construct cmv mito 4× gcamp6f  (Addgene inc)

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) HEK293 cells were transfected to express the Ca 2+ sensor ER-GCaMP6-210. Scale bar = 10 μm. ( B ) Resting [Ca 2+ ] ER values were defined as the steady-state fluorescence value after the medium conditioning step and before stimulation with 100 μM ATP + 100 μM CCh. Statistical analysis with unpaired t-test, p < 0.0001. ( C ) Values of [Ca 2+ ] ER after Ca 2+ release stimulated by ATP+CCh. Statistical analysis with unpaired t-test, p < 0.0001. ( D ) Decrease in [Ca 2+ ] ER after ATP+CCh stimulus. This decrease was calculated by subtracting the [Ca 2+ ] ER after stimulation with ATP+CCh from the resting [Ca 2+ ] ER . Statistical analysis with unpaired t-test and p = 0.0471. In ( B – D ), the [Ca 2+ ] ER values were monitored in different ROIs from 4 independent experiments. Total number of ROIs analyzed: n = 62 WT and n = 50 KO ROIs. The black line represents the mean of the data. ( E ) Measurement of [Ca 2+ ] i after Ca 2+ release from the ER in WT and STIM1-KO cells. Representative fluorescence profile of fura-2-loaded cells after stimulation with 100 μM ATP + 100 μM CCh in Ca 2+ -free Hank’s balanced salt solution (min = 1). The graph shows the mean F340/F380 ratio ± S.D. over time from 3 independent experiments for WT HEK293 (black line) and 4 experiments for STIM1-KO cells (red line). Total number of cells analyzed: n = 65 WT and n = 89 KO cells. ( F ) Analysis of the maximum increase in the F340/F380 ratio after ATP+CCh addition in Ca 2+ -free assay medium. The maximum values of the F340/F380 ratio of each cell were normalized relative to the resting ratio value to calculate the magnitude of the increase in [Ca 2+ ] i after ER Ca 2+ release. The mean of the data is represented by the black line. Statistical analysis with unpaired t-test. ( G ) Representative fluorescence image and ( H ) fluorescence profile of mito 4× -GCaMP6f after ATP+CCh stimulus. Cells stably transfected for the expression of the mitochondrial Ca 2+ sensor mito 4 × -GCaMP6f were cultured on collagen-coated coverslips and treated with 1 μg/ml doxycycline to induce expression of the Ca 2+ sensor. ER Ca 2+ release was triggered with ATP+CCh in Ca 2+ -free medium, as in ( E ) and ( F ). The ratios of fluorescence at each time point relative to resting fluorescence (F/F r ) were calculated in different ROIs. The graph shows the mean F/F r ± S.E.M. from 4 independent experiments for WT HEK293 ( n = 39 WT ROIs, black line) and STIM1-KO cells ( n = 40 KO ROIs, red line). ( I ) Analysis of the maximum fluorescence increase (F max /F r ) after ATP+CCh. The F max /F r of all ROIs was analyzed from 15 independent experiments for WT cells and 17 experiments for KO cells ( n = 160 WT and n = 169 KO ROIs). The mean of the data is represented by the black line. Statistical analysis with unpaired t-test, p < 0.0001. ( J ) Cells were transiently transfected to express the 4mtD3cpv sensor to measure basal [Ca 2+ ] mit . Data from n = 19 WT cells and n = 16 STIM1-KO cells from 4 independent experiments. The black line represents the mean of data. Statistical analysis with unpaired t-test, p < 0.0001. .

Article Snippet: The vector for the inducible expression of the Ca 2+ sensor mito 4× -GCaMP6f was generated by inserting the Bam HI- Apa I fragment from the construct CMV-mito 4× -GCaMP6f (Addgene #127870) into the Bam HI- Apa I sites of the pcDNA5-FRT/TO-FLAG vector.

Techniques: Transfection, Fluorescence, Stable Transfection, Expressing, Cell Culture

Journal: The EMBO Journal

Article Title: STIM1-containing contact sites promote direct calcium flux from the endoplasmic reticulum to mitochondria

doi: 10.1038/s44318-026-00700-8

Figure Lengend Snippet: ( A ) Schematic representation of STIM1 mutants with deletions in the 551-685 region. The specific sequences deleted between the amino acids 551 and 685 for each of the generated STIM1 mutants are shown. This diagram was created with BioRender.com. ( B ) Immunoprecipitation of GFP-tagged proteins was performed using 1 mg WCL from STIM1-KO HEK293 inducibly expressing STIM1-GFP, STIM1(Δ551-685)-GFP (mutant 3), STIM1(Δ551-642)-GFP (mutant 4), STIM1(Δ643-677)-GFP (mutant 7), STIM1(Δ668-674)-GFP (mutant 8), STIM1(Δ672-685)-GFP (mutant 9), or GFP (empty vector) as a control. Co-precipitation of GRP75 was assessed by immunoblotting. WCL (3 µg) from cells expressing STIM1-GFP was loaded as a positive control. Total immunoprecipitated GFP was used as a loading control. Total levels of STIM1 and GRP75 in WCL (30 μg protein/lane) were analyzed by immunoblotting (Fig. ). ( C ) Immunoprecipitation of GFP-tagged proteins was performed using 1 mg WCL from STIM1-KO HEK293 inducibly expressing STIM1-GFP, STIM1(Δ551-685)-GFP (mutant 3), STIM1(Δ551-642)-GFP (mutant 4), STIM1(Δ551-611)-GFP (mutant 5), STIM1(Δ582-642)-GFP (mutant 6), STIM1(Δ672-685)-GFP (mutant 9), or GFP only as a control. Other conditions as stated for ( B ). Total levels of STIM1 and GRP75 in WCL were analyzed by immunoblotting (30 μg protein/lane) (Fig. ). ( D ) STIM1-KO HEK293 cells inducibly expressing full-length (FL) STIM1-GFP or STIM1(Δ551-611)-GFP were fixed and incubated with rabbit anti-GRP75 and/or sheep anti-GFP antibodies. Negative controls consisted of cells incubated with single primary antibodies. Scale bar = 10 μm. ( E ) Quantification of the interactions per cell detected by PLA in ( D ). The number of cells evaluated is indicated in parentheses, and the mean is represented by the black line. Statistical analysis with unpaired t-test, p < 0.0001 and p = 0.5354 (n.s.). ( F ) STIM1-KO HEK293 cells expressing STIM1-GFP (full-length, FL) or STIM1(Δ551-611)-GFP were incubated with a rabbit anti-PTPIP51 and mouse anti-VAPB antibody for a PLA assay. Negative controls are shown in Fig. . Scale bar = 10 μm. ( G ) Quantification of the PLA assay (number of red dots per cell) shown in ( F ). The number of cells evaluated is indicated in parentheses, and the black line represents the mean of the data. Statistical analysis with unpaired t-test, p = 0.0016. ( H ) HEK293 cells expressing STIM1(full-length)-GFP were transfected for transient expression of a Flag-tagged STIM1 peptide encompassing residues 551-611. After 24 h of transfection, STIM1-GFP was immunoprecipitated and co-precipitated GRP75 was assessed by immunoblotting. Total levels of GRP75 and STIM1-GFP from WCL are shown in Fig. . ( I ) Quantification of co-precipitated GRP75 from 2 independent experiments and 3 technical replicates. Statistical analysis with unpaired t-test, p = 0.0072. Data are plotted as mean ± S.D. ( J ) HEK293 cells stably and inducibly expressing mito 4× -GCaMP6f were transiently transfected for the expression of mCherry-tagged STIM1 peptide corresponding to residues 551-611. The peptide was tagged with mCherry to select transfected cells, and the ratios F/Fr were calculated in mCherry-expressing cells only. The graph shows Fmax/Fr after the addition of ATP+CCh in Ca 2+ -free medium. Statistical analysis with unpaired t-test, p = 0.0407. Dots represent data from individual cells. ( K ) Analysis of the area under the curve (AUC) of the fluorescence profile after the ATP+CCh stimulus. A total of 16 independent experiments were performed in control (WT) cells ( n = 67 cells) and 17 experiments for cells expressing the peptide (551-611)-mCherry ( n = 44 cells). The mean of the data is represented by the black line. Statistical analysis with unpaired t-test, p = 0.0021. ( L ) HEK293 cells inducibly expressing the Flag-tagged STIM1(551-611)-peptide or HEK293 control cells (with no peptide expression) were fixed and incubated with anti-VAPB and anti-PTPIP51 antibodies and PLA reagents, as in ( F ). Scale bar = 10 μm. ( M ) Quantification of VAPB-PTPIP51 interactions per cell from ( L ). The number of cells evaluated is indicated in parentheses, and the black line represents the mean of the data. Statistical analysis with unpaired t-test, p < 0.0001. ( N ) Wild-type HEK293 cells inducibly expressing the Flag-STIM1(551-611) peptide (red line) and loaded with fura-2 were assessed for the extension of SOCE and compared to cells with no peptide expression (black line). A total number of 51 (WT) and 49 (WT + peptide) cells were analyzed from 3 independent experiments. Data are plotted as mean ± S.D. .

Article Snippet: The vector for the inducible expression of the Ca 2+ sensor mito 4× -GCaMP6f was generated by inserting the Bam HI- Apa I fragment from the construct CMV-mito 4× -GCaMP6f (Addgene #127870) into the Bam HI- Apa I sites of the pcDNA5-FRT/TO-FLAG vector.

Techniques: Generated, Immunoprecipitation, Expressing, Mutagenesis, Plasmid Preparation, Control, Western Blot, Positive Control, Incubation, Transfection, Stable Transfection, Fluorescence