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[Preprint]. 2023 Sep 19:2023.09.18.558356. [Version 1] doi: 10.1101/2023.09.18.558356

Figure 2: Fusing de novo designed JDMs with substrate binding domains dissolves intracellular condensates.

Figure 2:

a, Schematic of strategy to dissolve RIα condensates. Protein that drives condensation (here, RIα) is tethered with mCherry. JDM37 is fused to an mCherry nanobody and mT-Sapphire, and this tool is called condensate perturbator. b, Expression of engineered condensate perturbators dissolves mCherry-tagged RIα puncta in HEK293T cells (see Methods for details). Top: Representative epifluorescence images of the various conditions tested. Scale bar = 10μm. Bottom: Quantification of number of RIα puncta per cell. Each point represents a single cell (n=20 cells). c-d, Time-course imaging of HEK293T cells expressing mCherry-tagged RIα, either cAMP sensor ICUE3 (c) or PKA sensor AKAR4 (d), and either JDM37-based or JDM16-based condensate perturbators. In each condition, 10nM isoproterenol was added. Solid lines indicate representative average time with error bars representing standard error mean (SEM) (n=at least 15 cells per curve). e, Schematic of strategy to dissolve EML4-Alk oncogenic condensates. Protein that drives condensation (here, EML4-Alk) is tethered with mCherry. f, Expression of engineered condensate perturbators dissolves mCherry-tagged EML4-Alk puncta in Beas2B cells (see Methods for details). Top: Representative epifluorescence images of the various conditions tested. Scale bar = 10μm. Bottom: Quantification of number of EML4-Alk puncta per cell. Each point represents a single cell (n=20 cells). g, Raw FRET ratios of HEK293T cells expressing mCherry-tagged RIα, Ras sensor Ras-LOCKR-S, and either JDM37-based or JDM16-based condensate perturbators. Each point represents a single cell (n=18 cells). h, Cell growth curves of Beas2B cells expressing mCherry-tagged EML4-Alk with or without condensate perturbators (n=3 experiments). Line represents average from all 3 experiments. For the quantification of number of puncta per cell in b and f, cells only with sufficient expression of the condensate perturbator were chosen for analysis (see Methods for details).