Journal: bioRxiv

Article Title: MOSPD3 mediates the ER recruitment of ATG2A to promote autophagy

doi: 10.64898/2026.01.04.697522

Figure Lengend Snippet: a-f . Representative images of live BODIPY (blue)-stained COS7 cells transiently transfected with GFP-ATG2A (green) and Halo-VAPs (magenta; MOSPD1/2/3, VAPA and VAPB) with insets from at least 3 independent experiments. g. In cells as in ( a-f ), Pearson’s correlation coefficient of ATG2A vs different ER proteins including Halo-Sec61β (11 cells), Halo-MOSPD1 (13 cells), Halo-MOSPD3 (13 cells), Halo-MOSPD2 (14 cells), Halo-VAPA (13 cells), or Halo-VAPB (12 cells) in three independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. Scale bar, 10 μm in the whole cell images and 2 μm in the insets in (a-f).

Article Snippet: The African green monkey kidney fibroblast-like COS7 cell line (ATCC), human cervical cancer HeLa cells (ATCC), human embryonic kidney (HEK) 293T (ATCC) and human osteosarcoma U2OS ATG2A-KI cell line were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco).

Techniques: Staining, Transfection

Journal: bioRxiv

Article Title: MOSPD3 mediates the ER recruitment of ATG2A to promote autophagy

doi: 10.64898/2026.01.04.697522

Figure Lengend Snippet: a. Representative GFP-Trap assays demonstrating an interaction between endogenous ATG2A-GFP-KI and endogenous MOSPD3 from at least 3 independent experiments. b. Representative images of a live U2OS ATG2A-GFP KI cell transiently transfected with Halo-MOSPD3 (magenta) and mCh-LC3B (blue) with insets under EBSS stimulation. c. The percentage of endogenous ATG2A puncta that colocalized with MOSPD3 puncta (832 ATG2A-GFP puncta from 15 cells) from at least 3 independent experiments. Mean ± SD. d. Time-lapse montages of a COS7 cell transiently transfected with GFP-DFCP (green) and Halo-MOSPD3 (magenta) with white arrows denoting dynamic MOSPD3 puncta and yellow arrows denoting colocalization of GFP-DFCP1 puncta and Halo-MOSPD3 puncta from 3 independent experiments. e. The percentage of DFCP1 puncta relative to MOSPD3 puncta from 12 cells. Mean ± SD. f. The percentage of DFCP1 puncta stably associating with MOSPD3 puncta from 12 cells. GFP-DFCP1 puncta associating with Halo-MOSPD3 puncta over the imaging time window (∼15 min) were defined to be stable association. Mean ± SD. g. Membrane fractionation showing the ER association of ATG2A and ATG2B in WT and MOSPD3 KO HeLa cells in 3 independent experiments. Western blots were performed with antibodies against ATG2A, ATG2B, CANX (ER marker), Lamp1 (late endosome/PM marker), GM130 (Golgi marker) and TOM20 (mitochondrial marker). h. Membrane fractionation showing the ER association of ATG2A and ATG2B in HeLa cells treated with scrambled, VMP1, TMEM41B or VMP1+TMEM41B siRNAs in 3 independent experiments. Western blots were performed with antibodies against ATG2A, ATG2B, VMP1, TMEM41B, CANX (ER marker), Lamp1 (late endosome/PM marker), GM130 (Golgi marker) and TOM20 (mitochondrial marker). Scale bar, 10 μm in the whole cell image, 2 μm in the insets in (b, d).

Article Snippet: The African green monkey kidney fibroblast-like COS7 cell line (ATCC), human cervical cancer HeLa cells (ATCC), human embryonic kidney (HEK) 293T (ATCC) and human osteosarcoma U2OS ATG2A-KI cell line were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco).

Techniques: Transfection, Stable Transfection, Imaging, Membrane, Fractionation, Western Blot, Marker

Journal: bioRxiv

Article Title: MOSPD3 mediates the ER recruitment of ATG2A to promote autophagy

doi: 10.64898/2026.01.04.697522

Figure Lengend Snippet: a, b . Representative images of COS7 cells expressing GFP-ATG2A (green) along with either Halo-MOSPD3 (magenta; a ) or Halo-VMP1 or TMEM41B (magenta; b ) with insets. c. In cells as in ( a, b ), Pearson’s correlation coefficient of ATG2A vs TMEM41B (10 cells), VMP1 (10 cells) or MOSPD3 (10 cells) in three independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. d. Representative images of COS7 cells expressing GFP-ATG2A (green) along with either Halo-VMP1 (magenta; top panel) or TMEM41B (magenta; bottom panel) with insets upon EBSS starvation from three independent experiments. e. Representative images of COS7 cells expressing GFP-ATG2A-NT (green) with Halo-MOSPD3, Halo-VMP1, or TMEM41B-Halo (magenta) with insets. f. In cells as in ( e ), Pearson’s correlation coefficient of ATG2A-NT proteins vs VMP1 (10 cells), TMEM41B (10 cells), or MOSPD3 (10 cells) from three independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. Scale bar, 10 μm in the whole cell images and 2 μm in the insets in (a, b, d & e).

Article Snippet: The African green monkey kidney fibroblast-like COS7 cell line (ATCC), human cervical cancer HeLa cells (ATCC), human embryonic kidney (HEK) 293T (ATCC) and human osteosarcoma U2OS ATG2A-KI cell line were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco).

Techniques: Expressing

Journal: bioRxiv

Article Title: MOSPD3 mediates the ER recruitment of ATG2A to promote autophagy

doi: 10.64898/2026.01.04.697522

Figure Lengend Snippet: a. Representative images of a COS7 cell expressing GFP-ATG2A-ΔCT (green) and Halo-MOSPD3 (magenta) with two insets from at least 3 independent experiments. Yellow asterisks mark cells expressing both GFP-ATG2A-ΔCT and Halo-MOSPD3. b. Representative images of a COS7cell expressing GFP-ATG2A-NT (green) and Halo-MOSPD3 (magenta) with two insets from at least 3 independent experiments. Yellow asterisks mark cells transfected with both GFP-ATG2A-ΔCT and Halo-MOSPD3. c. In vitro pulldown assays using purified GFP-MOSPD3 and purified 14×His-ATG2A-NT or 14×His-ATG2B-NT from 3 independent experiments. d. AlphaFold Multimer prediction of the binding sites between ATG2A and MOSPD3. e. Conservation of the FFNT motif in ATG2A across species. f. The conserved FFNT motif within ATG2A is compared with homologous regions from the indicated human proteins (ANKLE2, CERT, VPS13D, and STARD3). Conserved residues are highlighted. g. Representative images of COS7 cells expressing GFP-ATG2A-NT (green; WT, T362A, T362D and the ΔFFN mutant) and Halo-MOSPD3 (magenta) with insets from at least 3 independent experiments. h. Pearson’s correlation coefficient of MOSPD3 vs. WT ATG2A-NT (10 cells), ATG2A-NT-ΔFFNT (11 cells), ATG2A-T362A (12 cells), or ATG2A-T362D (12 cells) in three independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. i. Membrane fractionation showing the ER association of WT GFP-ATG2A-NT, GFP-ATG2A-NT-T362A and GFP-ATG2A-NT-T362D from 3 independent experiments. Western blots were performed with antibodies against GFP, CANX (ER marker), Lamp1 (late endosome/PM marker), GM130 (Golgi marker) and TOM20 (mitochondrial marker). Scale bar, 10 μm in the whole cell images and 2 μm in the insets in (a, b, & g).

Article Snippet: The African green monkey kidney fibroblast-like COS7 cell line (ATCC), human cervical cancer HeLa cells (ATCC), human embryonic kidney (HEK) 293T (ATCC) and human osteosarcoma U2OS ATG2A-KI cell line were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco).

Techniques: Expressing, Transfection, In Vitro, Purification, Binding Assay, Mutagenesis, Membrane, Fractionation, Western Blot, Marker

Journal: bioRxiv

Article Title: MOSPD3 mediates the ER recruitment of ATG2A to promote autophagy

doi: 10.64898/2026.01.04.697522

Figure Lengend Snippet: a. Top: domain organization of MOSPD3. Bottom: representative images of a COS7 cell expressing GFP-ATG2A-NT (green) and Halo-MOSPD3 (magenta) with insets from at least 3 independent experiments. b. Representative images of a COS7 cell expressing GFP-ATG2A (green) and Halo-MOSPD3-ΔTM (magenta) with insets from at least 3 independent experiments. c. Representative images of a COS7 cell expressing GFP-ATG2A (green) and Halo-MOSPD3-MSP (magenta) with insets from at least 3 independent experiments. d. Representative images of a COS7 cell expressing either GFP-ATG2A-NT (green; top panel) or GFP-ATG2A (green; bottom panel) along with Halo-MOSPD3-ΔMSP (magenta) with insets from at least 3 independent experiments. e. Representative images of a COS7 cell expressing GFP-ATG2A-NT (green) and Halo-MOSPD3-ΔNT (magenta) with insets from at least 3 independent experiments. f. Representative images of a COS7 cell expressing GFP-ATG2A-NT (green) and Halo-MOSPD3-ΔPro (magenta) with insets from at least 3 independent experiments. g. In cells as in ( a-f ), Pearson’s correlation coefficient of ATG2A vs different MOSPD3 mutants including WT Halo-MOSPD3, Halo-MOSPD3-ΔNT, Halo-MOSPD3-Δ Pro, Halo-MOSPD3-ΔMSP, Halo-MOSPD3-ΔTM, Halo-MOSPD3-MSP. 10 cells for each group were analysed from three independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. Scale bar, 10 μm in the whole cell images and 2 μm in the insets in (a-f).

Article Snippet: The African green monkey kidney fibroblast-like COS7 cell line (ATCC), human cervical cancer HeLa cells (ATCC), human embryonic kidney (HEK) 293T (ATCC) and human osteosarcoma U2OS ATG2A-KI cell line were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco).

Techniques: Expressing

Journal: bioRxiv

Article Title: MOSPD3 mediates the ER recruitment of ATG2A to promote autophagy

doi: 10.64898/2026.01.04.697522

Figure Lengend Snippet: a. Representative images of COS7 cells transiently transfected with GFP-ATG2B (green) along with MOSPD1, MOSPD3, VAPA, VAPB or MOSPD2 (magenta) with an inset on the bottom from at least 3 independent experiments. b. In cells as in ( a ), Pearson’s correlation coefficient of ATG2B vs different VAP-related proteins including Halo-MOSPD1 (13 cells), Halo-MOSPD3 (14 cells), Halo-MOSPD2 (12 cells), Halo-VAPA (10 cells), or Halo-VAPB (11 cells) in three independent experiments. Ordinary one-way ANOVA with Tukey’s multiple comparisons test. Mean ± SD. c. AlphaFold Multimer prediction of the binding sites between ATG2B and MOSPD1 (left panel) or MOSPD3 (right panel). d. Conservation of the FFNT motif in ATG2B across species. e. The conserved FFNT motif within ATG2B is compared with homologous regions from the indicated human proteins (ATG2A, ANKLE2, ENTR1, CERT, VPS13D, and STARD3). Conserved residues are highlighted. f. GFP-Trap assays demonstrate interactions between ATG2B and MOSPD1/3 in HEK293 cells from three independent assays. g. Representative images of COS7 cells expressing GFP-ATG2B-ΔFFNT (green) along with either Halo-MOSPD1 (magenta; top panel) or Halo-MOSPD3 (magenta; bottom panel) with insets from at least 3 independent experiments. h. In cells as in ( g ), Pearson’s correlation coefficient of ATG2B-ΔFFNT vs Halo-MOSPD1 (12 cells), or Halo-MOSPD3 (13 cells) in three independent experiments. Unpaired student t test. Mean ± SD. i. Membrane fractionation showing the ER association of ATG2A and ATG2B in MOSPD1/3 double KO HeLa cells from 3 independent experiments. Western blots were performed with antibodies against ATG2A, ATG2B, MOSPD3, CANX (ER marker), Lamp1 (late endosome/PM marker), GM130 (Golgi marker) and TOM20 (mitochondrial marker). Scale bar, 10 μm in the whole cell images and 2 μm in the insets in (a & g).

Article Snippet: The African green monkey kidney fibroblast-like COS7 cell line (ATCC), human cervical cancer HeLa cells (ATCC), human embryonic kidney (HEK) 293T (ATCC) and human osteosarcoma U2OS ATG2A-KI cell line were grown in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco).

Techniques: Transfection, Binding Assay, Expressing, Membrane, Fractionation, Western Blot, Marker