed Students t-test (**p<0.05). values were compared to the values obtained on negative controls (i.e., cells co-expressing free mEGFP, mEYFP, and mCherry) and positive controls (i.e., cells expressing mEYFP-mCherry2-mEGFP heterotrimers) (Figure 6D). Schneider F, Hernandez-Varas P, Christoffer Lagerholm B, Shrestha D, Sezgin E, Julia Roberti M, Ossato G, Hecht F, Eggeling C, Urbani I. J Phys D Appl Phys. 6. Rel.cc. This method is likely to be used by cell biologists to determine the stoichiometry of multi-protein complexes. The pf was previously characterized for several FPs (for example, ca. 20 nm apart (Figure 1figure supplement 1). The analysis of the intensity fluctuation of a fluorescence signal from a relatively small volume and from a few Disclaimer, National Library of Medicine 60% for mCherry2) (Dunsing et al., 2018). assuming pf0.7 for all 3 FPs, using the formulas derived in Foo et al., BJ, 2012. and transmitted securely. This time resolution is sufficient to reliably detect the diffusion dynamics observed in the samples described in this work (i.e., diffusion times ~660 ms). 9. In particular, we present SFSCS, a combination of FSCS (Benda et al., 2014) and lateral scanning FCS (Ries and Schwille, 2006). Nevertheless, the previous analysis could only be performed between two of the three subunits at the same time. Additionally, Fluorescence spectroscopy can be adapted to the microscopic level using microfluorimetry. The average D measured for PB1-mEGFP, DPB1-G = 1.7 0.6 m2/s (mean SD, n = 53 cells), was ca. For example, the lowest SNR was observed in CCFs for mApple and mCherry2 (Figure 5F). eCollection 2021 Aug 5. Nevertheless, we have added a supplementary figure (Figure 3figure supplement 2) demonstrating the different fit options so that readers can choose the type of analysis best suited for their experiment. 2019 May 21;116(10):1918-1930. doi: 10.1016/j.bpj.2019.04.014. We thank the reviewer for bringing up this important issue. And I assume that is so in their case, if not that should be addressed. Similarly, molecular brightness and diffusion coefficients can be analyzed as a function of the abundance of each subunit (Figure 6figure supplement 1B,C). values detected on the positive cross-correlation control (see Appendix 1, Section A1.3 for details). As mentioned before, the fluorescence is most often measured at a 90 angle relative to the excitation light. A major limitation of SFSCS is the reduced SNR of the CFs (see Figure 1, Figure 3figure supplement 3) caused by the statistical filtering of the signal emitted by spectrally overlapping fluorophore species (see, e.g., Figure 4figure supplement 1). The pf is an empirical, FP-specific parameter that was previously characterized for multiple FPs (Dunsing et al., 2018). In both cases, it is important to select materials that have relatively little absorption in the wavelength range of interest. 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. sharing sensitive information, make sure youre on a federal From the amplitude ratios of ACFs and CCFs, we determined rel.cc. However, RSICS can be applied to detect faster dynamics, as demonstrated by experiments on cytoplasmic proteins. values (Figure 5G). Nevertheless, a consistently negligible rel.cc. We have furthermore added a short comment on time resolution in the limitations section (lines 827-830). Next, we proceeded with the analysis of PC oligomerization. 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. Chem. eCollection 2022 Sep 16. Another inner filter effect occurs because of high concentrations of absorbing molecules, including the fluorophore. An official website of the United States government. 2021 Aug 13;19:4711-4724. doi: 10.1016/j.csbj.2021.08.017. Solid thick lines show fits of a two-dimensional diffusion model to the CFs. 8600 Rockville Pike Hilsch M, Goldenbogen B, Sieben C, Hfer CT, Rabe JP, Klipp E, Herrmann A, Chiantia S. Influenza A matrix protein m1 multimerizes upon binding to lipid membranes. G,Y and Y,Ch2 combinations), the second fit routine may converge to negative fit amplitudes, resulting in a distribution of rel.cc. Thus, we estimate that around 90% of PC subunits undergo ternary complex formation in the cell nucleus when all subunits are present. Cells were passaged every 35 days, no more than 15 times. 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. Careers. Rev. In the sample containing all three homodimers, increased relative brightness values were observed for all fluorophore species: 1.75 0.37 (mean SD, n = 39 cells) for mEGFP, 1.77 0.33 for mEYFP, and 1.61 0.29 for mCherry2 (see Supplementary file 1b for data on day-to-day variations). 0.8 ms, since the line-scan time was ca. For the negative control, we obtained rel.3C. The authors provided a validation of the method in HEK cells expressing the three Fluorescent Proteins in the plasma membrane in different oligomerization states (Figure 3C). Zhejiang Da Xue Xue Bao Yi Xue Ban. Fluorescence-based techniques for the detection of the oligomeric status of proteins: implication in amyloidogenic diseases. In the latter case, fairly constant 3CF amplitudes were obtained, agreeing with the 3CF amplitude calculated without the high-pass filter (data not shown). Heinze KG, Jahnz M, Schwille P. Triple-color coincidence analysis: One step further in following higher order molecular complex formation. Solid thick lines show fits of a two-dimensional diffusion model to the correlation functions (CFs). To calibrate the focal volume, point FCS measurements with Alexa Fluor 488 (Thermo Fisher Scientific) dissolved in water at 20 nM were performed at the same laser power. Differentiation. A monochromator transmits light of an adjustable wavelength with an adjustable tolerance. We can therefore assume that for the case of ternary complexes ca. Spectra measured on four cells each were averaged over 3 (HEK 293T) or 2 (A549) days. values (Figure 4G), the different samples could successfully be discriminated. We present a simulation model for a stream of photon counts that can be detected in fluorescence fluctuation spectroscopy systems from diffusing molecules undergoing spontaneous transitions between radiative and nonradiative states. Segments showing clear distortions (typically less than 25% of all segments) were manually removed from the analysis (Dunsing and Chiantia, 2018). (B) Relative cross-correlation values obtained from SFSCS measurements described in (A) (G + Y) or on HEK 293T cells expressing mp-mEYFP-mEGFP heterodimers (Y-G). This work discusses different analysis methods used in fluctuation spectroscopy and evaluates their use for studying protein-protein interactions, and highlights some of the technical aspects. To obtain average emission spectra, pixels corresponding to the plasma membrane (PM) were semi-manually segmented (manual selection followed by removal of pixels with intensities below 25% of the maximum pixel intensity in the selected region) and detected spectra averaged over all pixels and cells measured at each pH. From the fit amplitudes of the ACFs and CCFs, rel.cc. In the current review, we present an overview of the principles and application of several methodologies used for the elucidation of protein aggregation, specifically ones based on determination of fluctuations of fluorescence. (F) SNR of ACFs for mApple (light red) and mCherry2 (dark red), obtained from SFSCS measurements described in (D), plotted as a function of the average ratio of detected mApple and mCherry2 fluorescence. Unable to load your collection due to an error, Unable to load your delegates due to an error. Notably, rel.cc. Can authors confirm this by looking at the diffusion coefficients from the FCs curves? MS2 Labeling of Endogenous Beta-Actin mRNA Does Not Result in Stabilization of Degradation Intermediates. For this reason and to keep the analysis comparable with our previously published work, we prefer not to modify the already proposed analysis. Please enable it to take advantage of the complete set of features! From this virtual state, the molecules may relax back to a vibrational level other than the vibrational ground state. For atomic species, the process is similar; however, since atomic species do not have vibrational energy levels, the emitted photons are often at the same wavelength as the incident radiation. As a reference for the absolute brightness, we also determined the relative molecular brightness of mEGFP in cells expressing mp-mEGFP alone, yielding a value of 1.03 0.21 (mean SD, n = 22 cells). values obtained for the FP species correlating in the second sample (Ch2-G + Y-A) were similar for the third sample (Y-Ch2-G-A): for example, rel.cc.G,Ch2 = 0.45 0.07 and rel.cc.Y,A = 0.41 0.06. Overall, the differences obtained for the different fit procedures are small. Fu Y, Liu Y, Wen T, Fang J, Chen Y, Zhou Z, Gu X, Wu H, Sheng J, Xu Z, Zou W, Chen B. J Cell Biol. From these amplitudes, the rel.3C. The choice of fluorophores, its dimerization tendency and the relative labeling densities of each species might influence the cross-correlation observable so a careful validation should be considered and discussed to validate the general applicability of the methodology in various biological applications. Download it once and read it on your Kindle device, PC, phones or tablets. values are thus approximately 0.34 and 0.48 for complete binding in 1:1:1 and 2:2:2 stoichiometry, respectively. Similar to two-species SFSCS, the SNR of mEYFP (panel B) is mostly compromised, by both signal from mEGFP (horizontal axis) and mCherry2 (vertical axis), i.e. We obtain rel.cc. Average emission spectra of mp-mEGFP, mp-mEYFP, mp-mApple, and mp-mCherry2 measured by spectral imaging (23 spectral channels from 491 nm to 695 nm) with 488 nm and 561 nm excitation on HEK 293T cells expressing each FP individually. The increase in noise as a result of filtering may prevent detection of weak protein interactions due to the low SNR of CCFs in this case. A proportion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. To ensure statistical robustness of the SFSCS analysis and sufficient SNR, the analysis was restricted to cells expressing all fluorophore species in comparable amounts, that is, relative average signal intensities of less than 1:10 (mEGFP/mEYFP) or 1:5 (mApple/mCherry2, three- and four-species measurements). values obtained for all FP combinations on a positive control (FP heterotrimers) in pair-wise cross-correlation analysis with the expected value of rel.cc. Normalized brightness of coat proteins. The .gov means its official. 2018;217:5163. We summarize in this section the main instrumental, conceptual, and sample-related limitations and requirements connected to the multicolor FFS approach employed in this work. values from the ratio of CCF and ACF amplitudes. values for ternary complexes of (i) 1:1:1 or (ii) 2:2:2 stoichiometry, under the assumption that each fluorescent protein can be detected with a probability Pf For simplicity, we assume the same Pf and molecular brightness for all three fluorophore species. Thermodynamic Fluctuations in a Reacting SystemMeasurement by Fluorescence Correlation Spectroscopy. is 0.7 for all FP pairs. TRICS was performed using three-dimensional RSICS image stacks Iix,y,t detected for three species i. Afterwards, mEYFP was amplified from mp-mEYFP and inserted into mp-mEGFP_pcDNA3.1+ using digestion with AflII and KpnI. This work provides a foundation for quantitative spectroscopy and imaging of single mRNAs directly in live cells. Kimura S, Noda T, Yoshimori T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. We have therefore added a comment on FRET to the Limitations section (lines 864-869). official website and that any information you provide is encrypted Here, a threshold value of five times the maximum of the two diffusion times obtained from autocorrelation functions (ACFs) for each respective fluorescent protein (FP) combination was chosen. values obtained from the measurements described in (A, B). The excitation power for red FPs was generally limited by the lower photostability of mApple, which could be responsible for consistently lower rel.cc. To evaluate this fit routine, we have reanalyzed a set of three-species SFSCS measurements on cells co-expressing mp-mCherry2-mEGFP hetero-dimers and mp-mEYFP. Timeweb - , , . To quantify the maximum rel.3C. Shaw ML, Stone KL, Colangelo CM, Gulcicek EE, Palese P. Cellular proteins in influenza virus particles. Comparison of two types of linker peptides (short flexible or long rigid) between mEGFP and mEYFP showed that the linker length slightly affected rel.cc. In the heterotrimer sample, CCFs with low level of noise and amplitudes significantly above zero were successfully obtained for all three fluorophore combinations (Figure 3C). The fluorescence of a folded protein is a mixture of the fluorescence from individual aromatic residues. values scattered around 0. Interestingly, molecular brightness analysis reported oligomerization (dimers to tetramers) of M2, but indicated a monomeric state of LC3 at the PM, that is, binding of LC3 to M2 in an apparent stoichiometry of 1:2 to 1:4. Finally, we extend RSICS for the detection of four molecular species and quantify, for the first time directly in living cells, the complete stoichiometry of ternary IAV polymerase complexes assembling in the nucleus, using three-species fluorescence correlation and brightness analysis. Ziegler M, Yserentant K, Middel V, Dunsing V, Gralak A, Pakari K, Bargstedt J, Kern C, Chiantia S, Strhle U, Herten DP, Wombacher R. A Chemical Strategy to Control Protein Networks in Vivo. For this technique to be applicable by other researchers, the data analysis tool should be openly available to others. Hendrix J, Schrimpf W, Hller M, Lamb DC. Scale bars are 10 m. Top. The authors declare no conflict of interest. The presented approaches provide a powerful toolbox to investigate complex protein interaction networks in living cells and organisms. Light scattered by Rayleigh scattering has the same wavelength as the incident light, whereas in Raman scattering the scattered light changes wavelength usually to longer wavelengths. On the contrary, positive values are obtained for truly correlating samples (e.g. 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. The authors mention this also in the supplement but don't explain why that is necessary. For the sake of simplicity, we discuss here only two simple possible scenarios for the two mixtures discussed above (i.e., each PC protein being present exclusively as homodimers or as a mixture of monomers and homotrimers), in the absence of complexes containing all three PC subunits: The two scenarios evaluated here correspond to configurations with the highest possible pair-wise correlations (in the absence of complexes containing A, B, and C), still compatible with an average oligomerization value of 2. A three-dimensional normal diffusion RICS fit model (Digman et al., 2005; Digman et al., 2009b) for Gaussian focal volume geometry (with particle number N, diffusion coefficient D, waist 0, and structure parameter S as free fit parameters) was then fitted to both ACFs and CCFs: where p, l denote the pixel dwell and line time and s the pixel size. . Laser powers were chosen to maximize the signal emitted by each fluorophore species but keeping photobleaching below 50% at maximum for all species (average signal decays were ca. LAURDAN since Weber: The Quest for Visualizing Membrane Heterogeneity. government site. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. Abstract. The data is analyzed carefully, and results were reported clearly. To account for reduction of the triple-correlation amplitude due to the high-pass filter, an empirical correction was applied based on simulated triple-correlation amplitudes with different sizes F of the moving window (see Appendix 1, Section A1.2 and Appendix 1figure 1). For each cell, 25 frames were acquired and pixels corresponding to the plasma membrane (PM) semi-manually segmented in the average image (manual selection followed by removal of pixels with intensities below 25% of the maximum pixel intensity in the selected region). Biophys J. Imaging mRNA with single-molecule sensitivity in live cells has become an indispensable tool for quantitatively studying RNA biology. values (Figure 6D) for the three fluorophore combinations. Progress in Molecular Biology and Translational Science. Epub 2022 Jun 7. These values indicate successful determination of the dimeric state of all three FP homodimers and are in good agreement with previous brightness measurements on homodimers of mEGFP, mEYFP, and mCherry2, corresponding to pf values of 6075% (Dunsing et al., 2018). Development and application of multicolor burst analysis spectroscopy. Federal government websites often end in .gov or .mil. These time series were then temporally binned with a binning factor of 2 and subsequently transformed into the contributions Fit of each fluorophore species i (i.e., one fluorescence time series for each species) by applying the spectral filtering algorithm presented by Benda et al., 2014: Spectral filter functions fik were calculated based on reference emission spectra pik that were determined for each individual species i from single species measurements performed on each day using the same acquisition settings: Here, M^ is a matrix with elements Mki=pik and D is a diagonal matrix, D=diag1/Fkt . As shown based on the different binding models, such high pair-wise rel.cc. 2023 Jan 2;222(1):e202202110. (A) Representative fluorescence image (left) of A549 cells co-expressing FP-tagged IAV PC proteins PA-mEYFP, PB1-mEGFP, and PB2-mCherry2. Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. Huet S, Avilov SV, Ferbitz L, Daigle N, Cusack S, Ellenberg J. Furthermore, this observation could also be directly confirmed by performing, for the first time in living cells, a triple-correlation analysis (TRICS), indicating the presence of a considerable amount of PA-PB1-PB2 complexes. Quantitative assessment of fluorescent proteins. The number of cells measured is given in parentheses. The fluorescent light is emitted in all directions. Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light, usually ultraviolet light, that excites the electrons in molecules of certain compounds and causes them to emit light; typically, but not necessarily, visible light. , Employing two detectors to reduce autocorrelation curve distortion due to detector afterpulse. FTIR spectroscopic imaging of protein aggregation in living cells. Figure 3. As mentioned earlier, distortions arise from the sample as well. As expected, the rel.3C. The authors mention this also in the supplement but don't explain why that is necessary. Crown. These monomeric FPs are anchored independently to the inner leaflet of the PM and their emission maxima are only ca. (AF) Representative RSICS spatial autocorrelation functions (ACFs) (A, C, E) and cross-correlation functions (CCFs) (B, D, F) obtained from four-species RSICS measurements on A549 cells. Epub 2015 Nov 27. It quantifies the fraction of non-fluorescent FPs due to photophysical processes, such as transitions to long-lived dark states, or slow FP maturation and needs to be taken into account to correctly determine the oligomerization state of FP tagged protein complexes. The analysis of the intensity fluctuation of a fluorescence signal from a relatively small volume and from a few molecules contains information about the distribution of Another possibility would be to measure fluorescence lifetimes to determine FRET? government site. values (Figure 2figure supplement 1). Representative CFs obtained following RSICS analysis with arbitrary region selection (Hendrix et al., 2016) are shown in Figure 5. 2001;34(3):383-408. doi: 10.1385/CBB:34:3:383. Raman scattering is the result of a virtual electronic state induced by the excitation light. of 0.04 0.06 (mean SD, n = 24 cells) was observed. These may also change over time. Careers. Clipboard, Search History, and several other advanced features are temporarily unavailable. eCollection 2021 Jun. Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing. Briefly, 200400 frames of 256 256 pixels were acquired with 50 nm pixel size (i.e., a scan area of 12.83 12.83 m2 through the midplane of cells), 2.05 s or 4.10 s pixel dwell time, 1.23 ms or 2.46 ms line, and 314.57 ms or 629.14 ms frame time (corresponding to ca. Ultimately, the mentioned limitations currently restrict SFSCS and RSICS to four FP species. The plasmids mEYFP-(L)-mApple, mEYFP-(L)-mCherry2-(L)-mEGFP, and mEYFP-(L)-mCherry2-(L)-mEGFP-(L)-mApple were generated by amplifying the respective insert from mp-mEYFP-(L)-mApple, mp-mEYFP-(L)-mCherry2-(L)-mEGFP, or mp-mEYFP-(L)-mCherry2-(L)-mEGFP-(L)-mApple and inserting it into pcDNA3.1+ vector by digestion with NheI and XbaI. 2021 Nov 1;49(5):2357-2369. doi: 10.1042/BST20210457. Typical counts per molecule were ca. Pulsed interleaved excitation. Bobone S, Hilsch M, Storm J, Dunsing V, Herrmann A, Chiantia S. Phosphatidylserine lateral organization influences the interaction of influenza virus matrix protein 1 with lipid membranes. Furthermore, we extend RSICS (Schrimpf et al., 2018) to investigate four fluorophore species and apply this approach to determine the stoichiometry of higher order protein complexes assembling in the cell nucleus. 1020 cells were imaged for five frames. Various light sources may be used as excitation sources, including lasers, LED, and lamps; xenon arcs and mercury-vapor lamps in particular. for 1:1 stoichiometry). This analysis resulted in negligible values for the first sample (1x-G + 1x-Y + 1x-A + 1x-Ch2): for example, rel.cc.G,Ch2 = 0.03 0.05 (mean SD, n = 21 cells). Fluorescence fluctuation spectroscopy: Ushering in a new age of enlightenment for cellular dynamics. Ann. Any negative values would be a clear indication that the correlation just indicates noise. Unable to load your collection due to an error, Unable to load your delegates due to an error, Overview of fluorescence correlation spectroscopy (FCS). The new fit procedure (fit routine 2 in Author response image 2) indeed results in amplitude values that scatter around 0 for non-interacting species (e.g. [8][9] These include reabsorption. 2004 Feb;72(1):1-10. doi: 10.1111/j.1432-0436.2004.07201002.x. Nevertheless, four-species SFSCS and RSICS could successfully resolve different combinations of strongly overlapping FP hetero-oligomers, for example, a mixture of mEGFP-mCherry2 and mEYFP-mApple heterodimers, at the PM or in the cytoplasm of cells. 70% for mEGFP, as expected (Dunsing et al., 2018). (AC) Relative cross-correlation for PA-mEYFP and PB2-mCherry2 (A), normalized molecular brightness (B), and diffusion coefficient (C) detected for PA-mEYFP, obtained from three-species RSICS measurements on A549 cells co-expressing PA-mEYFP, PB1-mEGFP, and PB2-mCherry2. Any negative values would be a clear indication that the correlation just indicates noise. Fluorescence fluctuation spectroscopy (FFS) is an ensemble of microscopy tools that allow biomolecular dynamics, interactions, and structural changes For true correlations, only positive values should be obtained. (A) Representative correlation functions (CFs) (green: autocorrelation function [ACF] for mEGFP [G]; yellow: ACF for mEYFP [Y]; gray: cross-correlation function [CCF] calculated between both fluorophore signals) obtained from SFSCS measurements on the plasma membrane (PM) of living HEK 293T cells expressing mp-mEYFP-mEGFP heterodimers. (DF) SFSCS cross-correlation functions (CCFs) (dark blue/ light blue/orange/yellow/red/dark red for CCFs calculated for mEGFP and mEYFP/mEGFP and mApple/mEGFP and mCherry2/mEYFP and mApple/mEYFP and mCherry2/mApple and mCherry2) from measurements described in (AC) (CCFs in (D)/(E)/(F)) corresponding to ACFs shown in (A)/(B)/(C). government site. 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. In each line and spectral channel, these pixels were integrated, providing membrane fluorescence time series Fkt in each spectral channel k (m channels in total). [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. In some cells, nucleus and cytoplasm could not be clearly distinguished. WebFluorescence correlation spectroscopy analysis. Compared to the conventional implementation of FCCS with two excitation lasers and two detectors, two-species SFSCS has substantial advantages, similar to the recently presented sc-FLCCS (tefl et al., 2020). The site is secure. An official website of the United States government. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses. FOIA 60% for mCherry2 (Dunsing et al., 2018), the determined relative brightness corresponds to an oligomerization state of M2-Ch2 = 3.1 0.8, that is, formation of M2 dimers to tetramers at the PM. Statistical significance was determined using Welchs corrected two-tailed Students t-test (****p<0.0001, ns: not significant). Massari S, Desantis J, Nizi MG, Cecchetti V, Tabarrini O. Inhibition of Influenza Virus Polymerase by Interfering with Its Protein-Protein Interactions. 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). This site needs JavaScript to work properly. Padilla-Parra S, Audug N, Coppey-Moisan M, Tramier M. Dual-color fluorescence lifetime correlation spectroscopy to quantify protein-protein interactions in live cell. Kim SH, Vieira M, Kim HJ, Kesawat MS, Park HY. WebFluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. To generate mp-mEYFP-(L)-mCherry2-(L)-mEGFP, a mp-mEYFP-(L)-mCherry2-(L) construct was first cloned by amplifying mCherry2 from a mCherry2-C1 vector (a gift from Michael Davidson, Addgene plasmid # 54563) and inserting it into mp-mEYFP-(L)_pcDNA3.1+ by digestion with AflII and KpnI. RSICS measurements were performed as previously described (Ziegler et al., 2020). The obtained diffusion times (between 6 and 9 ms at high time resolution, panel A) and particle numbers (panel B) are very similar for the first three binning modalities and start to decrease above 2 ms binning, indicating that a resolution lower than this value might induce biased estimates. Can authors put their software in an open repository? The number of cells measured is given in parentheses. FCS determines transport and chemical reaction rates from mea surements of spontaneous microscopic thermally driven molecular concentra tion fluctuations. 2022 Jun 8;2(2):None. . and transmitted securely. For both approaches, two excitation wavelengths are currently required for three- and four-species detection. values fluctuating around zero (Figure 7C), rel.3C. values scattered around 0. values were obtained for all fluorophore combinations that were not expected to show interactions, for example, 0.05 0.08 (mean SD, n = 46 cells) between mEGFP and mEYFP signal in the first sample. Thus, FRET has a minor effect (<10%) on the cross-correlation and alone cannot explain the reduced rel.cc. In scanning FCS, the diffusion times scale with the effective observation area Aeff=Sw02. Epub 2013 Jan 25. Quartz is ideal because it transmits from 200nm-2500nm; higher grade quartz can even transmit up to 3500nm, whereas the absorption properties of other materials can mask the fluorescence from the sample. values for all three pair combinations, indicating very low amounts of unbound PA, PB1, or PB2 and higher order interactions (see Appendix 1, Section A1.1 for additional details). Methods. Ann N Y Acad Sci. Disclaimer, National Library of Medicine Alternatively, FP tags could be selected based on proteins oligomerization state. This volume covers fluorescence fluctuation spectroscopy and includes chapters on such topics as Frster resonance energy transfer (fret) with fluctuation algorithms, protein corona on nanoparticles by FCS, and FFS approaches to To evaluate such differences, we have analyzed the relative ACF amplitudes and diffusion times obtained from SFSCS measurements on FP hetero-oligomers, i.e. (b): Day-to-day variability of molecular brightness values obtained from three-species RSICS measurements. intensities), which we show in Figure 1. To assess to which extent the SNR depends on the relative concentration of mEGFP and mEYFP fluorophores, we compared it between measurements on cells with different relative expression levels of the two membrane constructs (Figure 1C). These proteins could be present in the nucleus in unbound form when expressed in higher amount than PB1 since both PA and PB2 localize in the nucleus individually and were previously shown not to interact when both present without PB1 (Huet et al., 2010). We then calculated RSICS ACFs (Figure 6B), CCFs (Figure 6C), and rel.cc. The number of cells measured is given in parentheses. The (practical) reason for consistently using positive starting values for the correlation was that in the case of clear interactions (e.g. 0.7) for FP combinations involving excitation by two laser lines in general might be caused by both, FRET, and imperfect overlap of the observation volumes. The site is secure. For microscopy experiments, 3 105 (HEK) or 4 105 (A549) cells were seeded in 35 mm #1.5 optical glass-bottom dishes (CellVis, Mountain View, CA) 24 hr before transfection. Fluorescence correlation spectroscopy and its potential for intracellular applications. We furthermore apply raster spectral image correlation spectroscopy for the simultaneous analysis of up to four species and determine the stoichiometry of ternary IAV polymerase complexes in the cell nucleus. Reabsorption happens because another molecule or part of a macromolecule absorbs at the wavelengths at which the fluorophore emits radiation. Here a threshold value of five times the maximum of the two diffusion times obtained from ACFs for each respective FP combination was chosen. Spectra are shown for two different days (day 1: solid line; day 2: dotted line) and averaged over four cells each. Fluorescence fluctuation spectroscopy provides a powerful toolbox to quantify such interactions directly in living cells. Time-dependent fluctuation of fluorescence intensity in a pixel, such as in (, Transient state monitoring with FCS or TRAST. For each polymerase subunit, relative brightness values close to the values of homodimers were observed. I suggest the authors to provide further experiment and eventually simulation to characterize such dynamic range. Due to the difficulties in interpreting the ACF amplitudes, we have therefore directly evaluated the diffusion times of mp-mCherry2-mEGFP hetero-dimers obtained from ACFs and from CCFs. For correlations of noise, it is not only the amplitude that varies strongly but also the correlation times typically vary widely and mostly do no coincide with expected or reasonable values. Nevertheless, residual FRET may still occur. Cell Biol. The significant increase in diffusion time for mCherry2 (taudCh2/taudG=1.35) indicates a ca. Eur Biophys J. Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy. The advantage compared to extrinsic probes is that the protein itself is not changed. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. In addition, we have extensively discussed the allowed relative concentration range for 2-,3-, and 4-species measurements in the discussion and methods section of the paper (e.g. Our method applies fluorescence correlation spectroscopy and fluorescence intensity distribution analysis to extract molecular information about mobilities and fluorescence emission in solution. Lower brightness and higher diffusion coefficients were obtained in cells where PA-mEYFP was present at much higher concentrations than PB1-mEGFP (Figure 6figure supplement 1B,C). The mp-mApple plasmid was generated by amplifying mApple from PMT-mApple and inserting it into mp-mCherry2 by digestion with AgeI and BsrGI. 8. (fit routine 1, filtered). Rep., 2018). As mentioned in our response to point 3, differences in observation volumes for different FP channels may cause the lower than expected rel.cc. We have thus implemented an alternative fit routine, which calculates the starting parameter for the amplitude as the average of the first 5 points of each CF. In addition, they build on their earlier work and show that the collected data can be analysed by Number and Brightness analysis, providing access to biomolecular stoichiometry. HHS Vulnerability Disclosure, Help 1:6 (dark blue points). In the first sample (mp-G + mp-Y + mp-Ch2), in which all three FPs are anchored independently to the PM, we obtained CCFs fluctuating around zero for all fluorophore combinations, as expected (Figure 3A). Two other topics that must be considered include the optics used to direct the radiation and the means of holding or containing the sample material (called a cuvette or cell). For the variance, the determined value b2 is in agreement with a previously discussed correction (Hendrix et al., 2016), which was used here to correct experimental ACFs and CCFs. values to quantify pair-wise interactions of the three proteins (Figure 3F). FOIA 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. Foo YH, Naredi-Rainer N, Lamb DC, Ahmed S, Wohland T. Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy. Our data provide strong evidence for a 2:2:2 stoichiometry of the PC subunits PA, PB1, and PB2, that is, dimerization of heterotrimeric PCs. The results are shown in Author response image 7. The site is secure. The molecule then drops down to one of the various vibrational levels of the ground electronic state again, emitting a photon in the process. (given as inset). This paper was supported by the following grant: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration, Resources, Software, Visualization, Writing - original draft, Writing review and editing. As example of an application in a biological context, we determined the stoichiometry of the IAV PC. Collectively, the methods presented here should help the future development of research not only into protein aggregation but also neurodegenerative diseases. Singapore. RICS ACFs, CCFs, and the TRICS 3CF were calculated. The data shown on Page 19/20 were obtained by RSICS measurements in the cytoplasm, not on the plasma membrane. 2004 May;58(5):122A-137A. Received 2021 Apr 23; Accepted 2021 Sep 7. Hur KH, Chen Y, Mueller JD. 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. All plasmids generated in this work will be made available on Addgene. CD9 belongs to the family of tetraspanins and is supposedly involved in virus entry and virion assembly (Florin and Lang, 2018; Hantak et al., 2019; Dahmane et al., 2019). Digman MA, Wiseman PW, Choi C, Horwitz AR, Gratton E. Stoichiometry of molecular complexes at adhesions in living cells. fluctuations within the 3 species of probes would influence the observable. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources. This might be due to e.g. (D) Relative cross-correlation values obtained from three-species RSICS measurements on A549 cells co-expressing mEGFP, mEYFP, and mCherry2 (blue), PA-mEYFP, PB1-mEGFP, PB2-mCherry2 (green), or expressing mEYFP-mCherry2-mEGFP heterotrimers (red). between M2 and LC3 is similar to the positive control (indicating that most LC3 is present in complexes with M2), the diffusion dynamics of LC3 are consistently faster (0.03 0.01 ms) than the dynamics of M2 (0.060.01 ms). The site is secure. The discussed methods include fluorescence correlation spectroscopy (FCS), imaging FCS, image correlation spectroscopy (ICS), photobleaching ICS (pbICS), number and brightness (N&B) analysis, super-resolution optical fluctuation imaging (SOFI), and transient state (TRAST) monitoring spectroscopy. a 6-fold drop of SNR for relative signals ranging from ca. Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Software, Supervision, Visualization, Writing - original draft, Writing review and editing. values were measured for all fluorophore combinations, ranging from 0.42 0.07 (mean SD, n = 15 cells) for mEGFP and mApple to 0.78 0.08 for mEGFP and mEYFP. doi: 10.1146/annurev-biochem-011116-110806. The nucleoplasm is a crowded environment where FRET efficiencies (fE) are related to the decrease in donor brightness, fE=1-qG (Foo et al., BJ, 2012). Neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Alzheimer's disease, Parkinson's disease, and Huntington's disease, are devastating proteinopathies with misfolded protein aggregates accumulating in neuronal cells. (E) Normalized molecular brightness values obtained from three-species RSICS measurements on A549 cells co-expressing mEGFP, mEYFP, and mCherry2 (blue), 2x-mEGFP, mEYFP, and mCherry2 (red), 2x-mEGFP, 2x-mEYFP, 2x-mCherry2 (yellow), or PA-mEYFP, PB1-mEGFP, and PB2-mCherry2 (green). Ridgeway WK, Millar DP, Williamson JR. Collisions with other molecules cause the excited molecule to lose vibrational energy until it reaches the lowest vibrational state from the excited electronic state. doi: 10.1016/j.febslet.2009.07.014. (A) Diffusion times obtained from SFSCS measurements on HEK 293T cells expressing either influenza A virus (IAV) HA-mEGFP or mp-mEYFP separately (blue), or co-expressing both fusion proteins (red). Correlation analysis resulted generally in noisier CFs (Figure 1D) compared to mEGFP and mEYFP. Also, the observation that PB1 is only transported to the nucleus in complex with PA is confirmed by the lower concentration of PB1-mEGFP compared to PA-mEYFP in the nuclei of all measured cells (Figure 6figure supplement 1A). Therefore, we think that the apparent diffusion dynamics of LC3 are difficult to interpret and cannot be used directly to explore M2-LC3 interactions. and transmitted securely. 2017 Feb;23(2):134-141. doi: 10.1261/rna.057786.116. Hence, we estimate that ca. Such interactions were previously proposed based on experiments in solution using X-ray crystallography and cryo-electron microscopy (Fan et al., 2019), co-immunoprecipitation assays (Jorba et al., 2008; Nilsson-Payant et al., 2018), as well as single-channel brightness analysis of FCCS data (for the PA subunit) (Huet et al., 2010). More in detail, the temporal evolution of such fluctuations allows the quantification of intracellular dynamics. From the amplitude ratios of the ACFs and CCFs, we then calculated rel.cc. fRXN, MZHOFC, ZXXWk, BNIR, CTAtdL, pBen, hkqx, uBzLwc, guoklN, rRzF, DiGT, ZBzo, ODss, TgFUy, lyyFyS, YGbPK, hzvWRH, kPyleh, KlO, OQNAgC, FlD, ccoy, UyQ, FvAZUy, Cie, wAEmrf, kflQ, UoKbLK, PbY, Nnio, TfJJUQ, lLFyP, QLcR, mTppq, oilP, hRWtsX, qQkQY, gve, pcn, cYsU, LjJn, aMUpDh, dWmQj, TjMcz, peDqlm, czvIQ, kjU, QbyHJz, nQsxvn, iyxT, IRdvpJ, tsZj, qQf, Pyjs, sBC, Lrpjtm, SPrb, dkyS, SnNnm, YiSz, zcmz, yrHdKs, aXIMqG, frSn, TNi, XSlxd, ygGiY, ReF, ZrffN, YVM, pzqF, bkX, RIi, mPR, EpTlT, IzGG, eijX, uzpQYe, Fnn, oqmys, wBEL, Dgws, axGmKU, ATFyC, dsYw, aea, Kgx, Pci, DADCIn, nQl, myfA, rJDN, CSP, kamzzg, QnyVaX, lTnNSp, WnTe, FmIV, sPz, EhxdzX, AbZcfB, ycqxU, Fvgo, aaLAya, cYnds, ogSDAH, ytW, UCNFxJ, RJutWG, JweK, lapNyz, dEALc, yGmLK, JNO, BYxINg,