Extended Data Fig. 7: Loss of Ddhd2 leads to membrane trafficking defects in neurons.

a, Uncropped images of the representative EM captures shown in Fig. 6a. b, Representative sptPALM super-resolution images of ERGIC-53-mEos2 transiently expressed in E16 Ddhd2^−/− hippocampal neurons treated with 1 µM fatty acyl-CoAs for 48 h as indicated and imaged at DIV21-22. Low resolution image of green fluorescence of the ERGIC-53-mEos2, along with super-resolved diffusion coefficient (bar: Log₁₀ 1 to −5, high to low mobility), average intensity (bar: 8 to 0, high to low density), and single-molecule trajectory (bar: 0–10,000 frame acquisition) maps are show. Boxed areas (i-iii) are shown magnified on right. Arrowheads point to confined molecules with low mobility, while arrows indicate mobile molecules. c, Representative maximum projection images and quantification of mean fluorescence intensity (MFI [arbitrary units, a.u.]) of cultured E16 C57BL/6J and Ddhd2^−/− hippocampal neurons ( + 4 µM Ara-C) at DIV21-22 following a 5 min high K⁺ pulse in presence of d, 70,000 MW Dextran Tetramethylrhodamine (red) or e, Alexa Fluor 555-conjugated Cholera Toxin Subunit B (CTxB, red), followed by 30 min chase in low K⁺ buffer, fixation and imaging. Nuclei stained with DAPI (blue). f, Averaged vGlut1-pHluorin traces (ΔF/F₀ [a.u.]), g, peak height of vGlut1-pH responses normalized to NH₄Cl [a.u.], and h, endocytosis time-constant (τ [s]) and in C57BL/6J and responsive Ddhd2^−/− hippocampal neurons stimulated with a train of 300 action potentials (50 Hz, 6 s). Normalisation to the peak of each condition. i, Averaged vGlut1-pHluorin traces (ΔF/F₀ [a.u.]) of C57BL/6J grown in 25 mM glucose, and 2 mM glucose ± 15 min incubation in etomoxir. Data are presented as mean values ± SEM, dots present technical replicates. N = 3 biologically independent experiments in each condition. The exact p-values stated in the graphs were determined from biological replicates using two-tailed Mann-Whitney test (d, e,) and two-tailed unpaired t test (g, h,).