Feline Lindhout

VAP–SCRN1 interaction regulates dynamic endoplasmic reticulum remodeling and presynaptic function 121 4 SCRN1-F402A, SCRN1 shRNA, or VAPA/B shRNAs. Scale bars: 5 μm (full size) and 2 μm (zoom). C. Time-lapse of TagRFP-ER dynamics in hippocampal neurons (DIV16–18) co-expressing GFP, GFP-SCRN1, GFP-SCRN1-F402A, SCRN1 shRNA, or VAPA/B shRNAs. Intact and sTable ER structures (dark arrowheads), intact and dynamic ER structures (light arrowheads), and impaired non-dynamic ER structures (arrows) are indicated. Scale bars: 5 μm (full size) and 2 μm (zoom). D. Schematic illustration of the effect of VAP-SCRN1 interactions on ER structure and dynamics. E. ER nanostructures visualized with GFP-Sec61β in axons of hippocampal neurons (DIV18) immunostained for α-tubulin and co-expressed with pSuper empty vector, SCRN1 shRNA, or VAPA/B shRNAs, and subjected to expansion microscopy. Scale bars: 2 μm (full size) and 500 nm (zoom). F. FRAP experiment of TagRFP-ER in hippocampal neurons (DIV17–18) co-expressing GFP, GFP- SCRN1, and GFP-SCRN1-F402A. Scale bar: 2 μm. G. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV17–18) co-expressing GFP, GFP-SCRN1, and GFP-SCRN1-F402A. N = 2, n = 9 neurons. H. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T = 60 s in hippocampal neurons (DIV17–18) co-expressing GFP, GFP-SCRN1, and GFP-SCRN1-F402A. N = 2, n = 9 neurons. I. Average normalized fluorescent TagRFP-ER recovery after photo-bleaching in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, and VAPA/B shRNAs. N = 4, n = 6–12 neurons. J. Normalized fluorescent TagRFP-ER recovery after photo-bleaching at T = 60 s in hippocampal neurons (DIV18) co-expressing pSuper empty vector, SCRN1 shRNA, and VAPA/B shRNAs. N = 4, n = 6–12 neurons. Data information: Data represent mean ± SEM; n.s.: not significant; *P < 0.05; **P < 0.01, by Mann– Whitney U-test. type SCRN1 represented stabilized ER structures (Fig 4C,D, S4G). On the other hand, decreasing SCRN1 or VAP levels, or expressing dominant-negative SCRN1-F402A mutant, resulted in overall impaired dynamics of the dense ER patches that seemed partially discontinuous with the remaining ER structures (Fig 4C,D, S4G). To gain more detailed insights into the role of VAP-SCRN1 interactions on ER morphology, we next sought to visualize ER nanostructures in neurons using the recently developed expansion microscopy (ExM) technique (Tillberg et al, 2016). This ExM approach allows for a ~4.5-fold physical sample magnification by isotropic chemical expansion and has been validated to preserve the nanoscale organization within different biological specimens (reviewed in Wassie et al, 2019). Here, we successfully rtesolved the dense neuronal ER structures, which enabled us to distinguish individual ER tubules and sheets in neurons expressing ER membrane marker GFP-Sec61β (Fig 4E, S4H,I). Consistent with reported EM studies, we observed that the axonal ER network was comprised of 1 or 2 ER tubules per diameter and regularly formed tubular structures (Fig 4D; Terasaki, 2018; Wu et al, 2017; Yalcin et al, 2017). VAP or SCRN1 knockdown neurons showed marked differences on ER nanostructures.