Figure 1: The PAM2 switch regulates ATXN2’s behavior in stress and non-stress conditions.

(A) Stress granules form upon cellular stress. (B) ATXN2 is dispensable for arsenite-induced stress granule assembly. Average percentage of cells with stress granules is shown. n = 3 experiments with a total of [255–285] cells. Dashed lines highlight nuclei. EGFP is shown in inverse gray scale (see also Fig. S1A). (C) Phase separation of scaffold proteins and RNA drives stress granule assembly, with subsequent recruitment of non-essential client proteins, such as ATXN2. (D) Domain structure and disorder prediction of ATXN2. Metapredict⁹⁸ score: 1 = disordered, 0 = folded. (E) Deletion of PAM2 prevents homogeneous partitioning of ATXN2 in stress granules. (F) Quantification of the heterogeneity of ATXN2 deletion mutant distribution within the stress granule compartment (see STAR methods). One-way ANOVA. n = 30 stress granules. (G) PAM2 deletion drives spontaneous condensation of ATXN2 into small granules under non-stress conditions (see also Fig. S1B). (H) Wildtype ATXN2 can spontaneously condense upon overexpression. ΔPAM2 ATXN2 condensation is not an overexpression artefact (see also Fig. S2). Scatterplots show cells with diffuse or condensed ATXN2 localization. Average cytoplasmic EGFP intensity. Cells combined from 3 experiments. Mann-Whitney. * p-value < 0.05, **** p-value < 0.0001. Panel (B) shows HeLa cells. All other panels show U2OS cells. Every picture shows endogenous PABPC staining. EGFP-ATXN2 (mutants) are expressed from a plasmid.