Fig. 2: At higher stoichiometric ratios, Epsin1 destabilizes condensation of Eps15. | Nature Communications

Fig. 2: At higher stoichiometric ratios, Epsin1 destabilizes condensation of Eps15.

From: Epsin1 enforces a condensation-dependent checkpoint for ubiquitylated cargo during clathrin-mediated endocytosis

Fig. 2: At higher stoichiometric ratios, Epsin1 destabilizes condensation of Eps15.The alternative text for this image may have been generated using AI.

a Representative images of Eps15 condensates with varying stoichiometry of Epsin1. 16 μM Eps15 was used for all the experiments and Epsin1 was mixed with Eps15 from 8:1 to 1:1 molar ratio. The line profiles depict the intensity profile of green (Eps15) channels along the white dashed line. n = 3 independent replicates. A.U. represents arbitrary units. b The distribution of the Eps15 intensity ratio (Kapp) between the intensity inside the condensates and the solution with varying molar ratio of Epsin1. In total 60–70 condensates were analyzed under each condition. Data are mean ± standard deviation, calculated from n = 3 independent replicates. Statistical significance was tested using an unpaired, two-tailed student’s t test on GraphPad Prism. c Bar graph shows the melting temperature (Tm) of Eps15 and Eps15:Epsin1 mixed condensates, n = 3 independent replicates. The error bar represents ± 1 °C (deviation) of the melting temperature, which was the resolution of the temperature control system. All replicates resulted in the same melting temperature within this error. A.U. represents arbitrary units. d,e Representative images of protein condensates at increasing temperatures. Plots show fluorescence intensity of Eps15 measured along dotted lines in each image. n = 3 independent replicates. Condensates are formed from (d) 16 μM Eps15, (e) 16 μM Eps15 with 8 μM Epsin1. f Eps15 (16 μM) condensates mixed with 16 μM (1:1) of Epsin1ΔNPF, the line profile shows the intensity of Eps15 across the white dashed line. n = 3 independent replicates. A.U. represents arbitrary units. g Shows the cartoon of the Eps15 multimerization through intermolecular interaction between DPF motif and EH domains and how the addition of Epsin1 can destabilize Eps15 condensates. Epsin1 binds to the EH domain of Eps15 and destabilizes the condensates by preventing Eps15 multimerization. All condensate experiments were performed in 20 mM Tris-HCl, 150 mM NaCl, 5 mM TCEP, 1 mM EDTA and 1 mM EGTA at pH 7.5 with PEG8K, at room temperature, 24 °C. All scale bars equal 5 μm.

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