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• W2969293681 abstract "Article23 August 2019Open Access Source DataTransparent process VAP-SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function Feline W Lindhout Feline W Lindhout orcid.org/0000-0001-5075-5434 Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Yujie Cao Yujie Cao Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Josta T Kevenaar Josta T Kevenaar Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Anna Bodzęta Anna Bodzęta Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Riccardo Stucchi Riccardo Stucchi Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Maria M Boumpoutsari Maria M Boumpoutsari orcid.org/0000-0002-0349-9164 Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Eugene A Katrukha Eugene A Katrukha Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Maarten Altelaar Maarten Altelaar orcid.org/0000-0001-5093-5945 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Harold D MacGillavry Harold D MacGillavry Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Casper C Hoogenraad Corresponding Author Casper C Hoogenraad [email protected] orcid.org/0000-0002-2666-0758 Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Department of Neuroscience, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Feline W Lindhout Feline W Lindhout orcid.org/0000-0001-5075-5434 Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Yujie Cao Yujie Cao Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Josta T Kevenaar Josta T Kevenaar Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Anna Bodzęta Anna Bodzęta Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Riccardo Stucchi Riccardo Stucchi Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Maria M Boumpoutsari Maria M Boumpoutsari orcid.org/0000-0002-0349-9164 Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Eugene A Katrukha Eugene A Katrukha Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Maarten Altelaar Maarten Altelaar orcid.org/0000-0001-5093-5945 Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Harold D MacGillavry Harold D MacGillavry Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Casper C Hoogenraad Corresponding Author Casper C Hoogenraad [email protected] orcid.org/0000-0002-2666-0758 Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands Department of Neuroscience, Genentech, Inc., South San Francisco, CA, USA Search for more papers by this author Author Information Feline W Lindhout1, Yujie Cao1,‡, Josta T Kevenaar1,‡, Anna Bodzęta1,‡, Riccardo Stucchi1,2, Maria M Boumpoutsari1, Eugene A Katrukha1, Maarten Altelaar2, Harold D MacGillavry1 and Casper C Hoogenraad *,1,3 1Department of Biology, Cell Biology, Utrecht University, Utrecht, The Netherlands 2Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands 3Department of Neuroscience, Genentech, Inc., South San Francisco, CA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +31 030 2533894; Fax: +31 030 2513655; E-mail: [email protected] The EMBO Journal (2019)38:e101345https://doi.org/10.15252/embj.2018101345 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In neurons, the continuous and dynamic endoplasmic reticulum (ER) network extends throughout the axon, and its dysfunction causes various axonopathies. However, it remains largely unknown how ER integrity and remodeling modulate presynaptic function in mammalian neurons. Here, we demonstrated that ER membrane receptors VAPA and VAPB are involved in modulating the synaptic vesicle (SV) cycle. VAP interacts with secernin-1 (SCRN1) at the ER membrane via a single FFAT-like motif. Similar to VAP, loss of SCRN1 or SCRN1-VAP interactions resulted in impaired SV cycling. Consistently, SCRN1 or VAP depletion was accompanied by decreased action potential-evoked Ca2+ responses. Additionally, we found that VAP-SCRN1 interactions play an important role in maintaining ER continuity and dynamics, as well as presynaptic Ca2+ homeostasis. Based on these findings, we propose a model where the ER-localized VAP-SCRN1 interactions provide a novel control mechanism to tune ER remodeling and thereby modulate Ca2+ dynamics and SV cycling at presynaptic sites. These data provide new insights into the molecular mechanisms controlling ER structure and dynamics, and highlight the relevance of ER function for SV cycling. Synopsis This study describes a novel control mechanism, mediated by VAP and SCRN1, to tune ER integrity and remodeling. The ER-localized VAP-SCRN1 interactions are required to preserve Ca2+ homeostasis and synaptic vesicle cycling at presynaptic sites. VAP and VAP-associated protein SCRN1 are engaged in modulating SV cycling. VAPA and VAPB interact with SCRN1 at the ER membrane through a single FFAT-like motif. VAP or SCRN1 depletion reduces action-potential evoked Ca2+ responses. VAP-SCRN1 interactions modulate ER continuity and dynamics, presynaptic Ca2+ homeostasis, and SV cycling. Introduction The continuous and dynamic ER network is one of the most abundant organelles in cells. In neurons, somatodendritic domains contain both rough and smooth ER, whereas axons exclusively exhibit smooth ER. The smooth ER lacks ribosomes and is not involved in translation; instead, it is important for Ca2+ homeostasis, lipid synthesis and delivery, and signaling. The relevance of axonal ER in particular is highlighted by various human axonopathies caused by mutations in different generic ER proteins. More specifically, dysfunction of ER-shaping proteins such as atlastin-1, reticulon-2, receptor expression-enhancing protein 1 (REEP1), and receptor expression-enhancing protein 2 (REEP2) leads to hereditary spastic paraplegia (HSP), whereas mutations in ER receptor VAMP-associated protein B (VAPB) cause amyotrophic lateral sclerosis (ALS; Hazan et al, 1999; Zhao et al, 2001; Nishimura et al, 2004; Zuchner et al, 2006; Montenegro et al, 2012; Esteves et al, 2014; Yalcin et al, 2017). Together, these pathologies hint for an increased sensitivity for proper ER structure and function in axons. Recent ultrastructural three-dimensional analysis revealed that the ER in axons is comprised of a conserved and unique organization (Wu et al, 2017; Yalcin et al, 2017; Terasaki, 2018). The axonal ER structure consists of narrow ER tubules, which occasionally form cisternae at tubular branch points with comparably small lumen (Wu et al, 2017; Yalcin et al, 2017; Terasaki, 2018). This distinctive ER network extends throughout all axon branches with a relative constant density of only 1–2 narrow tubules per diameter, while remaining continuous with the rest of the ER network (Wu et al, 2017; Yalcin et al, 2017; Terasaki, 2018). At presynaptic terminals, the ER forms a local tubular network opposing the active zone. This presynaptic ER structure often wraps around mitochondria and is in close proximity to the plasma membrane, and it regularly forms tight membrane contact sites with these structures (Wu et al, 2017; Yalcin et al, 2017). Moreover, fast dynamics of axonal ER was observed in Drosophila neurons using fluorescent recovery after photo-bleaching (FRAP) analysis, suggesting that the neuronal ER network likely undergoes dynamic remodeling (Wang et al, 2016; Yalcin et al, 2017). However, the precise role of the dynamic ER network in axons and at presynaptic sites remains poorly understood. Emerging evidence implies that the presynaptic ER is engaged in modulating the tightly controlled Ca2+-induced SV cycle (Summerville et al, 2016; De Gregorio et al, 2017; de Juan-Sanz et al, 2017). In Drosophila neurons, it was reported that homologues of the HSP-associated ER-shaping proteins atlastin-1 and reticulon-1 are implicated in controlling neurotransmitter release at neuromuscular junctions, as loss of these proteins resulted in a marked decrease in SV cycling (Summerville et al, 2016; De Gregorio et al, 2017). In mammalian neurons, recent reports showed that presynaptic Ca2+ levels in the ER are locally elevated during evoked neuronal transmission, suggesting that the presynaptic ER buffers Ca2+ to modulate SV cycling (de Juan-Sanz et al, 2017). Moreover, the ER transmembrane protein VAP was originally identified as regulator of synaptic transmission in Aplysia californica, where it was specifically expressed in neuronal tissue (Skehel et al, 1995). Conversely, mammalian VAPA and VAPB are ubiquitously expressed in different cell types and its intracellular localization is restricted to ER membranes. VAPs act as key players in facilitating tight membrane contact sites between the ER and other intracellular membranes, which represent functional interactions through which Ca2+ exchange and lipid transfer occur (Muallem et al, 2017; Wu et al, 2018). VAP contains a C-terminal transmembrane domain which is inserted into the ER membrane, and a cytoplasmic N-terminal tail with a coiled-coil domain and a major sperm protein (MSP) domain. The MSP domain exhibits a FFAT(-like) binding site, which is unique for VAP proteins. Many VAP-associated proteins (> 100) with such a FFAT(-like) motif have been described (Murphy & Levine, 2016). This includes the cytoplasmic protein SCRN1, which contains a N-terminal C69 domain and a C-terminal coiled-coil domain and was predicted to have FFAT(-like) motifs (Murphy & Levine, 2016). The large number of FFAT-containing proteins typically localize to distinct subcellular structures, which has led to the general idea that VAP may act as a key ER receptor. In this study, we demonstrated that ER membrane protein VAP and cytoplasmic VAP-associated protein SCRN1 are important for Ca2+-driven SV cycling. We found that VAP interacts with SCRN1 at the ER membrane through a single FFAT-like motif. Decreasing these ER-localized VAP-SCRN1 interactions was accompanied by a number of phenotypes, including discontinuous ER tubules, impaired ER dynamics, elevated basal presynaptic Ca2+ levels, and decreased SV cycling. Together, these data point toward a model where ER remodeling, mediated by VAP-SCRN1 interactions is engaged in modulating Ca2+ dynamics and SV cycling at presynaptic sites. Results ER proteins VAPA and VAPB are involved in regulating SV cycling To determine whether the ER proteins VAPA and VAPB could be involved in modulating presynaptic function, we first mapped their subcellular localization in primary rat hippocampal neurons. Similar as reported previously in Drosophila neurons, we found that endogenous VAPA and VAPB appeared as punctae present along ER structures in axons which often co-localized with presynaptic marker synaptotagmin (Syt; Pennetta et al, 2002). At somatodendritic regions, endogenous VAPA and VAPB revealed a more diffuse patchy staining pattern that co-localized with expressed ER membrane protein Sec61β (Fig 1A). Exogenous HA-VAPA and HA-VAPB were observed at ER structures throughout neurons and also partially co-localized with presynaptic boutons (Fig EV1A). 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 efficiency at presynaptic sites (Fig 1B). Live neurons were incubated with antibodies recognizing the luminal side of SV membrane protein Syt, while neurons were briefly stimulated by bicuculline. Subsequently, neurons were fixed and the fluorescence intensity of internalized Syt at individual presynaptic boutons was measured. VAP was depleted from neurons by expressing shRNAs targeting VAPA and VAPB, which we have validated in the previous studies (Teuling et al, 2007; Kuijpers et al, 2013). Co-depletion of VAPA and VAPB showed a marked decrease (~ 50%) in Syt internalization compared to control cells (Fig 1C and D). In addition, VAPA and VAPB knockdown resulted in a slight decrease in bouton size and bouton density (Figs 1C, and EV1B and C). In summary, we observed that loss of function of ER proteins VAPA and VAPB was accompanied by decreased SV cycling and defects in bouton maintenance. Figure 1. VAP and VAP-associated protein SCRN1 modulate SV cycling 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). 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. 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). 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. Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non-neuronal tissues. Cereb., cerebellum. Hippoc., hippocampus. Spin., spinal. Pull-down assay of HEK293T cells co-expressing Myc-VAPA with BioGFP or BioGFP-SCRN1. Pull-down assay of HEK293T cells co-expressing Myc-VAPB with BioGFP or BioGFP-SCRN1. 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. 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). 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). 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. Source Data for Figure 1 [embj2018101345-sup-0006-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. VAPs modulate bouton maintenance and are associated with brain-enriched SCRN1 proteins Localization of exogenous HA-VAPA or HA-VAPB in neurons (DIV18) co-expressing GFP-Sec61β and immunostained for bassoon. Zooms represent (1) an axonal structure with bassoon-positive presynaptic boutons (arrowheads), and (2) a dendritic structure. Scale bars: 10 μm (full size) and 5 μm (zoom). Quantifications of bouton density in hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. N = 2, n = 80 boutons. Quantifications of bouton size in hippocampal neurons (DIV18) co-expressing RFP and pSuper empty vector or VAPA/B shRNAs. N = 2, n = 380–400 boutons. Western blot of endogenous SCRN1 expression in indicated adult rat neuronal and non-neuronal tissues. Cereb., cerebellum. Hippoc., hippocampus. Spin., spinal. 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. Selected candidates all showed > 10 enrichment of PSM compared to control. Localization of exogenous GFP-SCRN1 in hippocampal neurons (DIV18) immunostained for vGlut. Zoom represents an axon structure with presynaptic sites (arrowheads). Scale bars: 10 μm (full size) and 5 μm (zoom). COS7 cells expressing BioGFP-SCRN1, BioGFP-SCRN2, or BioGFP-SCRN3 and immunostained for SCRN1. Scale bar: 10 μm. Western blot of lysates from HEK293T cells expressing BioGFP-SCRN1, BioGFP-SCRN2, or BioGFP-SCRN2 and immunostained for indicated antibodies. Arrowheads represent (1) BioGFP-SCRN1 expression, (2) endogenous SCRN1 expression, (3) full-length BioGFP-SCRN proteins, and (4) N-terminal cleaved Bio GFP-SCRN2 and Bio GFP-SCRN3. Actin was used as loading control. Cortical neurons (DIV4) co-expressing RFP with pSuper empty vector (control) or SCRN1 shRNA #1. Scale bar: 10 μm. Quantifications of fluorescence intensity of endogenous SCRN1 in cortical neurons (DIV4) co-expressing RFP with pSuper empty vector (control) or SCRN1 shRNA #1, #2, or #3. N = 2, n = 13–14 cells. Data information: Data represent mean ± SEM; **P < 0.01; ***P < 0.001, by Mann–Whitney U-test. Source data are available online for this figure. Download figure Download PowerPoint VAPA and VAPB associate with brain-enriched SCRN1 proteins VAPs function as ER receptors for a large number of VAP-associated proteins containing a FFAT or FFAT-like motif (Murphy & Levine, 2016). To gain more insight into the underlying mechanism of VAP at presynaptic sites, we sought to identify the VAP interactor(s) that could be involved in controlling this function. In a recent study, many new VAP-associated proteins were identified by pull-down and mass spectrometry analysis, including the cytoplasmic protein SCRN1 (Murphy & Levine, 2016). Western blot analysis of lysates from different rat tissues using two different antibodies revealed that SCRN1 is abundantly enriched in brain tissues (Figs 1E and EV1D). This is consistent with the reported enriched expression of SCRN1 in the brain as described in various online expression databases (Protein Atlas, Expression Atlas, Alan Brain Atlas). We confirmed the association between VAP and SCRN1 with various biochemical assays. First, we conducted a pull-down experiment on lysates of HEK293T cells co-expressing biotinylated GFP (BioGFP) or GFP-SCRN1 (BioGFP-SCRN1) and Myc-VAPA or Myc-VAPB. Both Myc-VAPA and Myc-VAPB efficiently co-precipitated with BioGFP-SCRN1 (Fig 1F and G). Next, we examined the SCRN1 interactome using a more unbiased approach and performed BioGFP-SCRN1 pull-downs followed by mass spectrometry analysis using HEK293T cell lysates and adult rat brain extracts. The associations between SCRN1 and the VAPs were identified in both HEK293T lysates and brain extracts (Figs 1H and EV1E). Of all potential SCRN1-interacting proteins, both VAPA and VAPB showed the highest peptide-spectrum match (PSM) values in both datasets. Together, these biochemical data indicated that VAPs are associated with SCRN1. VAP-associated protein SCRN1 is involved in modulating SV cycling We next tested whether SCRN1 was present at presynaptic sites and if this protein could be engaged in modulating SV cycling. Similar to VAP, immunostaining for endogenous SCRN1 revealed a punctate pattern throughout the neuron and regularly co-localized with presynaptic marker Syt (Fig 1I). Exogenous GFP-SCRN1 showed a diffuse cytoplasmic signal, which also co-localized with presynaptic boutons (Fig EV1F). To conduct loss-of-function experiments, we next generated and validated three shRNA targeting SCRN1 and continued our depletion experiments with a single shRNA (Fig EV1G–J). We tested the role of SCRN1 in SV cycling by conducting the Syt uptake assay in neurons depleted from SCRN1. SCRN1 knockdown also showed a marked decrease (~ 40%) in relative Syt internalization compared to control cells, thereby phenocopying the effect of VAP knockdown (Fig 1J and K). The presynaptic phenotype in SCRN1 knockdown neurons was rescued by expressing wild-type GFP-SCRN1 (Fig 1J and K). Together, these results illustrate that SCRN1 depletion, similarly to VAP depletion, results in impaired SV cycling. SCRN1 does not exhibit autolytic protease activity To better understand the molecular function of VAP-associated protein SCRN1, we next tested whether its conserved proteolytic domain could be involved. Like all SCRN family members, SCRN1 contains a C69 protease domain and therefore belongs to the N-terminal nucleophile (Ntn) aminohydrolases superfamily (Pei & Grishin, 2003). Proteolytic activity in this superfamily relies on autolytic cleavage of the auto-inhibitory N-terminal of the precursor protein by the mature protein (Fig EV2A). This cleavage occurs right before the catalytic site of the protein, which is a cysteine residue in the SCRN family. Sequence alignment of the SCRN proteins revealed that the position of the predicted catalytic cysteine including the flanking residues is shared in SCRN2 and SCRN3, but not in SCRN1 (Fig EV2B). We analyzed N-terminal SCRN cleavage by conducting Western blotting of lysates from HEK293T cells expressing wild-type GFP-SCRN1, GFP-SCRN2, or GFP-SCRN3 (Fig EV2C). In lysates of GFP-SCRN2 and GFP-SCRN3 expressing cells, we identified a low molecular weight band corresponding to the predicted size of GFP fused to the N-terminal cleavage product. Conversely, this cleavage product was not observed in lysates of GFP-SCRN1 expressing cells. Moreover, mutant SCRN1, SCRN2, and SCRN3 constructs in which the predicted catalytic cysteine was replaced by a non-catalytic alanine residue also did not show a cleavage product (Fig EV2C). These data suggest that SCRN1, unlike its family members SCRN2 and SCRN3, does not exhibit autolytic protease activity. Click here to expand this figure. Figure EV2. SCRN1 does not exhibit proteolytic activity and its C-terminal is recruited to VAP MSP domain Schematic illustration of autolytic protease activation of C69 family members. Sequence alignment of predicted proteolytic sites of SCRN family members according to the MEROPS database. Western blot of lysates from HEK293T cells expressing BioGFP-SCRN1-WT, BioGFP-SCRN1-C9A, BioGFP-SCRN2-WT, BioGFP-SCRN2-C12A, BioGFP-SCRN3-WT, or BioGFP-SCRN3-C6A. Arrowheads represent (1) full-length BioGFP-SCRN proteins and (2) N-terminal cleaved BioGFP-SCRN2 and BioGFP-SCRN3. Pull-down assay of HEK293T cells co-expressing Myc-VAPA with BioGFP, BioGFP-SCRN1-WT, BioGFP-SCRN1-C9A, BioGFP-SCRN1-N, or BioGFP-SCRN1-C. Pull-down assay of HEK293T cells co-expressing Myc-VAPB with BioGFP, BioGFP-SCRN1-WT, BioGFP-SCRN1-C9A, BioGFP-SCRN1-N, or BioGFP-SCRN1-C. Hippocampal neurons (DIV16) co-expressing HA-VAPB with GFP-SCRN1-N or GFP-SCRN1-C. Scale bars: 10 μm (full size) and 2 μm (zoom). COS7 cells co-expressing GFP-SCRN1-C with HA-VAPA or HA-VAPB. Scale bars: 10 μm (full size) and 5 μm (zoom). COS7 cells co-expressing HA-SCRN1 with GFP-VAPB-TM, GFP-VAPB-MSP-CC, or GFP-VAPB-MSP. Scale bars: 10 μm (full size) and 5 μm (zoom). COS7 cells co-expressing GFP-SCRN1-N with HA-VAPA or HA-VAPB. Scale bars: 10 μm (full size) and 5 μm (zoom). Source data are available online for this figure. Download figure Download PowerPoint SCRN1 is recruited to VAP at the ER membrane To further examine the function of VAP-associated protein SCRN1, we next assessed whether the subcellular localization of SCRN1 could be controlled by VAP. This was addressed by conducting co-expression experiments of GFP-SCRN1 and HA-VAPA or HA-VAPB in cultured neurons and COS7 cells. In COS7 cells, the ER structures are relatively less compact and easier to visualize than in neurons. GFP-SCRN1 expression alone in neurons or COS7 cells showed a diffuse cytoplasmic distribution, which only partly coincided with ER structures (Fig 2A and B). In neurons, co-expression of GFP-SCRN1 with either HA-VAPA or HA-VAPB resulted in the formation of dense VAP/SCRN1-positive clusters at neurites (Fig 2C). COS7 cells co-expressing GFP-SCRN1 and HA-VAPA or HA-VAPB showed marked differences in SCRN1 localization, as it induced abundant SCRN1 recruitment to VAP at the ER membrane (Fig 2D). This observation suggests that enhancing the number of VAPs at the ER membrane allows for increased SCRN1 binding, presumably because it decreases the competition with other FFAT(-like)-containing proteins for the VAP-binding pockets (Fig 2E). Next, we assessed whether the observed recruitment to VAP at ER structures is shared within the SCRN family. Contrarily, we observed no change in GFP-SCRN2 or GFP-SCRN3 localization when co-expressed with HA-VAPB in COS7 cells (Fig 2F). Together, these data indicate that SCRN1, and not SCRN2 and SCRN3, is recruited to VAP at the ER membrane. Figure 2. SCRN1 is recruited to VAP at the ER membrane Localization of GFP-SCRN1 and TagRFP-ER in hippocampal neurons (DIV16–18). Scale bars: 10 μm (bottom panel, full size) and 5 μm (top panel; bottom panel, zoom). Localization of GFP-SCRN1 and ssRFP-KDEL in COS7 cells with normalized intensity plot of indicated line (dotted). Scale bars: 10 μm (full size) and 5 μm (zoom). Hippocampal neurons (DIV16) co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB. Scale bars: 10 μm (full size) and 5 μm (zoom). COS7 cells co-expressing GFP-SCRN1 with HA-VAPA or HA-VAPB with normalized intensity plot of indicated line (dotted). Scale bars: 10 μm (full size) and 5 μm (zoom). Schematic illustration of SCRN1 recruitment to ER membranes upon increasing VAP levels. COS7 cells co-expressing HA-VAPB with GFP-SCRN2 or GFP-SCRN3 with normalized intensity plot of indicated line (dotted). Scale bars: 10 μm (full size) and 5 μm (zoom). Download figure Download PowerPoint SCRN1 interacts with VAP through a single FFAT-like motif Next, we sought to determine the specific domains responsible for the interaction between VAP and SCRN1. We found that the C-terminal coiled-coil region of SCRN1 and the N-terminal major sperm protein (MSP) domain of VAPB are the minimal binding domains required for this interaction, as shown by co-expression experiments in COS7 cells and pull-down analysis of HEK293T lysates (Figs EV2D–I, 3A and E, and EV3B). The MSP domain of VAP contains a FFAT(-like) motif binding site, and FFAT(-like) motifs are found in the majority of the VAP-interacting proteins (Loewen et al, 2003; Murphy & Levine, 2016). Indeed, we found that the FFAT binding motif in VAP is responsible for the interaction with SCRN1. The VAP mutant VAP-K87D/M89D, in which FFAT binding is disrupted, was no longer able to recruit GFP-SCRN1 (Fig 3A, B and E; Kaiser et al, 2005). Next, we searched for FFAT(-like) motifs in SCRN1 using a previously reported algorithm and identified four potential FFAT-like motifs (Fig EV3A; Murphy & Levine, 2016). We generated SCRN1 constructs with single-point mutations for each single FFAT-like motif (Fig 3A). VAP association was disrupted when mutating the most C-terminal FFAT-like motif in SCRN1 (GFP-SCRN1-F402A), but not the other motifs, as shown by pull-down assays and co-expression experiments (Figs 3C–F, and EV3B–D). Sequence alignment of the SCRN family members revealed that this newly identified FFAT-like motif in SCRN1 is not shared with the other two SCRN family members, which is consistent with our observation that exogenous VAP is unable to recruit GFP-SCRN2 and GFP-SCRN3 (Figs 2F and 3G). Together, th" @default.
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• W2969293681 title "VAP‐SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function" @default.
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