We kindly thank Madlen Luckner for providing the plasmids for PA-mEYFP, PB1-mEGFP, and PB2-mCherry2 expression, Thorsten Wohland for providing the PMT-mApple plasmid, and Jelle Hendrix for fruitful discussion. ca. Singapore. We then calculated rel.cc. 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. values of the positive control, that is, the maximum pair-wise rel.cc. Fluorescence offered solutions to these difficulties, and in the early 1970s Magde, Elson, and Webb published seminal papers on the theory and application of fluorescence fluctuation analysis, specifically fluorescence correlation spectroscopy (FCS) (Elson and Magde 1974; Magde et al. 2001;34(3):383-408. doi: 10.1385/CBB:34:3:383. Differentiation. To explore the full potential of SFSCS, we extended the approach to systems containing three spectrally overlapping fluorophores. 2015 Mar 16;16(3):6076-92. doi: 10.3390/ijms16036076. Alternatively, FP tags could be selected based on proteins oligomerization state. For a 2:2:2 stoichiometry, we obtained an estimated rel.3C. This new volume of Methods in Enzymology continues the legacy of this premier serial by containing quality chapters authored by leaders in the field. We observed strong association of LC3 with M2, and consequent recruitment of LC3 to the PM (Figure 3figure supplement 4), in agreement with previous in vitro and localization studies (Beale et al., 2014). A more detailed analysis of the data confirmed this interpretation: the molecular brightness and diffusion coefficient of PA-mEYFP depended on the relative concentration of PB1-mEGFP and PA-mEYFP. Larsen JB, Taebnia N, Dolatshahi-Pirouz A, Eriksen AZ, Hjrringgaard C, Kristensen K, Larsen NW, Larsen NB, Marie R, Mndler AK, Parhamifar L, Urquhart AJ, Weller A, Mortensen KI, Flyvbjerg H, Andresen TL. This specific feature opens the possibility of a more in-depth analysis. (AC) Representative autocorrelation functions (ACFs) (green/yellow/orange/red for mEGFP [G]/mEYFP [Y]/mApple [A]/mCherry2 [Ch2]) obtained from four-species SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP, mp-mEYFP, mp-mApple, and mp-mCherry2 (A), mp-mCherry2-mEGFP heterodimers, mp-mEYFP, and mp-mApple (B), or expressing mp-mEYFP-mCherry2-mEGFP-mApple hetero-tetramers (C), as illustrated in insets. 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). of 0.60 was obtained with a short linker peptide between the two FPs, suggesting the presence of FRET (see Figure 1figure supplement 3). In our experiments, cross-talk-free SFSCS analysis with two species excited with a single excitation wavelength could be performed for relative intensity levels as low as 1:10 (mEGFP/mEYFP) or 1:5 (mApple/mCherry2). (G) Normalized molecular brightness values obtained from three-species SFSCS measurements on HEK 293T cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2 (blue), mp-2x-mEGFP, mp-mEYFP, and mp-mCherry2 (red), CD9-mEGFP, LC3-mEYFP, and M2-mCh2 (green), or expressing mp-mEGFP alone (yellow). 2015 Sep 16;6(9):1503-8. doi: 10.1021/acschemneuro.5b00176. 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. doi: 10.1242/dmm.046516. We thank the reviewer for this idea and have added a Limitations subsection at the end of the discussion, summarizing the main technical and conceptual limitations of the technique. This results in a better signal-to-noise ratio, and lowers the detection limit by approximately a factor 10000,[3] when compared to the 180 geometry. (solely based on pf, i.e. Error bars represent mean SD. Clipboard, Search History, and several other advanced features are temporarily unavailable. Negative initial values resulted in errors in the fit routine or fit curves that clearly did not provide a reliable fit to the CFs. The .gov means its official. To ensure statistical robustness of the RSICS 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 1:6 for all species (in all RSICS experiments). Some of these methodologies are classical protein aggregation analyses, while others are not yet widely used. The authors might be able to use that criterion to identify non-correlated data and thus avoid the positive amplitudes which are artifacts of the restrictions of the fitting parameters. For all fluorophore species, ACFs with amplitudes significantly above zero were obtained. To understand whether this analysis is sufficient to indicate the presence of heterotrimeric protein complexes for the specific case reported in this work, we investigated brightness and rel.cc. Devices that measure fluorescence are called fluorometers. 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. Briefly, all scan lines were aligned as kymographs and divided in blocks of 1000 lines. The .gov means its official. Finally, for best visualization, G3C is plotted for a and b values 1 (see Figure 7 and Appendix 1figure 2). In more detail, we performed four-species RSICS measurements on the following three samples: (i) cells co-expressing free mEGFP, mEYFP, mApple, and mCherry2 (1x-G + 1x-Y + 1x-A + 1x-Ch2), (ii) cells co-expressing mCherry2-mEGFP and mEYFP-mApple heterodimers (Ch2-G + Y-A), and (iii) cells expressing mEYFP-mCherry2-mEGFP-mApple hetero-tetramers (Y-Ch2-G-A). 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. By contrast, a xenon arc has a continuous emission spectrum with nearly constant intensity in the range from 300-800nm and a sufficient irradiance for measurements down to just above 200nm. values for all models are given in Appendix 1table 1 for = 1 or Pf = 0.7. 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). For the cloning of all following constructs, standard PCRs with custom-designed primers were performed, followed by digestion with fast digest restriction enzymes and ligation with T4-DNA-Ligase according to the manufacturers instructions. (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. and Mol. 0.7, see RSICS data in Figure 6 and previously published data in Dunsing et al., Sci. We show that SFSCS enables cross-talk-free SFCCS measurements of two protein species at the PM of living cells tagged with strongly overlapping fluorophores in the green or red regions of the visible spectrum, excited with a single excitation line. The four fluorescent protein (FP) species are denoted with G, Y, A, and Ch2. Data are pooled from two independent experiments. 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. Comm. Nicolai E, Garau S, Favalli C, D'Agostini C, Gratton E, Motolese G, Rosato N. New Microbiol. values from the ratio of CCF and ACF amplitudes. Published by Elsevier Inc. All rights reserved. To evaluate such differences, we have analyzed the relative ACF amplitudes and diffusion times obtained from SFSCS measurements on FP hetero-oligomers, i.e. Nevertheless, similar average D values were determined for different fluorophore species coupled as hetero-oligomers, for example, DG = 19.4 3.4 m2/s and DCh2 = 20 11 m2/s (mean SD, n = 23 cells) for mEGFP-mCherry2 heterodimers, and DG = 11.2 2.5 m2/s, DY = 11.6 2.6 m2/s, DA = 12.8 3.2 m2/s, DCh2 = 12.6 5.0 m2/s (mean SD, n = 20 cells) for hetero-tetramers. 2005 Sep;89(3):2077-90. doi: 10.1529/biophysj.104.052779. Most of the intrinsic fluorescence emissions of a folded protein are due to excitation of tryptophan residues, with some emissions due to tyrosine and phenylalanine; but disulfide bonds also have appreciable absorption in this wavelength range. For allowing anisotropy measurements, the addition of two polarization filters is necessary: One after the excitation monochromator or filter, and one before the emission monochromator or filter. Filters and/or monochromators may be used in fluorimeters. 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. (AD) Average emission spectra of GPI-mEGFP (A), GPI-mEYFP (B), GPI-mApple (C), and GPI-mCherry2 (D) measured by spectral imaging (23 spectral channels from 491 nm to 695 nm) using 488 nm and 561 nm excitation on HEK 293T cells supplemented with buffer at different pH values, ranging from pH 5.0 to pH 9.2. of 0.48, that is, only slightly higher than the average value determined experimentally for IAV PCs. Error bars represent mean SD. and transmitted securely. Page 11: Why are correlation amplitudes limited to positive values? We address the issue of imperfect overlap below, in the response to question 5. In some cells, nucleus and cytoplasm could not be clearly distinguished. Therefore, tryptophan fluorescence can be a very sensitive measurement of the conformational state of individual tryptophan residues. 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). In addition, molecular brightness and cross-correlation analysis are compromised by FP maturation. 2016;572:51-64. doi: 10.1016/bs.mie.2016.02.015. Read reviews from worlds largest community for readers. This process of re-emitting the absorbed photon is "resonance fluorescence" and while it is characteristic of atomic fluorescence, is seen in molecular fluorescence as well.[2]. assuming pf0.7 for all 3 FPs, using the formulas derived in Foo et al., BJ, 2012. Surprisingly, we found that a high The technique was independently developed by Watt Webb and Rudolf Rigler during the early 1970s. We have evaluated the diffusion dynamics of M2 and LC3 (see Author response image 8). Overview of fluorescence correlation spectroscopy, Overview of fluorescence correlation spectroscopy (FCS). 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). Exemplary 3CFs for the two simulated scenarios are shown in Appendix 1figure 2. We obtain rel.cc. 1A) microscope, which enables selective imaging of the To explore the interaction of four different FP-tagged proteins, four-species FFS may substantially reduce the experimental effort because all pair-wise interactions can be quantified in a single measurement (instead of six separate conventional two-species FCCS measurements). Correction of all these instrumental factors for getting a standard spectrum is a tedious process, which is only applied in practice when it is strictly necessary. Then, mEGFP was inserted from mEGFP-(L)_pcDNA3.1+ (see below) by digestion with KpnI and BamHI. Palmer AG, Thompson NL. 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. Several applications might aim to study interaction within proteins of different abundance, it is important to understand the relative concentration range where the method can be used and provide reliable results. 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 different species Note that the combinations (1=2=0,1=2,2=0) and (c1=2=0,) would also result in a = 2 and b = 0, but these values were not included since they refer to a correlation between identical pixel positions (e.g., 2=0,2=0) between two FP channels and would be influenced by shot-noise artifacts (see above). Assuming pf values of 75, 77, and 61% (as calculated from the determined relative brightness values of homodimers) for mEGFP, mEYFP, and mCherry2, respectively, pf corrected normalized brightness values of PB1-G = 2.1 0.7 (mean SD, n = 53 cells), PA-Y = 1.8 0.6, and PB2-Ch2 = 2.2 0.7 were obtained (see Materials and methods for details). official website and that any information you provide is encrypted 5.9 W (561 nm). 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). As mentioned before, the fluorescence is most often measured at a 90 angle relative to the excitation light. For this technique to be applicable by other researchers, the data analysis tool should be openly available to others. intensities), which we show in Figure 1. (, Principles of a typical number and brightness (N&B) analysis. The site is secure. values of 7080% measured for mEGFP and mEYFP coupled in FP hetero-oligomers compared to 4560% observed for mEGFP and mCherry2. Figure 1: It seems like the time resolution of the measurements are on the order of several milliseconds. sharing sensitive information, make sure youre on a federal 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). Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E. Measuring fast dynamics in solutions and cells with a laser scanning microscope. Zhejiang Da Xue Xue Bao Yi Xue Ban. FPs linked by a short peptide displayed lower rel.cc., probably due to fluorescence resonance energy transfer (FRET), as previously reported (Foo et al., 2012). Four-dimensional image stacks Ix,y,t,k (time-lapse images acquired in k spectral channels) were imported in MATLAB (The MathWorks) from CZI image files using the Bioformats package (Linkert et al., 2010) and further analyzed using custom-written code (Dunsing and Chiantia, 2021). Nevertheless, the previous analysis could only be performed between two of the three subunits at the same time. Of note, the RSICS approach presented here provides for the first time simultaneous information on molecular interactions, molecular brightness (and thus stoichiometry), diffusion dynamics, and concentration for all three complex subunits. The free parameter 0 (starting value = 13 pixels) was used to determine which CCFs were too noisy (i.e., 0> 4 pixels) to obtain meaningful parameters (typically in the absence of interaction). 10% for mEGFP, 30% for mEYFP, 40% for mApple, and 20% for mCherry2). We have added the figure as supplement (Figure 3figure supplement 3) and refer to it now in the discussion (lines 696-697, 728, 730, 734). Thus, FRET has a minor effect (<10%) on the cross-correlation and alone cannot explain the reduced rel.cc. government site. We then compared the diffusion times measured by SFSCS to the values obtained on cells expressing each of the two constructs separately (Figure 2A). More precisely, one would expect a slightly larger observation volume for mCherry2 than for mEGFP (due to the higher wavelength) and thus an amplitude ratio GCh2/GG smaller than 1. = pf2 = 0.49 (1:1:1 stoichiometry) and rel.3C. This LAURDAN since Weber: The Quest for Visualizing Membrane Heterogeneity. 1 Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. 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. Higher normalized values (up to 1.19, see Appendix 1table 1) can be obtained only in the presence of hetero-complexes involving all three PC subunits, which we calculated for comparison for the two mixtures (i.e., AA-BB-CC, or A-B-C in mixtures with AAA-BBB-CCC) and both Pf values. For FPs with a probability pf to be fluorescent, the expected rel.cc. Surprisingly, we found that a high fluorescence background resulted from inefficient dimerization of fluorescent protein (FP)-labeled MS2 coat protein (MCP). Stefl et al. To directly confirm that IAV PC subunits form ternary complexes in the cell nucleus, we implemented a triple-correlation analysis (TRICS) to detect coincident fluctuations of the signal emitted by mEGFP-, mEYFP-, and mCherry2-tagged proteins. for heterotrimers is likely due to the approximated interpolation of the amplitude value from only the first five points of the 3CF. Conformational analysis of misfolded protein aggregation by FRET and live-cell imaging techniques. Disclaimer, National Library of Medicine Reviewer #3 (Recommendations for the authors): This manuscript is a carefully conducted study of multi-color fluorescence fluctuation spectroscopy as applied to plasma membranes. of 0.72 0.12 (mean SD, n = 22 cells) in the latter sample compared to a vanishing rel.cc. 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). Due to imperfect optical overlap, experimentally detectable rel.3C. for 1:1 stoichiometry heterodimers (A-B/A-C/B-C) or 1:1:1 stoichiometry heterotrimers (A-B-C), resulting in values of 1 (for Pf = 1) and 0.7 (for Pf = 0.7). To this aim, a polygonal region of interest (ROI) was selected in the time- and channel-averaged image frame containing a homogeneous region in the cytoplasm (four-species measurements on FP constructs) or nucleus (three-species measurements on polymerase complex and related controls) of cells. 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. To generate GPI-mApple and GPI-mCherry2, mApple and mCherry2 inserts were amplified from PMT-mApple and mCherry2-C1, respectively, and inserted into GPI-mEYFP using restriction by AgeI and BsrGI. AU - Mller, Joachim D. N1 - Funding Information: This work was supported by grants from the National Institutes of Health (GM64589) and the National Science Foundation (MCB-0110831). From SFSCS analysis of measurements in the latter sample, we obtained a normalized molecular brightness of 1.64 0.36 (mean SD, n = 21 cells) for mp-2x-mEGFP, relative to the brightness determined in the monomer sample (n = 19 cells). 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. Weidemann T, Mcksch J, Schwille P. Fluorescence fluctuation microscopy: a diversified arsenal of methods to investigate molecular dynamics inside cells. Stem-loop RNA labeling can affect nuclear and cytoplasmic mRNA processing. But the diffusion time measured are on the order of ~9 ms. With the time resolution relatively close to the measured diffusion time, did the authors check whether diffusion times are biased? 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. In this range, the increase in noise due to statistical filtering was moderate, benefitting from the fairly large spectral separation of green/yellow and red emission (Figure 3figure supplement 3). This is due to the exposure of the tryptophan to an aqueous environment as opposed to a hydrophobic protein interior. National University of Singapore, doi: 10.2174/1568026611212220014. The obtained rel.cc. The .gov means its official. 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). Firstly, the distortion arising from the instrument is discussed. The discussed methods include fluorescence correlation spectroscopy (FCS), imaging FCS, image correlation spectroscopy (ICS), photobleaching ICS Massari S, Desantis J, Nizi MG, Cecchetti V, Tabarrini O. Inhibition of Influenza Virus Polymerase by Interfering with Its Protein-Protein Interactions. values of mEGFP or mEYFP with mApple than with mCherry2. values normalized to that of the positive control (i.e., the pair-wise rel.cc. 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. Shown are average photon weights from five SFSCS acquisitions each. Both types use the following scheme: the light from an excitation source passes through a filter or monochromator, and strikes the sample. The obtained value and the autocorrelation function (ACF) amplitude value (also corrected for the decay induced by the high-pass filter) were used to calculate the relative triple-correlation value rel.3C. A more detailed analysis of our data showed that in the analyzed cells (i.e., cells showing clear membrane recruitment of LC3, Figure 3figure supplement 4A,B), the PM concentration of LC3 was on average only 30% compared to that of M2 (Figure 3figure supplement 4C), although both proteins were expressed in comparable amounts in the sample in general. 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). values obtained from the measurements described in (A, B). [14] Recent advances in computer science and machine learning have even enabled detection of bacterial contamination of water [15], Rendell, D. (1987). Careers. [13] Proteins that lack tryptophan may be coupled to a fluorophore. (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). Nevertheless, this procedure requires to set an arbitrary threshold for filtering of the diffusion times, which may be subjective and depend on the specific sample dynamics. This confirms that the previously obtained low cross-correlation does not result from artefacts in the spectral filtering. 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). Epub 2021 Apr 5. First, the spatial 3CF was calculated: where 1,2 denote spatial lags along lines and 1,2 along columns of the image stacks. In contrast, we observed substantial CCFs when analyzing measurements on cells expressing mp-mEYFP-mEGFP heterodimers (Figure 1figure supplement 3A). The new PMC design is here! The mp-mEYFP-(L)-mApple construct was cloned beforehand by inserting mApple from mEGFP-(L)-mApple into mp-mEYFP-(L)_pcDNA3.1+ using restriction by BamHI and EcoRI. 70% for mEGFP, as expected (Dunsing et al., 2018). Thus, if the observation volumes were the same, the relative amplitudes should be close to 1, as shown indeed in Author response image 6. Characterization of ternary protein systems in vivo with tricolor heterospecies partition analysis. In the case of interactions, the fit converges to positive values. that can be expected in an experiment, we calculate rel.3C. We then calculated RSICS ACFs (Figure 6B), CCFs (Figure 6C), and rel.cc. Here a threshold value of five times the maximum of the two diffusion times obtained from ACFs for each respective FP combination was chosen. 2022 Aug 6;23(15):8750. doi: 10.3390/ijms23158750. As a consequence, the overlap of excitation volumes of the two laser lines might be limited, thus reducing the maximum achievable rel.cc., as previously discussed for standard FCCS (Foo et al., 2012). 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). The analysis of the intensity fluctuation of a fluorescence signal from a relatively small volume and from a few The axes a and b indicate shifts in the x and y direction, respectively, across the three detection channels, as described in Materials and methods. KTH Royal Institute of Technology, See this image and copyright information in PMC. On the contrary, mCherry2 (3-fold change of SNR, panel C) and mEGFP (2-fold change of SNR, panel A) are only moderately affected. 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 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). The nucleoplasm is a crowded environment where Can authors confirm this by looking at the diffusion coefficients from the FCs curves? 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. For correlated data, for example, G,Ch2, both fit routines converged to comparable positive values. From these amplitudes, the rel.3C. Clipboard, Search History, and several other advanced features are temporarily unavailable. In addition, tryptophan is a relatively rare amino acid; many proteins contain only one or a few tryptophan residues. 2008;1130:320-6. doi: 10.1196/annals.1430.040. presented raster spectral image correlation spectroscopy (RSICS), a powerful combination of RICS with spectral detection and statistical filtering based on the emission spectra of mEGFP, mVenus, and mCherry fluorophores (Schrimpf et al., 2018). SFSCS and RSICS were performed on a Zeiss LSM880 system (Carl Zeiss, Oberkochen, Germany) using a 40, 1.2 NA water immersion objective. This indicates residual FRET between, e.g. Can authors confirm this by looking at the diffusion coefficients from the FCs curves? Error bars represent mean SD. Our method significantly extends the potential of far-field FFS, including for the noninvasive investigation of molecular reactions at higher concentrations. (A) Representative correlation functions (CFs) (green: autocorrelation function [ACF] for mEGFP [G]; yellow: ACF for mEYFP [Y] gray: cross-correlation function [CCF] calculated for both fluorophore species) obtained from SFSCS measurements on the PM of HEK 293T cells co-expressing mp-mEGFP and mp-mEYFP. For the other FP combinations requiring excitation with two laser lines, the obtained rel.cc. This method is likely to be used by cell biologists to determine the stoichiometry of multi-protein complexes. -, Klaips C.L., Jayaraj G.G., Hartl F.U. (C) Molecular brightness of LC3-mEYFP obtained from three-species scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements shown in Figure 3 as a function of the ratio of LC3-mEYFP to M2-mCherry2 expression at the PM, in units of protein monomers. 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. The fluorescence of a folded protein is a mixture of the fluorescence from individual aromatic residues. fluctuations within the 3 species of probes would influence the observable. 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. 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. Notably, the technical approaches can be carried out on a standard confocal microscope, equipped with a spectral photon counting detector system. 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. fluctuations within the 3 species of probes would influence the observable. Sweden Clipboard, Search History, and several other advanced features are temporarily unavailable. 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. Fluorescence Fluctuation Spectroscopy (Ffs), Part a book. Typically, tryptophan has a wavelength of maximum absorption of 280nm and an emission peak that is solvatochromic, ranging from ca. 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. Therefore, unless otherwise noted, similar long rigid linkers were inserted in all constructs used in this study that contain multiple FPs (see Supplementary file 1a). 1 The authors provided a validation of the method in HEK cells expressing the three Fluorescent Proteins in the plasma membrane in different oligomerization sates (Figure 3C). These extensions do not further compromise the SNR of CFs detected for mEGFP and mEYFP (see Figure 3figure supplement 3A,B), but may additionally reduce the SNR of CFs corresponding to red FPs (in particular when mEGFP and/or mEYFP concentration is much higher than that of red FPs, Figure 3figure supplement 3C). Having the tags on M2 and CD9 on different sides of the plasma membrane (EC/IC) should minimize potential hindrance in the interactions. FCS determines transport and chemical reaction rates from mea surements of spontaneous microscopic thermally driven molecular concentra tion fluctuations. The number of cells measured is given in parentheses. Then, all aligned line scans were averaged over time and fitted with a Gaussian function. In contrast, significantly higher, positive rel.3C. Before 2023 Jan 2;222(1):e202202110. FTIR spectroscopic imaging of protein aggregation in living cells. Unable to load your collection due to an error, Unable to load your delegates due to an error, Normalized brightness of coat proteins. The emission spectra of the FPs utilized in this study did not depend on cell lines or subcellular localization (Figure 5figure supplement 1) and showed no (mEGFP, mEYFP) or little (mApple, mCherry2) variation with pH over a range of 5.09.2 (Figure 5figure supplement 2). The number of monomers was calculated by dividing the signal detected for PB1-mEGFP and PA-mEYFP in scanning fluorescence spectral correlation spectroscopy (SFSCS) measurements by the average molecular brightness detected for mEGFP and mEYFP fluorophores in the monomeric reference sample (cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2). HHS Vulnerability Disclosure, Help and transmitted securely. Page 19/20: The diffusion coefficient for the hetero-tetramer is only about half of the one determined for hetero-dimers. There remain mainly technical questions and some points that should be discussed to ensure consistency of the results. Pathways of cellular proteostasis in aging and disease. This is the case when measuring the quantum yield or when finding the wavelength with the highest emission intensity for instance. Methods. Fluorescence correlation spectroscopy (FCS) is a powerful technique for quantification of molecular dynamics, and it has been widely applied in diverse fields, e.g., biomedicine, biophysics, and chemistry. 2002 Aug;83(2):605-18. doi: 10.1016/S0006-3495(02)75195-3. . National University of Singapore, via the TMDs should not be affected. 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. As shown in Author response image 5, the data spread for the diffusion times of the hetero-trimer is too large to draw definitive conclusions. In this context, spectral information could be further exploited to separate low signal levels of endogenously expressed, fluorescently tagged proteins from autofluorescence background. This article is distributed under the terms of the, GUID:22105F3E-6D92-4F8B-951D-6DC572B6F6CF, GUID:EEDABAFC-36DA-4CE8-91E7-1CF6F22F3F00, GUID:231433D0-A16D-4237-ABFC-F63B141FE379, GUID:116C4987-25AA-4CB9-9694-1265F76CA8E3, GUID:075E40C9-5DFE-42CF-A098-C90143454C8F, GUID:CAD6AC5B-DD0E-4308-B62B-3FEC612AE21F, GUID:00FF800F-E30A-456E-8422-A7755BA60705, GUID:8B04CD14-80AD-4C68-A7B8-C94C34811362, GUID:6E7AE3F1-1109-4DED-BB84-87AE1F3BBB5E. Finally, we used these improvements to study endogenous -actin mRNA, which has 24xMS2 binding sites inserted into the 3' untranslated region. Notably, a significant rel.cc. As an example, we investigate the interactions of influenza A virus (IAV) matrix protein 2 with two cellular host factors simultaneously. In the case of uncorrelated data, that is, for CFs fluctuating around zero, this constraint can generate low, but positive correlation amplitudes due to noise. This in now stated in lines 1024-1029. This limitation applies to all FFS methods that discriminate different fluorophore species based on spectral (e.g., FSCS [Benda et al., 2014], RSICS [Schrimpf et al., 2018]) or lifetime patterns (e.g., sc-FLCCS [tefl et al., 2020]). Fluorescence correlation spectroscopy (FCS) measures fluctuations of fluorescence intensity in a sub-femtolitre volume to detect such parameters as the diffusion time, number of molecules or dark states of fluorescently labeled molecules. Biol Reprod. Protein homeostasis as a therapeutic target for diseases of protein conformation. The https:// ensures that you are connecting to the For example, it is possible to quantify the relative cross-correlation of two subunits, e.g. Before Fluorescence fluctuation spectroscopy: Ushering in a new age of enlightenment for cellular dynamics. By using two-wavelength excitation and spectral detection in a scanning mode the authors show that they can determine the cross-correlation between up to four different probes in a single measurement. We have furthermore added a short comment on time resolution in the limitations section (lines 827-830). J Vis Exp. Bookshelf 2016 Apr 1;98:10-17. doi: 10.1016/j.ymeth.2015.11.024. An official website of the United States government. 70% of the expected value (Figure 6D). Figure 7A and B show representative 3CFs for the negative control and the PC sample, respectively. and transmitted securely. At low concentrations the fluorescence intensity will generally be proportional to the concentration of the fluorophore. of 0.53 0.10 was also determined for mApple and mCherry2 signals, that is, from the CCFs exhibiting the lowest SNR. They show a fairly large variation due to noise but the majority of data points report similar diffusion dynamics (0.070.06 ms) as M2 oligomers, which would be expected for binding of a membrane associated to a transmembrane protein. 35% difference in size and a slight shift of observation volumes (since the diffusion time obtained from the CCF does not fall in between the values obtained from ACFs for taudCh2 and taudG, see the discussion in Foo et al., 2012). CCFs obtained from measurements on cells co-expressing mp-Cherry2-mEGFP hetero-dimers and mp-mEYFP were either fitted with the same positive initial value for the amplitude (fit routine 1), or with the average of the first five points of each CCF (fit routine 2). Data are plotted as a function of the ratio of PB1-mEGFP to PA-mEYFP, in units of protein monomers, and pooled from four independent experiments (n = 53 cells). Molecules have various states referred to as energy levels. 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 the study of receptors in live cells. HHS Vulnerability Disclosure, Help We obtain rel.3C. Epub 2022 Jun 7. To detect hetero-interactions between CD9, LC3, and M2, we co-expressed the fluorescent fusion proteins CD9-mEGFP, LC3-mEYFP, and M2-mCherry2 (i.e., M2 carrying an mCherry2 tag at the extracellular terminus) in HEK 293T cells (Figure 3D) and performed three-species SFSCS measurements at the PM (Figure 3E). We thank the reviewer for bringing up this important issue. The authors use a range of protein constructs that include between one to four fluorescent proteins, which they use in different compositions to demonstrate that they can analyse all possible interactions of four probes in a single measurement. Sample size is given in parentheses in each graph. For three- and four-species measurements, both laser lines were used. Such framework allows the minimization of cross-talk artifacts in FCCS measurements performed in living cells (Padilla-Parra et al., 2011). As these are membrane probes with the fluorescent proteins not expected to interact with the membrane, is there an explanation for the factor 2? We furthermore verified that SFSCS analysis results in correct estimates of protein diffusion dynamics. . FFS utilizes the fluctuating fluorescence signal when fluorescently labeled molecules move through a subfemtoliter observation volume, allowing various physical and biological systems to be studied at the single-molecule level. values for CD9-mEGFP with LC3-mEYFP or M2-mCherry2 (rel.cc.CD9-G,LC3-Y = 0.09 0.13, rel.cc.CD9-G,M2-Ch2 = 0.07 0.09, mean SD, n = 19 cells) were similar to those of the negative cross-correlation control (i.e., cells co-expressing mp-mEGFP, mp-mEYFP, and mp-mCherry2, see previous paragraph). (A) Representative fluorescence image (left) of A549 cells co-expressing FP-tagged IAV PC proteins PA-mEYFP, PB1-mEGFP, and PB2-mCherry2. -, Labbadia J., Morimoto R.I. We normalized the obtained values to the average values determined by RSICS on cells co-expressing monomeric mEGFP, mEYFP, and mCherry2, measured on the same day. For the second sample (Ch2-G + Y-A), values significantly above zero, that is, rel.cc.G,Ch2 = 0.46 0.09 (mean SD, n = 23 cells) and rel.cc.Y,A = 0.300.10, were only observed for two fluorophore pairs. 2012;12:26232640. FEBS Lett. As a simple approximation, we assume therefore that each species, independently of its participation in hetero-complexes, is either (i) exclusively dimeric or (ii) present as a well-defined mixture of monomers and homotrimers. (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. Cranfill PJ, Sell BR, Baird MA, Allen JR, Lavagnino Z, de Gruiter HM, Kremers GJ, Davidson MW, Ustione A, Piston DW. For all three protein species (PA-mEYFP, PB1-mEGFP, PB2-mCherry2, referred here simply as A, B, and C), normalized brightness values close to the values of FP-homodimers were observed in this work. Bookshelf 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. Huet S, Avilov SV, Ferbitz L, Daigle N, Cusack S, Ellenberg J. The .gov means its official. , : , 196006, -, , 22, 2, . Here, we applied fluorescence fluctuation spectroscopy to quantitatively characterize and enhance the MS2 system. We establish fluorescence fluctuation spectroscopy (FFS) with nanoscale detection volumes generated by stimulated emission depletion. 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). values are lower than expected (e.g. Investigation of the underlying causes of neurodegeneration requires the establishment and selection of appropriate methodologies for detailed investigation of the state and conformation of protein aggregates. In this range, SFSCS not only enabled the quantification of protein interactions via cross-correlation analysis, but also yielded correct estimates of protein diffusion dynamics and oligomerization at the PM. 0.4 ms and a 2-line binning was performed. Dunsing V, Chiantia S. SpectralFFS. values (Figure 2figure supplement 1). Fluorescence correlation spectroscopy: novel variations of an established technique. The average D measured for PB1-mEGFP, DPB1-G = 1.7 0.6 m2/s (mean SD, n = 53 cells), was ca. In addition to differences in observation volumes, the amplitudes are also affected by pf (e.g. values were obtained for the polymerase samples, rel.3C. The average laser power used in two-photon excitation is much higher than in the single-photon excitation scheme. Calculating the effective overlapping observation area from the cross-correlation diffusion time (taudG,Ch2=1.38*taudG), the determined FRET efficiencies (see q factors in response to point 3, qG=0.8, qR=1.3) and keeping the pf values (0.7 for both) into account, we calculated an expected rel.cc. In particular, we present scanning fluorescence spectral correlation spectroscopy (SFSCS), combining SFCS and FSCS. Foust DJ, Godin AG, Ustione A, Wiseman PW, Piston DW. An improvement of the allowed relative concentration range can be achieved by using brighter or more photostable fluorophores, for example, organic dyes, compensating for reduced SNR due to statistical filtering. Intermolecular interactions in the PC are hypothesized to be required for the initiation of vRNA synthesis during replication of the viral genome (Fan et al., 2019; Chen et al., 2019). values were calculated for all cross-correlation combinations: where Gi,j(0) is the amplitude of the CCF of species i and j, and Gi(0) the amplitude of the ACF of species i. Fluorescence correlation spectroscopy (FCS) was created to measure these molecular dynamics; it is sensitive to fluctuations in fluorescence intensity observed from a small open volume element (on the order of 10 15 liter) containing only a few molecules.Fluctuations may be due to molecules diffusing in and out of the volume or to chemical transitions between A further interesting example of FFS analysis relevant in the field of cell biology is represented by scanning F(C)CS (SF(C)CS). In analytical chemistry, fluorescence detectors are used with HPLC. In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses. 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. Also, significantly large amplitudes were observed for all six CCFs for the hetero-tetramer sample, albeit with different levels of noise. 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). 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. 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). rel.cc.=0.72 for mEYFP-mEGFP hetero-dimers in 2-species SFSCS, Figure 1). about navigating our updated article layout. The tdMCP-FP allowed uniform RNA labeling and provided quantitative measurements of endogenous mRNA concentration and diffusion. Int J Mol Sci. Image stacks were further processed with a high-pass filter (with a moving four-frame window) to remove slow signal variations and spatial inhomogeneities. Mller BK, Zaychikov E, Bruchle C, Lamb DC. diffusion of FP tetramer about 2.5 times slower than diffusion of monomers, are in good agreement with previous studies (e.g. First in vitro approaches to perform FCS on more than two species exploited quantum dots (Burkhardt et al., 2005) or fluorescent dyes with different Stokes shifts excited with a single laser line in one- (Hwang et al., 2006) or two-photon excitation (Heinze et al., 2004; Ridgeway et al., 2012a), coupled with detection on two or more single photon counting detectors. All data generated or analysed during this study are included in the manuscript and supporting files. 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. SFSCS analysis followed the scanning FCS scheme described previously (Ries and Schwille, 2006; Dunsing and Chiantia, 2018), combined with spectral decomposition of the fluorescence signal by applying the mathematical framework of FLCS and FSCS (Benda et al., 2014; Bhmer et al., 2002). 2017 Feb;23(2):134-141. doi: 10.1261/rna.057786.116. doi: 10.1146/annurev-biochem-060614-033955. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources. To quantify the maximum rel.3C. Our method To remove shot noise contributions, the correlation at zero lag time was omitted from the analysis. RSICS analysis followed the implementation introduced recently (Schrimpf et al., 2018), which is based on applying the mathematical framework of FLCS and FSCS (Benda et al., 2014; Bhmer et al., 2002) to RICS. Since it is a new technique all the limitations of the technique can be discussed in a "limitations" subsection. observed for pairs of green/yellow and red FPs. Epub 2020 Feb 13. Afterwards, RICS spatial ACFs and pair-wise CCFs were calculated for each image stack and all combinations of species i, j (e.g., G and Y, G and Ch2, Y and Ch2 for three species), respectively (Schrimpf et al., 2018; Hendrix et al., 2016): ACF amplitudes were corrected as described in Hendrix et al., 2016 to account for the effect of the high-pass filter. For measuring excitation spectra, the wavelength passing through the emission filter or monochromator is kept constant and the excitation monochromator is scanning. Normalized brightness of coat proteins. They show that the influenza A virus (IAV) matrix protein 2 (M2) interacts more strongly with LC3 compared to CD9, both host cell factors. Data are pooled from four independent experiments. 2022 Nov 4;23(21):13511. doi: 10.3390/ijms232113511. Therefore, we think that the apparent diffusion dynamics of LC3 are difficult to interpret and cannot be used directly to explore M2-LC3 interactions. WebFluorescence correlation spectroscopy analysis. Dunsing V, Luckner M, Zhlke B, Petazzi RA, Herrmann A, Chiantia S. Optimal fluorescent protein tags for quantifying protein oligomerization in living cells. Statistical significance was determined using Welchs corrected two-tailed Students t-test (****p<0.0001, ns: not significant). doi: 10.1073/pnas.2201103119. Also, the emission spectra of these FPs are shifted by less than 20 nm (Figure 1figure supplement 1, spectral filters are shown in Figure 1figure supplement 2B). ( A ) Schematic of the coat protein, Normalized brightness of mRNA. 20 nm apart (Figure 1figure supplement 1). Careers. Fluorescence fluctuation spectroscopy (FFS) approaches provide a powerful toolbox that fulfills this aim (Jameson et al., 2009; Weidemann et al., 2014; Petazzi et al., 2020). Resultingly, only a small percentage of the excitation light reaches the fluorophores that are visible for the detection system. 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