Figure 7.

Ddx4^N0-N4 biomolecular condensates are modulated by ion binding and condensate protein charge

(A) FRAP experiment showing the signal originating from Ddx4^YFP1 (in condensates otherwise composed of Ddx4^N1) pre- and post-bleach. Molar ratio of the two proteins is indicated. Scale bar 5 μm.

(B) Mean fits of Ddx4^YFP1 FRAP in the presence of additional NaCl (30 mM) or CaCl₂ (10 mM). Errors indicate SEM.

(C) Images (xz plane) of Ddx4^N1 condensates sitting on siliconized glass coverslips. Addition of Ca²⁺ ions (10 mM), but not Na⁺ ions (30 mM), changed Ddx4^N1 condensate contact angle from >90° to <90°. Contrast is derived from Alexa 488 fluorescence. Scale bar 5 μm.

(D) Contact angles of Ddx4^N0-N4 condensates in the absence and presence of additional multivalent ions (CaCl₂ and MgCl₂ (10 mM), YCl₃ (0.5 mM)), indicated in box inset. Error bars represent SD.

(E) Partitioning of free Cy5 dye (white bars) and Cy5-labeled 24-mer single-stranded RNA (ssRNA) (top), free Alexa 488 dye (middle), and YFP (bottom) into Ddx4^N0-N4 charge series condensates. Error bars represent SD. Same color scheme as in (D).

(F) Ddx4^N0-N4 charge series implemented in the context of Ddx4^YFP (Ddx4^YFP0–YFP4). Triangles (and corresponding construct numbers) indicate the C-terminal residue of the Ddx4 sequence immediately preceding mCitrine. White circle indicates C-terminal peptide tag.

(G) Fluorescence originating from Ddx4^YFP4 and Ddx4^YFP0 in HeLa cell nuclei (A, condensates; B, nucleoplasm). Scale bar, 10 μm.

(H) Stability of Ddx4^YFP0–YFP4 and Ddx4^YFP1 with C-terminal peptide tags. Error bars indicate SD.