Extended Data Fig. 7: YF mutation alters material properties of AKAP95 condensates.
From: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis
a, OD450 at different time after TEV protease treatment of 50 µM MBP-AKAP95 (101–210) WT, YS, and YF in 150 mM NaCl, as mean ± SD of readings after subtracting that of MBP at each time (constant at 0.07–0.08) from n = 3 independent assays. Dashed lines show half of the maximum turbidity and time (τ1/2) to reach it. b, Ratio of protein concentration inside the droplets over sum of inside and outside for (101–210) WT and YF at increasing protein concentrations and in 30 mM NaCl, as mean ± SD from n = 6 randomly picked droplets each, in one representative assay from 3 repeats based on Fig. 7a. c, Confocal microscopy images of GFP-AKAP95 (101–210) WT and YF at increasing protein concentrations, all in 150 mM NaCl and 10% of PEG 6000 after TEV protease treatment for 20 min. Repeated 2 times with similar results. d, DIC and fluorescence microscopy images for 20 µM MBP-AKAP95 (101–210) YF spiked with Oregon-green-labelled same protein (molar ratio 10:1), after TEV protease treatment for 30 min. Changes in NaCl concentration is indicated. Images were taken 5 min after salt adjustment. Repeated 2 times with similar results. e, Different extent of droplet fusion (arrows) by AKAP95 (101–210) WT and YF, both at 50 µM and in 30 mM NaCl and 10% of PEG 6000 after TEV protease treatment for 30 min. A similar trend was observed in 5 fusion events (or attempted fusion for YF) for each. Also see Supplementary Videos 4–6. f, Equation for Line Raster Scan Image Correlation fitting autocorrelation G(Ψ), which depends on G(0)=γ/N (γ: beam profile, N = number of mobile particles), diffusion coefficient D (µm2/s), line scan time tl, pixel size Ψ, radial beam waist w0 and axial beam waist wz. The radial waist w0 (0.218 µm) was calibrated with sub-diffraction beads (0.1 µm) diluted solution as reported before72. The axial waist wz was considered equal to 3*w0. g, Fluorescence confocal microscope image of HeLa cell expressing full-length AKAP95 WT or YF fused to GFP. Arrow indicates the scanned region for in vivo Line RICS. h, Fluorescence confocal microscope image of GFP-AKAP95 (101–210) WT and YF in 150 mM NaCl and 10% of PEG 6000 after TEV protease treatment for 20 min, for in vitro Line RICS experiments. i, Line Fluorescence carpet formed by ~104 lines (each line is composed by 128 pixels, 50 nm/pixel) acquired with 0.101 s line scan time and 32.8 µs/pixel. Line RICS autocorrelation curves were computed on 64 sections of 128 lines followed by averaging all the curves. The sectioning of the line carpet permitted to avoid the effect of the movement of the condensates on the measurement of the autocorrelation curves. (λex=488 nm). j, Line RICS autocorrelation curves for experimental data and fitted. k, Residuals of the fitting. Experiments in g–i were repeated 3 times with similar results. Scale bar, 2 µm for c and h, 5 µm for d, e, and g.
