Bergsma T et al. (2024), Imaging-based quantitative assessment of biomol...

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J Biol Chem 2024 Feb 24;3012:108130. doi: 10.1016/j.jbc.2024.108130.

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Imaging-based quantitative assessment of biomolecular condensates in vitro and in cells.

Bergsma T , Steen A , Kamenz JL , Otto TA , Gallardo P , Veenhoff LM .

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The formation of biomolecular condensates contributes to intracellular compartmentalization, and plays an important role in many cellular processes. The characterization of condensates is however challenging, requiring advanced biophysical or biochemical methods that are often less suitable for in vivo studies. A particular need for easily accessible yet thorough methods that enable the characterization of condensates across different experimental systems thus remains. To address this, we present PhaseMetrics, a semi-automated FIJI-based image analysis pipeline tailored for quantifying particle properties from microscopy data. Tested using the FG-domain of yeast nucleoporin Nup100, PhaseMetrics accurately assesses particle properties across diverse experimental setups, including particles formed in vitro in chemically defined buffers or in Xenopus egg extracts, and in cellular systems. Comparing the results with biochemical assays, we conclude that PhaseMetrics reliably detects changes induced by various conditions, including the presence of polyethylene glycol, 1,6-hexanediol, or a salt gradient, as well as the activity of the molecular chaperone DNAJB6b and the protein disaggregase Hsp104. Given the flexibility in its analysis parameters, the pipeline should also be applicable to other condensate-forming systems and we show it application for detecting TDP-43 particles. By enabling the accurate representation of the variability within the population and the detection of subtle changes at the single-condensate level, the method complements conventional biochemical assays. Combined, PhaseMetrics is an easily accessible, customizable pipeline that enables imaging-based quantitative assessment of biomolecular condensates in vitro and in cells, providing a valuable addition to the current toolbox.

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Species referenced: Xenopus laevis

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	Figure 1. Quantitative characterization of the chemical properties of Nup100FG particles formed in vitro.A, representative images of fluorescein-5-maleimide-labeled Nup100FG (Nup100FG-5 MF) particles formed in the absence or presence of 10% PEG3350 (1h) (glass-slide setup; see Experimental procedures). The images are selected using the predesigned excel sheet for the selection of the particle that is most representative for the overall particle population, based on the analysis parameters quantified in (B-F). B-F, mean fluorescence intensity, intensity skewness, size, perimeter and circularity of Nup100FG-5 MF particles exemplified in (A). Graphs show median ± interquartile range of 300 particles per condition (n = 3). 100 particles were analyzed for each independent replicate. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. G,H, background fluorescence intensity and partition coefficient of Nup100FG-5 MF protein mixtures (droplet-in-chamber setup; particles exemplified in Fig. S1D). I, representative images of Nup100FG-5 MF particles, formed in the presence of 10% PEG3350 (1h) (droplet-in-chamber setup), in the absence or upon exposure to 5% 1,6-HD, for either 10 min after an hour of particle formation (t10 m), or immediately from the start (t0). J-L, mean fluorescence intensity, size and circularity of Nup100FG-5 MF particles exemplified in (I). Graphs show median ± interquartile range of ≥750 particles per condition (n = 2). M and N, background fluorescence intensity and partition coefficient of Nup100FG-5 MF protein mixtures exemplified in (I). Graph shows median ± interquartile range of 19 images per condition (n = 2). ∗∗p < 0.01, ∗∗∗∗p < 0.0001.
	Figure 2. Quantitative assessment of Nup100FG particles using imaging and sedimentation assays.A, graphical summary of experimental setup and representative images of Nup100FG-5 MF particles formed by mixing preformed particles in indicated PEG3550 conditions (glass-slide setup). In control samples Nup100FG-5 MF was kept in a constant concentration of 0%, 0,25%, 2,5%, 5% or 10% PEG3550 for 1 h. P: pellet fraction, SN: supernatant fraction. B-F, relative mean fluorescence intensity, intensity skewness, size, perimeter and circularity of Nup100FG-5 MF particles exemplified in (A) (B–E, relative to median control at 0% PEG3350). Graphs show median ± interquartile range of 350 particles per condition (n = 4). ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. G, relative background fluorescence intensity of supernatant fraction after centrifugation of Nup100FG-5 MF protein mixtures (relative to median control at 0% PEG3350) (droplet-in-chamber setup) (n = 3). H and I, sedimentation assay to assess the supernatant and pellet fractions from SDS-PAGE Gels. Band intensities of pellet (H) and supernatant (I) are quantified relative to the average control at 0% PEG3350. Mean ± SEM (n = 4). ∗∗∗∗p < 0.0001. J, supernatant fractions of control samples with 0%, 0,25%, 2,5%, 5% and 10% PEG3350 after centrifugation 10 min 13,000 rpm. (droplet-in-chamber setup). K, comparison between the supernatant fractions quantified by SDS-PAGE (from I) and quantified by imaging (total fluorescence intensity - summed particles and background - relative to median control at 0% PEG3350) exemplified in (Fig. S2D). The bold lines plus shades show the median ± 95% CI from three replicates. Illustrations in A created in BioRender. Bergsma, T. (2025) https://BioRender.com/ j44i528.
	Figure 3. Quantitative assessment of salt effects on Nup100FG particles formed in vitro in absence of a crowding agent.A, representative images of Nup100FG-5 MF particles, formed in the absence of a crowding agent, in response to varying salt concentrations (1h) (glass-slide setup). Insets correspond to images with 2x enhanced brightness, to highlight low-intensity particles. B-F, mean fluorescence intensity, intensity skewness, size, perimeter and circularity of Nup100FG-5 MF particles exemplified in (A). All graphs show median ± interquartile range of ≥225 particles per condition (0, 150, 500 mM, 1-3M (n = 3), 200mM-400 mM (n = 2)). ≥75 particles were analyzed for each independent replicate. The means of each condition were compared to the mean of the 0 mM condition. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 ∗∗∗∗p < 0.0001.
	Figure 4. Quantitative assessment of salt effects on Nup100FG particles formed in vitro in the presence of a crowding agent.A, representative images of Nup100FG-5 MF particles, formed in the presence of 10% PEG3350, in response to varying salt concentrations (1h) (glass-slide setup). Insets correspond to images with 2× enhanced brightness, to highlight low-intensity particles. B-G, mean fluorescence intensity, intensity skewness, size, perimeter, circularity, and frequency distribution of the circularity of Nup100FG-5 MF particles exemplified in (A). All graphs show median ± interquartile range of ≥250 particles per condition (n = 3). ≥75 particles were analyzed for each independent replicate. The means of each condition were compared to the mean of the 0 mM condition. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p =< 0.0001. H-J, overlapping intensity, size and circularity distributions between particles observed in absence and presence of 10% PEG3350 in the low- and high-salt regimes. In all graphs, the bold lines plus shades show the median and ± 95% CI of all measurements from three replicates.
	Figure 5. Quantitative assessment of changing Nup100FG particle properties in vitro in the presence of a phase state modulator.A, representative images showing changing properties of Nup100FG-5 MF particles, formed in the presence of 10% PEG3350, over prolonged timeframe in the absence and presence of DNAJB6b (glass-slide setup). B-D, mean fluorescence intensity, size and circularity of Nup100FG-5 MF particles exemplified in (A), relative to the mean of the control at t0. The bold lines plus shades show the median ± 95% CI of all measurements from three replicates. 100 particles were analyzed for each time point for each of the independent replicate experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. E, comparison between our previously published FTA dataset (52) (relative signal intensity to mean control at t15) and relative mean fluorescence intensity of Nup100FG-5 MF particles (relative to mean control at t0) formed in the presence of DNAJB6b exemplified in (A). The bold lines plus shades show the median ± 95% CI from three replicates. F, representative images showing Nup100FG-5 MF particles in the absence or presence of DNAJB6b-AF594 (1h) (molar ratio 1:1) (glass-slide setup). G, mean fluorescence intensity outside the particles for Nup100FG-5 MF ± DNAJB6b protein mixtures exemplified in Fig. S4A (droplet-in-chamber setup). Graph shows median ± interquartile range of 18 images per condition (n = 3). ∗p=<0.05. H-J, mean fluorescence intensity, size and circularity of Nup100FG-5 MF particles exemplified in (F). Mixed: Nup100FG particles that are colocalized with a DNAJB6b particle, Nup100FG-only: Nup100FG particles that are not colocalized with a DNAJB6b particle. Graphs show median ± interquartile range of 300 particles per condition (n = 3). 100 particles were analyzed for each independent replicate. ∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. K, comparison of mean fluorescence intensity in intersected and no-overlap regions of Nup100FG-5 MF + DNAJB6b-AF594 particles. Graph shows the relative intensity in the intersect over no-overlap regions for a total of 150 particles (n = 3; 50 particles were analyzed for each independent replicate). The individual particles were colored according to their relative intensity values as indicated in the heat map, in which blue reflects a decrease and pink reflects an increase in intensity within the intersected region as compared to the no-overlap region. Different symbols were used to highlight the individual datapoints belonging to each of the independent replicates.
	Figure 6. Quantitative assessment of changing Nup100FG particle properties in Xenopus egg extracts over time.A, representative images of Nup100FG-5 MF particles in Xenopus egg extracts (XEE), analyzed at the indicated time points. Insets represent Zoom-in panels illustrating the most-representative particle for each time point. B-F, mean fluorescence intensity, skewness, size, perimeter and circularity of Nup100FG-5 MF particles exemplified in (A). Graphs show median ± interquartile range of 300 particles per condition (n = 3). The means of each timepoint were compared to the mean of the t0 timepoint. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. G, comparison of time-dependent changes in circularity of Nup100FG-5 MF particles exemplified in (A) (XEE) and Fig. 4A (in vitro, formed in the presence of 10% PEG3350). The bold lines plus shades show the median and ± 95% CI of all measurements from three replicates.
	Figure 7. Quantitative assessment of changing Nup100FG particle properties in yeast cells over time.A, representative images of eGFP-Nup100FG particles in yeast cells, analyzed at the indicated time points. Cells are outlined with colored circles aligning with the different subcategories as specified in (B). B and C, distributions of eGFP-Nup100FG particle subpopulations per cell, using (B) manual and (C) plugin-based quantification. AB: amyloid-like body, AA: amorphous aggregate. Graphs B-C show mean ± SEM of ≥450 cells per condition (n = 3). 200 to 400 particles were counted for each independent replicate. D-G, mean fluorescence intensity, size, perimeter and circularity of eGFP-Nup100FG particles exemplified in (A). Graphs D-G show median ± interquartile range of ≥750 particles per condition (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.
	Figure 8. Quantitative assessment of changing Nup100FG particle properties in yeast cells in the presence of a phase state modulator. A, representative images of eGFP-Nup100FG particles in yeast cells in absence (mCherry as negative control) or presence of mCherry-DNAJB6b. B and C, distributions of eGFP-Nup100FG particle subpopulations per cell, using (B) manual and (C) plugin-based quantification. AB: amyloid-like body, AA: amorphous aggregate. Graphs B and C show mean ± SEM of ≥500 cells per condition (n = 3). 250 to 350 particles were counted for each independent replicate. D-G, mean fluorescence intensity, size, perimeter and circularity of eGFP-Nup100FG particles exemplified in (A). ∗p < 0.05, ∗∗∗∗p < 0.0001. H, Mean intensity of eGFP-Nup100FG soluble fraction. Graphs D-H show median ± interquartile range of ≥800 particles per condition (n = 3). ∗∗∗p < 0.001. I, sedimentation assay to assess the supernatant and pellet fraction of eGFP-Nup100FG in the absence or presence of DNAJB6b. T: total protein, P: pellet fraction, SN: supernatant fraction. The positions of molecular weight markers (in kilodaltons (kDa)) are indicated. J, quantification of the pellet fraction of eGFP-Nup100FG. Represented band intensities are relative to the average intensity of the control. Mean ± SEM (n = 5). ∗p =< 0.05. K, filter trap assay to assess aggregated fraction of eGFP-Nup100FG in the absence or presence of DNAJB6b. L, quantification of the band intensities of eGFP-Nup100FG on filter trap. Represented band intensities are relative to the average intensity of the control. Mean ± SEM (n = 5). ∗∗∗p < 0.001.
	Figure 9. Quantitative assessment of altered Nup100FG particle properties in yeast cells in a ΔHsp104 background.A, representative images of eGFP-Nup100FG particles in a WT and ΔHsp104 background. B, distributions of eGFP-Nup100FG particle subpopulations per cell. Graph shows mean ± SEM of ≥1000 cells per condition (n = 3). 375 to 600 particles were counted for each independent replicate. C-G, mean fluorescence particle intensity, soluble intensity, size, perimeter and circularity of eGFP-Nup100FG particles exemplified in (A). Graphs show median ± interquartile range of ≥1500 particles per condition (n = 3). ∗∗∗p < 0,001, ∗∗∗∗p < 0.0001. H, frequency distribution of the circularity of GFP-Nup100FG particles.
	Figure 10. Overview of different types of particles and imaging setups to study particle formation.A and B, imaging setups to study particle formation. A, in the glass-slide setup, proteins are left to phase separate for a pre-defined period of time in low-protein binding tubes and mounted on an untreated glass-slide with coverslip right before imaging. B, in the droplet-in-chamber setup, proteins are directly pipetted onto the surface of a chambered coverslip and resuspended in a droplet of assay buffer, followed by microscopy analysis. Illustrations created in BioRender. Bergsma, T. (2025) https://BioRender.com/ t52y602.
	Figure 11. PhaseMetrics image analysis pipeline for quantitative assessment of biomolecular condensates. Plugin design with single channel module, and multi-channel module with object-based colocalization functionality. The flowchart describes the sequence of steps performed by the pipeline to extract multiple parameters from fluorescence microscopy images and allows for the analysis of biomolecular condensate properties. The yellow dashed box represents the bounding rectangle, red stars represent the centroids.