Fig. 4: LNPK-enriched ER junctions recruit lysosomes and boost translation.
From: Secretome translation shaped by lysosomes and lunapark-marked ER junctions
a, Schematic of PLA. Anti-LNPK antibody (via oligo-labelled anti-rabbit, blue) and organelle marker antibodies (via oligo-labelled anti-mouse, green) generated proximity signals amplified by rolling circle amplification and visualized with a fluorescent probe (orange). b, Representative images from PLA experiment, showing DNA, PLA puncta (orange) and merged views of LNPK antibody with EEA1 (early endosome), LAMP1 (lysosome) or TOMM20 (mitochondria). Scale bars, 5 µm. c, Quantification of PLA spots per cell for LNPK with EEA1, LAMP1 and TOMM20, and for LAMP1 with REEP5 (control). Data from 10 fields of view, 3 replicates. Statistical comparisons versus LNPK–LAMP1. Dunnett’s test, ****P < 0.0001. d, Schematic of the optogenetic ER–lysosome recruitment tool comprising iLID–Halo–LAMP1 (lysosome) and sspB–mCherry–Sec61β (ER). mCh, mCherry. e, Representative images of iLID–Halo–LAMP1 (magenta) and sspB–mCherry–Sec61β (green) during 488 nm activation in control and LNPK-KO cells. Scale bars, 1 µm. f, Quantification of ER signal within lysosomal mask over time following activation. Control cells: t1/2 = 7.9 s; LNPK-KO cells: t1/2 = 26 s. Shaded areas represent 95% confidence intervals. Orange dashed line indicates a normalized intensity of 1. g, Representative images of SunTag, LAMP1, cytERM-SunTag–MS2 (mRNA) and merged views. Arrows mark translating mRNAs. Scale bar, 1 µm. h, Top, relative SunTag intensity versus distance to lysosomes (binned at 500 nm) for cytERM–SunTag–MS2 in control and LNPK-KD cells. Normalized to intensity at 4.25 µm. Shaded areas represent 95% confidence intervals. Orange dashed line indicates a relative SunTag intensity of 1. Multiple unpaired t-tests were done, ****P < 0.0001, **P < 0.01. Bottom, schematic showing how lysosome proximity increases SunTag intensity, reflecting higher ribosome occupancy.
