Akt-mediated phosphorylation of MICU1 regulates mitochondrial Ca2+ levels and tumor growth (2018)
Figure 1

Figure 1. MICU1 phosphorylation at the Ser124 position increases the mitochondrial basal Ca2+ levels

Potentially phosphorylated residues in the MICU1 (NM_144822) sequence were detected using the Scansite 3 software (http://scansite3.mit.edu). The different values refer to the surface accessibility scores (Scansite) or the phosphorylation scores, which were obtained with both NetPhos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) and NetPhorest (http://www.netphorest.info) software.

HeLa cells overexpressing the HA-tagged MICU1 WT, MICU1 S124D or MICU1 S124A mutants were stained for HA or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm.

Representative images of the 2mt-GCaMP6m 474/410 ratio of ShMICU1 HeLa stable cells expressing an empty vector (ctrl) or the MICU1 WT, MICU1 SD, and MICU1 SA. Scale bar 10 μm.

Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA control (plko) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=5 independent experiments; 55-67cells).

Potentially phosphorylated residues in the MICU1 (NM_144822) sequence were detected using the Scansite 3 software (http://scansite3.mit.edu). The different values refer to the surface accessibility scores (Scansite) or the phosphorylation scores, which were obtained with both NetPhos 3.1 (http://www.cbs.dtu.dk/services/NetPhos/) and NetPhorest (http://www.netphorest.info) software.

HeLa cells overexpressing the HA-tagged MICU1 WT, MICU1 S124D or MICU1 S124A mutants were stained for HA or HSP60 (mitochondrial marker). Merged images are indicated (merge). Scale bar 10 μm.

Representative images of the 2mt-GCaMP6m 474/410 ratio of ShMICU1 HeLa stable cells expressing an empty vector (ctrl) or the MICU1 WT, MICU1 SD, and MICU1 SA. Scale bar 10 μm.

Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial targeted GCaMP6m, in ShRNA control (plko) or ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs (n=5 independent experiments; 55-67cells).

(E and F) Representative kinetics (E) and analysis (F) of aequorin-based [Ca2+]m measurements in ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 20 μM 2,5-di-tert-butylhydroquinone (TBHQ) in the absence of extracellular Ca2+ (n=3 independent experiments).

(G and H) Representative kinetics (G) and analysis (H) of aequorin-based [Ca2+]m measurements in intact ShRNA MICU1 HeLa stable clone cells transfected with the indicated constructs and challenged with 10 μM cyclopiazonic acid (CPA) in the presence of 100 μM EGTA (n=3 independent experiments).

(I) Western blot analysis for the presence of MICU1 in clones arising from single cells generated by CRISPR/Cas9-mediated genome editing. The results for 12 of the 36 clones that were examined are shown.

(J) Resting mitochondrial calcium levels, evaluated through ratiometric imaging of the mitochondrial-targeted GCaMP6m, in MICU1-KO cells generated using the CRISPR/Cas9 technique and transfected with the indicated constructs (n=3 independent experiments; 30-56 cells).

(K and L) Representative kinetics (K) and analysis (L) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 20 μM TBHQ in the absence of extracellular Ca2+ (n=3 independent experiments.).

(M and N) Representative kinetics (M) and analysis (N) of aequorin-based [Ca2+]m measurements in intact MICU1-KO cells generated using the CRISPR/Cas9 technique, transfected with the indicated constructs and challenged with 10 μM CPA in the presence of 100 μM EGTA (n=3 independent experiments).

Data information: (D, F, H, J, L, N) Means ± SEMs. ***p<0.001; ****p<0.0001 (one-way ANOVA).