Figure S6

ANXA11 Acts as an Adaptor between RNA Granules and Lysosomes, Related to Figure 6

(A) Additional example of a time-lapse imaging sequence of U2OS cells expressing LAMP1-HaloTag, Opto-ANXA11 and mEmerald-G3BP1 after 0.2% 488nm light activation to initiate Opto-ANXA11 oligomerization. U2OS cells were exposed to heat shock (43 ^oC) for 15 minutes prior to light activation. Here, G3BP1-labeled RNA granules (green) associate with ANXA11 puncta (red) in the cytoplasm before redistributing onto the surface of LAMP1-labeled lysosomes (white) as merged puncta. Scale bar: 1 μm.

(B) Quantification of the percentage of RNA granules co-localizing with LAMP1 over 300s with or without light activation in (A). N=11. Paired t-test, ^∗∗, p<0.01. Error bars = SEM.

(C) Percentage of co-localization between ANXA11 and LAMP1, G3BP1 and LAMP1, G3BP1 and ANXA11. N=20. Error bars = SEM.

(D) Q-PCR shows relative ANXA11 mRNA level in control shRNA or ANXA11 shRNA transduced neurons. N=3. Error bars = SEM.

(E) Frequency of LAMP1-labeled vesicles in axons expressed control shRNA or ANXA11 shRNA. N=36(sh control), 35 (sh ANXA11), t-test, ns, not significant. Error bars = SEM.

(F) Effect of ALS-associated ANXA11 mutants on RNA granule-lysosome association in heat shocked cells. U2OS cells were transfected with mEmerald tagged ANXA11 or ANXA11 ALS-related mutant constructs. Cells were heat shocked (43^oC) for 30 minutes, fixed, followed by immunostaining with antibodies against G3BP1 and LAMP1 to examine the effects of ANXA11 mutants on RNA granule-lysosome contacts compared to those in WT ANXA11-mEmerald expressing cells. Arrows point to G3BP1-labeled RNA granule(megenta) contacting with lysosomes(red). Scale bar: 30 μm.

(G) Percentage of G3BP1-labeled granules co-localized with LAMP1-labeled lysosomes in (E). n= 20 (WT), 25 (D40G), 28 (R235Q), 28 (R346C). One-way ANOVA, ns, not significant. ^∗∗∗∗p < 0.0001. Error bars = SEM.

(H) U2OS cells were transfected with mEmerald tagged ANXA11 or ANXA11 ALS-related mutant constructs. Cells were heat shocked (43^oC) for 30 minutes, fixed, hybridized with Cy3-Oligo dT(30) followed by immunostaining with antibodies against LAMP1 to examine the effects of ANXA11 mutants on RNA granule-lysosome contacts compared to that in WT ANXA11-mEmerald expressing cells. Graphs on the right represent intensity profiles across the dotted line, revealing the ANXA11 mutants R235Q and R346C show decreased colocalization with RNA granules and lysosomes and affected RNA granule-lysosome contact. Scale bar: 1μm.