ZFIN Figure: Liao et al., 2019, Figure S4

Figure S4

ALS-Associated Mutations in ANXA11 Disrupt RNA Granule Interactions, Related to Figure 4

(A) A schematic map of ANXA11 protein with the position of ALS-associated mutants.

(B) Quantification show the temporal evolution of the integrated fluorescence intensity from the expressed Opto-mCherry, ANXA11 full-length, NTD or CTD proteins during 300 seconds of light activation, n=11 (Opto-mCherry), 17 (ANXA11 full-length), 20(ANXA11 NTD), 20(ANXA11 CTD). Error bars = SEM.

(C) U2OS were transfected with Opto-mCherry (CRY2olig-mcherry), Opto-ANXA11, Opto-ANXA11 NTD or Opto-ANXA11 CTD for 24hrs. Cells with similar Opto-ANXA11 expression levels were exposed to 0.2% 488nm light to initiate oligomerization. Scale bar: 30 μm.

(D) Co-localization of ANXA11 or ANXA11 ALS-associated mutants with TDP43 and mRNA labeled by Oligo-dT. U2OS cells expressing mEmerald labeled ANXA11 or ANXA11 ALS-associated mutants were heat shocked for 30 mins, fixed, and then hybridized with Cy3-Oligo dT(30) and immunostained with antibodies against TDP43 to label RNA granules. The extent of co-localization of ANXA11 or the ALS-associated mutants with the RNA granules is plotted in the line-scans to the right.

(E) Quantification of percentage of area of ANXA11 structures co-localizing with TDP43-labeled RNA granules in (D). One-way ANOVA, ns, not significant. ^∗∗∗∗p < 0.0001. Error bars = SEM. N=12 (WT), 7 (D40G), 6 (R235Q), 7(R346C).

(F) Co-localization of ANXA11 or ANXA11 ALS-associated mutants with RNA granules labeled by TDP43 and mRNA labeled by Oligo-dT after heat shock (HS). U2OS were heat shocked for 30 mins and then moved to 37^oC for 4 hrs to allow recovery. The cells were then fixed, hybridized with Cy3-Oligo dT(30) followed by immunostaining with antibodies against TDP43 to label RNA granules. Linescan analysis show the related intensity profiles of ANXA11 or ALS-associated mutations with mRNA (Cy3 Oligo-dT) and TDP43. Scale bar: 30 μm.

(G) Co-localization of ANXA11 or ALS-associated ANXA11 mutants with RNA granules labeled by G3BP1 and mRNA (right panel) after heat shock (HS). U2OS were heat shocked for 30 mins and then moved to 37^oC for 4 hrs to allow recovery. The cells were then fixed, hybridized with Cy3-Oligo dT(30) followed by immunostaining with antibodies against G3BP1 (right panel) to label RNA granules. Linescan analysis show the related intensity profiles of ANXA11or ALS-associated ANXA11 mutants with mRNA (Cy3 Oligo-dT) and G3BP1. Scale bar: 30 μm.

(H) U2OS cells expressing mCherry-G3BP1 to label RNA granules were co-transfected with ANXA11-mEmerald, ANXA11(D40G)-mEmerald, ANXA11(R235Q)-mEmerald or ANXA11(R346C)-mEmerald for 24 hrs. Cells were heat shocked (43^oC) for 30 min, A single G3BP1-positive puncta in each of the different transfected cells was photobleached and recovery of fluorescence into the puncta was monitored by time-lapse imaging. Scale bar: 1 μm.

(I) Quantification of H. N=7(WT), 9(D40G), 8(R235Q), 7(R346C). Error Bars=SEM.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

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Reprinted from Cell, 179, Liao, Y.C., Fernandopulle, M.S., Wang, G., Choi, H., Hao, L., Drerup, C.M., Patel, R., Qamar, S., Nixon-Abell, J., Shen, Y., Meadows, W., Vendruscolo, M., Knowles, T.P.J., Nelson, M., Czekalska, M.A., Musteikyte, G., Gachechiladze, M.A., Stephens, C.A., Pasolli, H.A., Forrest, L.R., St George-Hyslop, P., Lippincott-Schwartz, J., Ward, M.E., RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether, 147-164.e20, Copyright (2019) with permission from Elsevier. Full text @ Cell