Figure 4

Figure 4

Effects of ALS-Associated ANXA11 Mutations on RNA Granule Interactions

(A) U2OS cells expressing mEmerald-tagged ANXA11 (WT, D40G, R235Q or R346C) were heat shocked (43^oC) for 30 minutes. A single ANXA11-positive puncta in each of the different transfected cells was photobleached and recovery of fluorescence was monitored by time-lapse imaging. Scale bar: 1 μm.

(B) Quantification of the FRAP experiments in (A), n=21. Error bars = SEM.

(C) Phase partitioning characteristics of ANXA11 ALS-associated mutants in vitro. Purified WT, D40G, R346C forms of ANXA11 were temperature transitioned between 4^oC and 25^oC through multiple cycles. ANXA11(p.D40G) formed both large spherical droplets and smaller, non-fusing gelled condensates, with the condensates unable to reform after one round of temperature shift (middle panel). ANXA11(p.R346C) formed irregularly-shaped solid and spiculated gelled condensates with few, if any, liquid droplets capable of disassembly/reassembly during temperature shifts (bottom panel). Scale bar: 5 μm.

(D) U2OS cells expressing similar levels of Opto-mCherry (CRY2olig-mCherry), Opto-ANXA11 or Opto-ANXA11 ALS-associated mutant were exposed to 0.2% 488nm light to initiate oligomerization. Scale bar: 30 μm. See also Figure S4B.

(E) Quantification of integrated fluorescence intensity of Opto-labeled proteins in (D) during 300 seconds of 488 nm light activation, n=20. Error bars = SEM. See also Figure S4C.

(F) Quantification of the number of Opto-labeled puncta present 30 minutes after the 488 nm light was turned off. n=17-19. One-way ANOVA, ns, not significant. ^∗∗∗∗p < 0.0001. Error bars= SEM.

(G) Immunostaining of mEmerald-tagged wild-type and mutant ANXA11 with G3BP1 and mRNA labeled by Oligo-dT in U2OS cells following 30 minutes of heat shock. Co-localization of ANXA11 with individual RNA granules is plotted in the line scans to the right. Scale bar: 30 μm. See also Figure S4D.

(H) Quantification of area of ANXA11 structures co-localizing with G3BP1-labeled RNA granules in (G). n=28-31. One-way ANOVA, ns, not significant. ^∗∗∗∗p < 0.0001. Error bars = SEM.