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. 2021 Jul 23;12:4493. doi: 10.1038/s41467-021-24713-5

Fig. 7. Lysosome fission and translocation associated to ER – lysosome contact sites at the pre-axonal region.

Fig. 7

a Representative still images of DIV6 neurons co-transfected with Sec61β-GFP (green) and TRIM46-BFP and labeled for lysosomes with SirLyso (magenta) prior to imaging for 120 s every 1.5 s. Lysosome translocation along an ER tubule (top panels); Lysosome fission at contact sites followed by lysosome translocation (middle panels); lysosome fission and co-translocation with an ER tubule (bottom panels). See also Supplementary Movie 11. b Schematic representation of the reversible GB/RA contact assay used to visualize ER – lysosome contacts in live cells. GB and RA modules were tagged to RAB7 and protrudin, respectively. Only proximity of the two proteins enables reconstitution of fluorescent protein at contact sites (magenta). c Representative still images of ER–lysosome contacts (magenta) at pre-axonal region from neurons co-transfected with control pSuper or shRNAs targeting RTN4 plus DP1 or shRNAs targeting P180 together with LAMP-GFP, GB-Rab7, RA-Protrudin, and TRIM46-BFP (blue) or labeled with NF640 (blue) prior imaging. d Representative still images of fusion events from neurons co-transfected and labeled as in (c). e Schematic representation of our proposed model of ER tubules regulating axonal lysosome availability at the pre-axonal region. Scale bars represent 1 µm in (a) and (d) and 5 µm in (c).