ZFIN Figure: Liao et al., 2019, Figure 6

Figure 6

ANXA11 Acts as an Adaptor between RNA Granules and Lysosomes

(A) Time-lapse imaging of U2OS cells expressing LAMP1-HaloTag, Opto-ANXA11 and mEmerald-G3BP1 after 488nm light exposure to induce Opto-ANXA11 oligomerization. U2OS cells were exposed to heat shock (43 ^oC) for 15 minutes prior to light activation to form visible G3BP1 stress granules. Stress granules (green) associate with LAMP1-labeled lysosomes (white) at sites where ANXA11 puncta (red) are localized. Scale bar: 1 μm. See also Figure S6A, Video S3.

(B) Live cell confocal imaging of U2OS expressing LAMP1-HaloTag, ANXA11-mEmerald and mCherry-G3BP1 following 30 minutes of heat shock (43^oC). Quantification of the intensity profiles of the different probes across midline of stress granules (dotted line) is shown to right. n=6, Error bars = SEM. Scale bar: 1μm.

(C) Schematic of in vitro RNA granule liposome reconstitution assay.

(D) Stress granule cores were purified from cultured cells, and incubated with PI3P containing liposomes +/– recombinant ANXA11 +/– Ca²⁺. Upper panel: + ANXA11 only, Middle panel: + Ca²⁺only, Bottom panel: + both ANXA11 and Ca²⁺. Scale bar=10 μm.

(E) Quantification of mean intensity of stress granule binding to PI3P containing liposomes in (D). n>300, One-way ANOVA, ^∗∗∗p < 0.001. Error bars = SEM.

(F) Co-localization of ANXA11, lysosomes, and RNA granules in axons. Rat cortical neurons were transduced with LAMP1-HaloTag to label lysosomes, ANXA11-mEmerald to label ANXA11, and mCherry-G3BP1 to label RNA granules. Arrows indicate areas of ANXA11, lysosome and RNA granule co-localization. Scale bar: 5 μm. See also Figure S7A, Video S4, 5.

(G) ANXA11 knockdown perturbs mRNA/lysosome co-trafficking in axons. Kymographs of mRNA (actin-24xMBS/ MCP-NLS-2xEGFP) and lysosome (LAMP1-HaloTag) trafficking in axons is shown. Rat neurons expressed control or ANXA11-targeting shRNAs. Scale bar: 5 μm.

(H) Quantification of (G). n=22-36, t-test, ^∗∗p < 0.01. Error bars = SEM.

(I) smFISH of beta-actin in growth cones from neuron expressing control shRNA (left panel) or ANXA11 shRNA (right panel). Black colored spots represent the signal from beta-actin smFISH probes, red signal represents membrane stain of growth cones. Scale bar: 1 μm.

(J) Quantification of average number of beta-actin mRNA molecules in (I). N=44-96, t-test, ^∗∗p < 0.01. Error bars = SEM.

Expression Data

Expression Detail

Antibody Labeling

Phenotype Data

Phenotype Detail

Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image.

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