Figure 3

ANXA11 Interacts with Both RNA Granules and Lysosomes in Living Cells

(A–D) ANXA11 interact with RNA granules in cells

(A) ANXA11-mEmerald redistributes from the cytoplasm into dispersed puncta immediately following heat shock (43^oC) in U2OS cells. Scale bar: 20 μm. See also Figure S3A.

(B) Heat shock induced ANXA11-mEmerald puncta in U2OS cells are motile and undergo fusion (upper panel), and recover rapidly after photobleaching (i.e., FRAP)(bottom panel). Scale bar: 1 μm. See also Figure S3B.

(C) Quantification of FRAP experiment in (B), n=23. Error bars = SEM. See also Figure S3C.

(D) Immunostaining of mEmerald-tagged ANXA11 with RNA granule markers (Cy3-Oligo dT(30), anti-G3BP1) before, during and 4 hours after heat shock (HS) in U2OS cells. Line scans show the related intensity profiles of ANXA11 with mRNA (Cy3 Oligo-dT) and with G3BP1. Scale bar: 30 μm. See also Figure S3D.

(E) Immunostaining of mEmerald-tagged ANXA11 full-length, N-terminal or C-terminal domain and G3BP1 following 30 minutes of heat shock (43^oC) in U2OS cells. Line scans show the related intensity profiles of ANXA11 with G3BP1. Scale bar: 30 μm. Right panels show the quantification of ANXA11 tructation area overlap with G3BP1(relative to ANXA11 area). One-way ANOVA, ^∗p < 0.05, ^∗∗∗∗p < 0.0001, n=10. Error bars = SEM. Scale bar: 30 μm. See also Figures S3E, S3F.

(F–K) ANXA11 puncta interact with lysosomes in cells

(F) Panel 1: Live cell imaging of U2OS cells expressing ANXA11-mEmerald and LAMP1-HaloTag following heat shock at 43^oC in U2OS cells. Panels 2-4: Enlarged areas of panel 1, with arrows pointing to ANXA11/lysosome contact sites (Scale bar: 1 μm). See also Figure S3H.

(G) Rat cortical neurons expressing LAMP1-HaloTag and ANXA11-mEmerald imaged after heat shock. ANXA11 puncta co-localized with lysosomes in different neuronal regions: soma (1,2), dendrite (3,4) and axon (5). Arrows point to contact sites between lysosomes and ANXA11 puncta. Scale bar: 5 μm

(H) Live imaging of rat cortical neuron axons expressing LAMP1-HaloTag and ANXA11-mEmerald. The corresponding kymograph shows ANXA11 puncta (green) either co-trafficking or co-localizing with lysosomes (magenta). Scale bar: 5 μm. See also Figure S3G.

(I) Immunostaining of mEmerald-tagged ANXA11 full-length, N-terminal or C-terminal domain with LAMP1-positive lysosomes in U2OS cells following 30 minutes of heat shock (43^oC). Line scans show related intensity profiles of ANXA11 and LAMP1. Scale bar: 30 μm. Far right panel shows the quantification of ANXA11 trucations and LAMP1 co-localization (relative to ANXA11 area). One-way ANOVA, ^∗p < 0.05, ^∗∗∗∗p < 0.0001, n=10. Error bars = SEM. Scale bar: 30 μm.

(J) FLIM-FRET analysis of the interaction between ANXA11 and lysosomes and its regulation by lysosomal Ca²⁺ and PI(3,5)P₂. Human i³Neurons were transduced with ANXA11-mCerulean3 (FRET donor) and LAMP-YFP (FRET acceptor). FLIM-FRET images were acquired for the same neurons before and after treatment with ML-SA1, BAPTA-AM or YM201636, and the lifetime of the ANXA11-mCerulean3 signal was determined. Left vertical panels show intensity images of LAMP1-YFP with the various treatments. Middle and right panels show ANAX11-mCeurlean3 lifetimes before and after drug treatment.

(K) Quantification of FLIM-FRET lifetime measurements from (H). N=31 (NT), 24 (ML-SA1), 20 (BAPTA-AM), 24 (YM201636). One-way ANOVA, ^∗p < 0.05. Error bars = SEM.