Feline Lindhout

VAP–SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function 123 4 respectively. Zooms represent typical boutons. Scale bars: 5 μm (full size) and 2 μm (zoom). B. Quantifications of fluorescence intensity of internalized endogenous Syt at individual presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA, or SCRN1 shRNA with GFP-SCRN1-F402A. N = 2, n = 201–300 boutons. C. Representative time-lapse of cytosolic GCaMP6f upon electric field stimulation (50 APs, 20 Hz) in axons of hippocampal neurons (DIV21) co-expressed with RIM1a-mCherry to visualize presynaptic sites (arrowheads). Scale bar: 5 μm. D. Time-lapses of cytosolic GCaMP6f upon electric field stimulation (50 APs, 20 Hz) in axons of hippocampal neurons (DIV21) co-expressing pSuper control, SCRN1 shRNA, or VAPA/B shRNAs. Scale bar: 5 μm. E. Average normalized response of GCaMP6f fluorescent intensity at presynaptic boutons upon stimulation (50 APs, 20 Hz) in hippocampal neurons (DIV21) co-expressing pSuper empty vector, SCRN1 shRNA, or VAPA/B shRNAs. N = 5–6, n = 15–27. F. Average normalized peak response of GCaMP6f at presynaptic boutons upon stimulation (50 APs, 20 Hz) in hippocampal neurons (DIV21) co-expressing pSuper empty vector, SCRN1 shRNA, or VAPA/B shRNAs. N = 5–6, n = 15–27. G. Representative time-lapse of cytosolic GCaMP6f before (F 0 ) and after (F 0 )) ionomycin treatment at axons of hippocampal neurons (DIV18) co-expressed with mRFP and pSuper empty vector or VAPA/B shRNAs. Arrowheads mark presynaptic boutons. Scale bar: 5 μm. H. Basal GCaMP6f fluorescence (F) normalized to the maximum GCaMP6f fluorescence (F max) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing mRFP with pSuper empty vector or VAPA/B shRNAs. N = 2, n = 47–50. I. Average basal GCaMP6f fluorescence (F 0 ) normalized to the max GCaMP6f fluorescence intensity (F max ) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing mRFP with pSuper empty vector or VAPA/B shRNAs. N = 2, n = 47–50. J. Representative time-lapse of cytosolic R-GECO1 before (F 0 ) and after (F max ) ionomycin treatment at axons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1, or GFP-SCRN1-F402A. Arrowheads mark presynaptic boutons. Scale bar: 5 μm. K. Basal R-GECO1 fluorescence (F) normalized to the maximum R-GECO1 fluorescence (F max ) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1, or GFP-SCRN1-F402A. N = 3, n = 70–89. L. Average basal R-GECO1 fluorescence (F 0 ) normalized to the maximum R-GECO1 fluorescence intensity (F max ) after ionomycin treatment at presynaptic boutons of hippocampal neurons (DIV18) co-expressing GFP, GFP-SCRN1, or GFP-SCRN1-F402A. N = 3, n = 70–89. Data information: Data represent mean ± SEM; n.s.: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, by Mann–Whitney U-test. More specifically, these neurons showed severe discontinuity of ER tubules in axons, and overall less dense and irregular ER structures in axons, dendrites, and soma (Fig 4E, S4H,I). To quantitatively validate the role of VAP-SCRN1 interactions on ER continuity and dynamics, we conducted FRAP experiments on the previously observed characteristic dense ER patches in TagRFP-ER expressing neurons. Consistent with previous indications,