Fig. 5. E-Syt1/Syt7 heterodimer mediated ER fragmentation directly by lysosomal enzyme.

a Domain architecture of Syt7. b Co-IP of E-Syt1 and Syt7. 293 T cells transfected with HA-Esyt1 and indicated plasmids of Syt7 mutants. Syt7 mutant proteins were immunoprecipitated using anti-mCherry antibody and the precipitates were analyzed using anti-HA antibody. c 786 O/OE-SR cells stably expressing GFP-Esyt1 were infected with mCherry or mCherry-tagged mutants with Syt7 by lentivirus, and nucleus stained with Hoechst 33342. Representative confocal images were shown. Scale bar 50 µm. Enlargement images, scale bar 10 µm. d Indicated plasmids of Syt7 mutants were transfected in 786 O/OE-SR cells with or without knockdown of E-Syt1. Lysates of cells were tested by WB. e 786 O/OE-SR cells transfected with Syt7 or empty vector. Representative TEM images showed that in some ER-Lysosome contact sites, ER was fragmented and engulfed by autophagosomes. Red area depicted the lysosome, yellow area indicated autophagosome, blue area indicated ER, light blue area indicated ER fragment. Scale bar 20 µm. Enlargement images, scale bar 4 µm. f 786 O/OE-SR cells transfected with Syt7 or mutated vector were treated with ER Tracker (yellow) and nuclear dye Hoechst 33342 (blue). ER morphology was assessed by confocal microscopy. Scale bar 20 µm. Scale bar 20 µm. g Co-localization of Esyt1 and CTSB in 786 O/OE-SR cells after siRNA mediated knockdown of Syt7. Scale bar 10 µm. Thirty cells were randomly selected for each treatment. The number of binding sites between E-Syt1 and CTSB in each cell was counted and the experiment was repeated 3 times (h) 786 O/OE-SR cells transfected with Syt7 or empty vector were treated with CA-074Me (10 μM) or DMSO for 24 h and then cells were incubated with ER Tracker (yellow) and nuclear dye Hoechst 33342 (blue). ER morphology was assessed by confocal microscopy. Scale bar 20 µm.