Feline Lindhout

VAP–SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function 147 4 Supplementary Figure 4. SCRN1 oligomerizes and regulates ER remodeling together with VAP A. Quantifications of reticular localization of expressed VAPB in COS7 cells (%). Cells with non-re- ticular ER structures contained < 30% detecTable ER tubules in cytoplasm. Gray bars represent non-reticular localization accompanied with impaired VAP-SCRN1 interaction. Left: co-expression GFP-SCRN1 with HA-VAPB or HA-VAPB-K87D/M89D (N = 2–3, n = 44–46). Right: co-expression of HA-VAPB with GFP or GFP-SCRN1, GFP-SCRN1-N, GFP-SCRN1-C, GFP-SCRN1-Y40A, GFP- SCRN1-F144A, GFP-SCRN1-F153A, or GFP-SCRN1-F402A (N = 2–3, n = 41–64). B. Quantifications of reticular localization in COS7 cells (%) co-expressing HA-VAPA with GFP or GFP-SCRN1, GFP-SCRN1-N, GFP-SCRN1-C, GFP-SCRN1-Y40A, GFP-SCRN1-F144A, GFP- SCRN1-F153A, or GFP-SCRN1-F402A. Cells with non-reticular ER structures contained < 30% detecTable ER tubules in cytoplasm. Gray bars represent non-reticular localization accompanied with impaired VAP-SCRN1 interaction. N = 2–3, n = 39–62 cells. C. Quantifications of reticular localization of TagRFP-ER co-expressed with GFP, GFP-SCRN1, GFP-SCRN1-N, GFP-SCRN1-C, GFP-SCRN1-F402A, or GFP-SCRN1-C9A in COS7 cells. Gray bars represent non-reticular localization accompanied with impaired VAP-SCRN1 interaction. Cells with non-reticular ER structures contained < 30% detecTable ER tubules in cytoplasm. N = 2–4, n = 58–120. D. Live COS7 cells co-expressing TagRFP-ER with GFP, GFP-SCRN1, or GFP-SCRN1-F402A. Scale bars: 10 μm (full size) and 2 μm (zoom). E. Pull-down assay of HEK293T cells co-expressing HA-SCRN1-N with BioGFP and BioG- FP-SCRN1. F. Pull-down assay of HEK293T cells co-expressing HA-SCRN1-N with BioGFP, BioGFP-SCRN1-N, BioGFP-SCRN1-C, and BioGFP-SCRN1-F402A. G. Time-lapse of COS7 cells co-expressing TagRFP-ER and GFP, GFP-SCRN1, or GFP-SCRN1- F402A. Arrowheads mark ER tubule remodeling event, and arrows mark ER remodeling artifacts. Scale bar: 2 μm. H. ER nanostructures in somatic structures of hippocampal neurons (DIV18) co-expressing GFP- Sec61β with pSuper empty vector, SCRN1 shRNA, or VAPA/B shRNAs, and subjected to expan- sion microscopy. Left panels show maximum intensity projections of all Z-planes. Individual ER tubules and perinuclear sheets are shown in zooms of Z-plane #1 and Z-plane #2, respectively. Scale bars: 5 μm (full size) and 500 nm (zoom). I. ER nanostructures visualized with GFP-Sec61β in dendrites of hippocampal neurons (DIV18) im- munostained for α-tubulin and co-expressed with pSuper empty vector, SCRN1 shRNA, or VAPA/B shRNAs, and subjected to expansion microscopy. Scale bars: 5 μm (full size) and 1 μm (zoom). Data information: *P < 0.05; **P < 0.01; ***P < 0.001, by chi-square test ( A – C ) with post hoc analy- sis including Bonferroni correction ( A , B ). Source data are available online for this Figure.