Journal: BMC Molecular and Cell Biology

Article Title: The mitochondrial outer membrane protein SYNJ2BP interacts with the cell adhesion molecule TMIGD1 and can recruit it to mitochondria

doi: 10.1186/s12860-020-00274-1

Figure Lengend Snippet: TMIGD1 localizes to mitochondria and cell-cell contacts in HK-2 cells. a Sparsely grown HK-2 cells were fixed and stained with polyclonal antibodies against TMIGD1 (#HPA021946) and markers for mitochondria (MitoTracker), the trans-Golgi network (p230TG) and early endosomes (EEA1). Note that TMIGD1 localizes specifically to mitochondria. Scale bars: 5 μm. b HK-2 cells were grown as single cells or were grown and maintained at confluency for different periods of time, as indicated. Cells were fixed and stained with antibodies against TMIGD1 (#HPA021946) and MitoTracker (sparsely grown cells), or with antibodies against TMIGD1 (#HPA021946) and the cell-cell contact marker ZO-1 (cells grown to confluency). Right panels: Quantification of TMIGD1 signal intensities at cell-cell contacts (top bar graph) and of fraction of cells with TMIGD1-positive cell-cell contacts at different days of confluency (bottom bar graph). Quantification of TMIGD1 signal intensities at cell-cell contacts (top bar graph) was performed as described in the Methods section. Statistical analysis was performed using unpaired Student’s t-test. Data were obtained from three independent experiments and are presented as arithmetic means ± SEM; ** P < 0.01, *** P < 0.001. Scale bars: 10 μm. c HK-2 cells stably transfected with the pInducer10-mir-RUP-PheS plasmid vector that allows expression of both a TMIGD1-specific shRNA and turboRFP (tRFP) under a doxycycline-regulated promoter were grown to low confluency (sparse) or high confluency (confluent), fixed with PFA and methanol, respectively, and analyzed by fluorescence microscopy with antibodies against TMIGD1 (#HPA021946) and the cell contact marker β-catenin, as indicated. In sparsely grown cells, the tRFP signal encoded by the pInducer10 vector was used as marker for shRNA-expressing cells. Note that the pInducer10 vector is based on a TetON system, i.e. shRNA expression is induced by doxycycline. Scale bars: 10 μm

Article Snippet: The following antibodies were used in this study: rabbit pAb anti-hSYNJ2BP (Sigma-Aldrich #HPA000866); rabbit pAb anti-TMIGD1 (Sigma-Aldrich #HPA021946); rabbit pAb anti-TMIGD1 (Novus #NBP1–80672); mouse anti-EEA1 (Beckton-Dickinson (BD)-TL #610154); mouse anti-P230 transGolgi (BD-TL #611280); mouse anti-GM130 (BD-TL #610822), mouse mAb anti β-catenin (BD-TL #610456); mouse mAb anti-ZO-1 (BD-TL #610966); mouse mAb anti-α-Tubulin (Sigma-Aldrich, clone B-5-1-2, #T5168); mouse mAb anti-Flag M2 (Sigma-Aldrich #F1804); rabbit pAb anti-Flag (Sigma-Aldrich #F7425); goat pAb anti-Myc (SantaCruz #sc-789G); mouse mAb anti-Myc 9E10 [ ]; goat pAb anti GST (GE Healthcare #27–4577-01).

Techniques: Staining, Marker, Stable Transfection, Transfection, Plasmid Preparation, Expressing, shRNA, Fluorescence, Microscopy

Journal: BMC Molecular and Cell Biology

Article Title: The mitochondrial outer membrane protein SYNJ2BP interacts with the cell adhesion molecule TMIGD1 and can recruit it to mitochondria

doi: 10.1186/s12860-020-00274-1

Figure Lengend Snippet: TMIGD1 interacts with SYNJ2BP. a Schematic presentation of murine Synj2bp isoforms. The TMIGD1-interacting fragment isolated from the yeast-two hybrid library (indicated in red) encodes AA 1–105 of Synj2bp and encompasses the entire PDZ domain (AA 13–100). Abbreviations: TM, transmembrane, MT-IM, mitochondrial intermembrane ( b ) TMIGD1 interacts with SYNJ2BP through its PDZ domain-binding motif. Lysates of SYNJ2BP-transfected HEK293T cells were incubated with GST-fusion proteins containing the entire cytoplasmic domain of TMIGD1 (GST-TMIGD1) or the cytoplasmic domain lacking the PDZ domain-binding motif (GST-TMIGD1/Δ5) or with GST alone (GST-). Precipitates obtained with GST fusion proteins were immunoblotted with anti-SYNJ2BP antibodies (Sigma #HPA000866, top panel, 90% of precipitates) or with anti-GST antibodies (bottom panel, 10% of precipitates). SYNJ2BP precipitated with GST-TMIGD1 but not with GST-TMIGD1/Δ5. Abbreviations: IB, immunoblotting; Lys, lysate. c SYNJ2BP co-immunoprecipitates with TMIGD1. Immunoprecipitates obtained with anti-TMIGD1 polyclonal antibodies (Affi1611) from HEK293T cells transfected with empty vector (pFlag vector), Flag-TMIGD1 and/or Flag-SYNJ2BP as indicated were immunoblotted with anti-SYNJ2BP antibodies (Sigma #HPA000866, left panel, 90% of precipitates) or with anti-Flag antibodies (right panel, 10% of precipitates). Note that SYNJ2BP is present in TMIGD1 immunoprecipitates. Abbreviations: endog., endogenous; IB, immunoblotting; IP, immunoprecipitation; Lys, lysate. d SYNJ2BP localizes at mitochondria in sparsely seeded cells but to cell-cell contacts in confluent cells. HK-2 cells were either sparsely seeded or grown to confluency for 3d, 7d, or 12 d as indicated, then fixed and stained for SYNJ2BP and either Mitotracker or ZO-1 as indicated. Scale bars: 10 μm

Article Snippet: The following antibodies were used in this study: rabbit pAb anti-hSYNJ2BP (Sigma-Aldrich #HPA000866); rabbit pAb anti-TMIGD1 (Sigma-Aldrich #HPA021946); rabbit pAb anti-TMIGD1 (Novus #NBP1–80672); mouse anti-EEA1 (Beckton-Dickinson (BD)-TL #610154); mouse anti-P230 transGolgi (BD-TL #611280); mouse anti-GM130 (BD-TL #610822), mouse mAb anti β-catenin (BD-TL #610456); mouse mAb anti-ZO-1 (BD-TL #610966); mouse mAb anti-α-Tubulin (Sigma-Aldrich, clone B-5-1-2, #T5168); mouse mAb anti-Flag M2 (Sigma-Aldrich #F1804); rabbit pAb anti-Flag (Sigma-Aldrich #F7425); goat pAb anti-Myc (SantaCruz #sc-789G); mouse mAb anti-Myc 9E10 [ ]; goat pAb anti GST (GE Healthcare #27–4577-01).

Techniques: Isolation, Binding Assay, Transfection, Incubation, Western Blot, Plasmid Preparation, Immunoprecipitation, Staining