Article Figures & Data
Figures
- Figure 1.
Structural model of mouse SynDIG1 membrane-associated region. A, Transmembrane view of ribbon (top) and surface (bottom) representation of the Rosetta model. Membrane segments colored by rainbow color scheme—from the N-terminal (blue) to the C-terminal (red) region. Side chains of N- and C-terminal residues and residues within the I236–G239 loop are labeled. Side chains of C191 and C192 are shown in space-filling representation; all other residues are shown in stick representation. Carbon, nitrogen, oxygen, and sulfur atoms are colored gray, blue, red, and yellow, respectively. B, Zoomed-in view of ribbon (left) and surface (right) representation of the model near C191. C, Zoomed-in view of ribbon (left) and surface (right) representation of the model near C192. D, Ramachandran plot of the distribution of amino acid backbone conformations in the structural model in A. Each residue is a dot in the graph of ϕ vs ψ backbone angles. Probability contours based on a reference set of high-resolution proteins are shown on the plot as green lines.
- Figure 2.
SynDIG1 palmitoylation is required for clustering and stability in COS cells. A, Hippocampi from 1-month-old mice were lysed and subjected to ABE assay. Palmitoylated (ABE) SynDIG1 and total protein represented by 10% of the input sample in the presence or absence of HAM were measured by immunobloting with anti-SynDIG1 antibodies (SD1). B, Identification of SynDIG1 palmitoylation sites. COS cells were transfected with HA-tagged WT or mutant forms of SynDIG1. After 24 h, cells were lysed and subjected to ABE assay. Palmitoylated (ABE) SynDIG1 and total protein represented by 10% of the input sample in the presence or absence of HAM were measured by immunoblotting with anti-SD1 antibodies. C, Stability of WT and palmitoylation-deficient SynDIG1 mutant C191,192A in COS cells was investigated by treatment with 100 μg/ml CHX for indicated times. Immunoblotting for β-tubulin (β-tub) served as a loading control. D, Graph depicts the percentage of SynDIG1 detected by immunoblotting from lysates isolated after CHX treatment normalized to samples at 0 h. E, SynDIG1 clustering in heterologous cells requires palmitoylation of C192. COS cells were transfected with WT or mutant forms of SynDIG1, fixed after 24 h, and labeled with anti-SD1 (green) and anti-EEA1 (red) antibodies. Bottom, Zoomed-in image of the boxed region in the top panel. Scale bars: E, 10 μm; inset, 2 μm. F, SynDIG1 lacking palmitoylation is retained in the secretory pathway. COS cells were transfected with HA-tagged WT or mutant forms of SynDIG1. After 24 h, cells were treated with BFA or vehicle (Control) for 30 min; fixed; and labeled with anti-HA (green), anti-GM130 (blue), and anti-calreticulin (CR; red) antibodies. Nuclei are indicated by Hoechst stain (magenta) in the merged image. Scale bar, 5 μm.
- Figure 3.
SynDIG1 localization in neurons requires C192 palmitoylation. A, Hippocampal neurons were transfected at 5 DIV with HA-tagged WT or mutant forms of SynDIG1, fixed at 9 DIV and immunostained with anti-HA (red), anti-EEA1 (green), and anti-GM130 (blue) antibodies. Scale bar, 20 μm. B, C, Graphs represent enrichment of SynDIG1 colocalization with subcellular markers. Similar results were obtained in two independent experiments, n = 10 cells for each condition. Error bars, mean ± SEM. ***p < 0.001. D, Representative stretches of hippocampal neurons treated at 11 DIV with vehicle (DMSO) or 50 μm 2-BP for 4 h; fixed; and stained with antibodies against PSD-95 or SynDIG1 (SD1), VGluT1, and MAP2. Scale bar, 10 μm. E, F, Graphs represent the puncta size and ID of PSD-95 and SD1 upon 2-BP treatment compared with vehicle. Data are the average of two independent experiments, n = 25 cells for each condition. Error bars, mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 4.
SynDIG1 palmitoylation is regulated by neuronal activity. A, Blocking synaptic activity with TTX leads to increased SynDIG1 and PSD-95 palmitoylation. Hippocampal slice cultures were treated with 2 μm TTX or vehicle for 16 h and subjected to ABE assay. B, Hippocampal neurons were transfected at 5 DIV with WT or C191A; treated with 2 μm TTX or vehicle at 10 DIV; fixed at 12 DIV; and immunostained with antibodies against HA, PSD-95, and VGluT1. Synapses were defined as the colocalization of PSD-95 and VGluT1 clusters. Untransfected control (Crtl) neurons are shown for comparison. Scale bar, 10 μm. C, D, Graphs represent percentage synapses containing WT or C191A in vehicle- and TTX-treated neurons (C) or synapse density upon overexpression of WT or C191A compared with untransfected Crtl neurons in vehicle-treated samples (D). Data are the average of the following two independent experiments: vehicle: WT, n = 27, C191A, n = 30; TTX: WT, n = 30, C191A, n = 29 (C); and Ctrl: n = 25; WT: n = 30; C191A: n = 30 (D). Error bars, mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.
