ZFIN Image: Liao et al., 2019, Figure S3

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Figure Caption

Figure S3

ANXA11 Exhibits Phase Condensate Properties and Interacts with Both RNA Granules and Lysosomes in Living Cells, Related to Figure 3

(A) ANXA11’s amino acid sequence 1-185 was tagged with mEmerald and expressed in U2OS cells. Small ANXA11 positive puncta appeared in cells that had not been heat shocked.

(B) Live cell imaging of puncta from (A) reveal these structures are motile and undergo fusion (upper panel). Upon photobleaching, ANXA11 fluorescence within the puncta quickly returned from free cytoplasmic pools (bottom panel). Scale bar: 1 μm.

(C) Quantification of the FRAP experiment in (B), n=7.

(D) Co-localization of ANXA11 puncta with RNA granule markers before, during and after heat shock (HS). U2OS cells under normal culture conditions (before HS), under heat shock (HS) or 4 hours after heat shock (after HS) were fixed, hybridized with Cy3-Oligo dT(30) followed by immunostaining with antibody against TDP43. Linescans show the related intensity profiles of ANXA11 with mRNA (Cy3 Oligo-dT) and with TDP43. Scale bar: 30 μm.

(E) Co-localization of ANXA11 full-length, N-terminal or C-terminal domain with RNA granules. U2OS cells were fixed after 30 minutes of heat shock (43^oC), followed by immunostaining with antibody against TDP43. Line scans show the related intensity profiles of ANXA11 with TDP43. Scale bar: 30 μm. One-way ANOVA, ^∗∗∗∗p < 0.0001, n=20 (FL), 11(NTD), 19(CTD), Error bars=SEM.

(F) Co-localization of ANXA11 full-length, N-terminal or C-terminal domain with RNA granule. U2OS cells were fixed after 30 minutes of heat shock (43^oC), followed by hybridizing with Cy3-Oligo dT(30). Linescans show the related intensity profiles of ANXA11 with TDP43. Scale bar: 30 μm. One-way ANOVA, ^∗∗∗∗p < 0.0001, n=30 (FL), 30(NTD), 27(CTD), Error bars=SEM.

(G) Rat cortical neurons were transduced with ANXA11-mEmerald. A single labeled ANXA11 puncta was photobleached, then the recovery of fluorescence into the bleached region-of-interest was examined over time. Scale bar: 2 μm.

(H) Time-lapse imaging showing the interaction of ANXA11 puncta (red) with LAMP1-labeled lysosomes (white) in U2OS cells after heat-shock. Scale bar: 1 μm.

(I) Quantification of LAMP1 labeled lysosomes co-localizing with ANXA11(relative to number of lysosomes) in U2OS or rat neuron. N=25(U2OS), 10(neuron).

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