Feline Lindhout

VAP–SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function 111 4 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 Ca 2+ -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 Ca 2+ levels in the ER are locally elevated during evoked neuronal transmission, suggesting that the presynaptic ER buffers Ca 2+ to modulate SV cycling (de Juan-Sanz et al, 2017). Moreover, the ER transmembrane protein VAPwas 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 Ca 2+ exchange and lipid transfer occur (Muallemet al, 2017;Wu et al, 2018). VAPcontains aC-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 & Levi-e, 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 Ca 2+ -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 Ca 2+ levels, and decreased SV cycling. Together, these data point toward a model where ER remodeling, mediated by VAP-SCRN1 interactions is engaged in modulating Ca 2+ dynamics and SV cycling at presynaptic sites.