Fig 2: The TMCC3 mutant substituting alanine for serine 15 is prone to localize at three-way junctions against overexpression of 14-3-3γ. HA-14-3-3γ or a control vector was transfected into U2OS-GFP-TMCC3 cells and U2OS-GFP-TMCC3-S15A cells. Six hours after transfection, the cells were immunostained with the anti-HA mAb. GFP-TMCC3 and GFP-TMCC3-S15A were detected by GFP fluorescence. Representative images of six independent experiments are shown. Scale bars, 20 μm. The boxed areas are enlarged to highlight GFP-TMCC3 puncta and GFP-TMCC3-S15A puncta and shown below each image. Arrowheads indicate representatives of GFP-TMCC3 puncta and GFP-TMCC3-S15A puncta. Scale bars, 10 μm. Sixty transfected cells were randomly chosen, and the number of cells losing GFP-TMCC3 puncta was counted. The ratio of the cells losing GFP-TMCC3 puncta or GFP-TMCC3-S15A puncta to the transfected cells is expressed as a percentage and shown in the bottom panel. The error bars represent SD of six independent experiments. Statistical analysis was performed using Student’s t test. ∗∗∗p < 0.001. TMCC3, transmembrane and coiled-coil domain family 3.

Fig 3: The negative regulation of TMCC3 by 14-3-3γ is involved in formation of the reticular ER network. The siRNA targeting TMCC3 was transfected into U2OS cells to allow endogenous TMCC3 to be knocked down. HA-TMCC3, HA-TMCC3-S15A, or the control vector was then transfected to the TMCC3-knockdown cells. As a negative control, the control siRNA was transfected into U2OS cells, followed by transfection of the control vector. The samples were subjected to immunostaining with the anti-HA mAb, the anti-CLIMP-63 pAb, and the anti-α-tubulin mAb. Representative images of five independent experiments are shown in the top and middle panels. Scale bars, 20 μm. The boxed areas are enlarged to highlight the ER sheet expansion and shown below each image. Scale bars, 10 μm. Twenty transfected cells were randomly chosen, and the pixel areas of CLIMP-63 and the total cell were measured in each cell. The ratio of CLIMP-63 area to the total cell area is shown in the bottom panel. Each dot indicates the ratio of CLIMP-63 area to the total cell area of a single cell. The five independent experiments are indicated by different colors. The mean value from each experiment is shown with rhombus. The bars represent mean ± SD from the five independent experiments. Statistical analysis was performed using Student’s t test. ∗∗∗p < 0.001. ER, endoplasmic reticulum; TMCC3, transmembrane and coiled-coil domain family 3.

Fig 4: Phosphorylated serine 15 in the deduced 14-3-3 binding motif is required for potent binding to 14-3-3γ. A, the N-terminal region of TMCC3 highlighting the deduced 14-3-3 binding motifs. Serine residues being the possible phosphorylation sites in three deduced 14-3-3 binding motifs are indicated in red. Arginine, serine, and proline residues at −3, 0, and +2 positions, respectively, in the deduced 14-3-3 binding motif predicted with the highest score are underlined. B, serine 15 is required for potent binding to 14-3-3γ. Indicated combinations of HA-TMCC3-D1, HA-TMCC3-D1-S15A, HA-TMCC3-D1-S15/25/46A, and Flag-14-3-3γ were transfected into HEK293 cells, followed by immunoprecipitation with the anti-Flag mAb. The samples were immunoblotted with the anti-HA mAb and the anti-Flag pAb. The arrowheads indicate the bands of interest. The asterisks indicate the nonspecific bands from the light chain of IgG. C, serine 15 is phosphorylated. HA-TMCC3-D1, HA-TMCC3-D1-S15A, and HA-TMCC3-D1-S15/25/46A were transfected into HEK293 cells, followed by immunoprecipitation with the anti-HA mAb. The samples were immunoblotted with the anti-phospho-14-3-3 binding motif pAb and the anti-HA pAb. The asterisk indicates the nonspecific bands from the light chain of IgG. TMCC3, transmembrane and coiled-coil domain family 3.

Fig 5: Overexpression of 14-3-3γ reduces localization of TMCC3 to three-way junctions. A, overexpression of 14-3-3γ reduces localization of GFP-TMCC3 to three-way junctions. HA-14-3-3γ or mCherry was transfected into U2OS-GFP-TMCC3 cells. Six hours after transfection, the cells were fixed and permeabilized. The cells transfected with HA-14-3-3γ were subjected to immunostaining with the anti-HA mAb. GFP-TMCC3 and mCherry were detected by GFP fluorescence and mCherry fluorescence, respectively. Representative images of three independent experiments are shown in the left panels. Scale bars, 20 μm. The boxed area are enlarged to highlight GFP-TMCC3 puncta and shown below each image. Arrowheads indicate the representatives of GFP-TMCC3 puncta. Scale bars, 10 μm. Sixty transfected cells were randomly chosen, and the number of cells losing GFP-TMCC3 puncta was counted. The ratio of the cells losing GFP-TMCC3 puncta to the transfected cells is expressed as a percentage and shown in the right graph. The error bars represent SD of three independent experiments. Statistical analysis was performed using Student’s t test. ∗∗∗p < 0.001. B, overexpression of 14-3-3γ reduces localization of endogenous TMCC3 to three-way junctions. HA-14-3-3γ or a control vector was transfected into U2OS cells, followed by immunostaining with the anti-TMCC3 pAb, the anti-PDI mAb, and the anti-HA mAb. Representative images of three independent experiments are shown. Scale bars, 20 μm. The boxed areas are enlarged to highlight localization of TMCC3 and shown below each image. The red and green images are merged in the rightmost column. Arrowheads indicate localization of TMCC3 at three-way junctions. Scale bars, 10 μm. C, effect of overexpression of 14-3-3γ on the protein level of TMCC3. HA-14-3-3γ or the control vector was transfected into U2OS cells. The total cell lysates were subjected to immunoblotting with the anti-TMCC3 pAb, the anti-HA mAb, and the anti-GAPDH. TMCC3, transmembrane and coiled-coil domain family 3.