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VAP–SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function 113 4 Figure 1. VAP and VAP-associated protein SCRN1 modulate SV cycling A. Endogenous localization of VAPA or VAPB and Syt in hippocampal neurons (DIV16) expressing GFP-Sec61β. Zooms represent (1) an axonal structure with presynaptic boutons (arrowheads), and (2) a dendritic structure. Scale bars: 10 μm (full size) and 5 μm (zoom). B. Schematic illustration of the Syt antibody uptake assay: live neurons were stimulated with bicuculline and incubated with primary Syt antibodies, and next neurons were fixed and stained with secondary antibodies. C. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co- expressing RFP and pSuper empty vector or VAPA/B shRNAs. Yellow and gray arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 μm (full size) and 2 μm (zoom). D. Quantifications of fluorescence intensity of internalized endogenous Syt at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. N = 2, n = 288–541 boutons. E. Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non-neuronal tissues. Cereb., cerebellum. Hippoc., hippocampus. Spin., spinal. F. Pull-down assay of HEK293T cells co-expressing Myc-VAPA with BioGFP or BioGFP-SCRN1. G. Pull-down assay of HEK293T cells co-expressing Myc-VAPB with BioGFP or BioGFP-SCRN1. H. Scaled representation of SCRN1-associated proteins identified with pull-down assay followed by mass spectrometry analysis of purified BioGFP or BioGFP-SCRN1 from HEK293T cell lysates with adult rat brain extracts. All candidates showed > 10 enrichment of PSM compared to control. I. Endogenous localization of SCRN1 and Syt in cortical neurons (DIV18) expressing GFP. Zoom represents an axon structure with presynaptic boutons (arrowheads). Scale bars: 10 μm (full size) and 5 μm (zoom). J. Representative image of Syt antibody uptake at axons of hippocampal neurons (DIV18) co- expressing RFP and pSuper empty vector, SCRN1 shRNA, or SCRN1 shRNA with GFP-SCRN1. Yellow and gray arrowheads mark presynaptic boutons with and without internalized Syt, respectively. Zooms represent typical boutons. Scale bars: 5 μm (full size) and 2 μm (zoom). K. Quantifications of fluorescence intensity of internalized endogenous Syt at single presynaptic boutons of hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector, SCRN1 shRNA alone, or SCRN1 shRNA with GFP-SCRN1. N = 2, n = 201–300 boutons. Data information: Data represent mean ± SEM; ***P < 0.001, by Mann–Whitney U-test. Source data are available online for this Figure. Exogenous HA-VAPA and HA-VAPB were observed at ER structures throughout neurons and also partially co-localized with presynaptic boutons (Fig S1A). Thus, VAP is abundantly present at ER structures throughout the cell including at presynaptic sites. To test whether VAP could be engaged in regulating synaptic functions, we next investigated whether VAPA and VAPB are engaged in modulating the SV cycle. This was addressed using the Syt antibody uptake assay, which provides a quantifiable read-out of exo- and endocytosis