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. 2022 Mar 16;221(5):e202112024. doi: 10.1083/jcb.202112024

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© 2022 Wang et al.

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Figure 3. — The material properties of TFEB, TFEB(bHLH-B8A), and PGL-3 droplets. (A) Schematic for the measurement of the interfacial tension of droplets. Negative pressure is used to draw out the surface of a droplet. The function for calculating the interfacial tension is shown underneath. Droplets with a diameter of ∼5–10 μm diluted in pre-prepared saturated protein solutions were used for the experiments. (B and C) Representative pictures of the micropipette experiments for TFEB (B) and TFEB(bHLH-B8A) (C) droplets formed in 500 mM NaCl buffer. 1,764 and 196 Pa of negative pressure was used to draw out the surface of TFEB and TFEB(bHLH-B8A) droplets, respectively. The direction of suction is indicated by arrows. The calculated interfacial tension is shown underneath as mean ± SEM (n = 11 and 10 for TFEB and TFEB(bHLH-B8A) droplets, respectively). (D) Schematic for the single-AuNR tracking experiments. The AuNRs are packaged into the droplets during LLPS induction. The trajectories of the translational and rotational Brownian diffusion of the AuNRs are recorded and analyzed to calculate the viscosity both on the surface and inside the droplets. (E) Trajectories of translational movements of a single AuNR in the interior and on the surface of the same TFEB droplet formed in 500 mM NaCl buffer. The AuNR moves more freely in the interior than on the surface of the droplet. (F–H) Autocorrelation decay curves (solid lines) reflect the rotational behavior of AuNRs in the droplets. The calculated apparent viscosity, which is corrected with the viscosity of the solution, is shown at the top. Rotational tracks of the AuNRs in the interior and on the surface were obtained from the same droplets for TFEB (F) and TFEB(bHLH-B8A) (G) formed in 500 mM NaCl buffer, and PGL-3 droplets (H) formed in 150 mM NaCl buffer. (I) Schematic for the AFM experiments. A droplet (blue hemisphere) adsorbed onto a glass coverslip is tapped by an elastic probe with a known spring constant. The cantilever of the probe bends in response to the force when the probe taps the droplet, and the degree of bending is detected by a laser beam and converted to digital signals. Droplets with a diameter of ∼5–10 μm were used for the experiments. (J–L) The force–indentation curves measured on TFEB (J) and TFEB(bHLH-B8A) (K) droplets formed in 500 mM NaCl buffer and PGL-3 droplets formed in 150 mM NaCl buffer (L) with the approach and retraction curves as solid lines. The cyan dashed lines show the fitting of the Hertz model to the approach curves. (M–O) The distribution of the measured elastic modulus E of TFEB (M) and TFEB(bHLH-B8A) (N) droplets formed in 500 mM NaCl buffer and PGL-3 droplets formed in 150 mM NaCl buffer (O). The calculated elastic moduli are E = 11.8 ± 4.8, 4.0 ± 1.9, and 1.6 ± 0.4 kPa for TFEB, TFEB(bHLH-B8A), and PGL-3 droplets, respectively. Data are shown as mean ± SEM (n = 21, 25, and 20 for TFEB, TFEB(bHLH-B8A), and PGL-3 droplets, respectively).