Journal: Cell reports
Article Title: MICU2 controls mitochondrial calcium signaling and migration in neurons during development
doi: 10.1016/j.celrep.2025.116583
Figure Lengend Snippet: (A) Generation of Micu2 −/− (KO) mice. The Cre transgene under the EIIa promoter was used to target exon 3 of the floxed Micu2 (WT) gene for removal on the C57BL/6J background, as described previously. (B) Micu2 −/− mice showing Mendelian distribution. (C) Abundance of different mtCU subunits (MICU1, MICU2, MICU3, MCU, and EMRE) quantified by western blot in lysates from DIV11–15 WT and KO cortical neurons under reducing conditions normalized to the mitochondrial mass loading control, TOMM20, and WT. Significant differences ( p < 0.05) between conditions are highlighted in bold. Statistical analysis between the groups was determined using Student’s t test or Welch’s t test if criteria for equal variances are not met; n = 8 (embryos) for each group. (D) Immunodetection of MICU dimers and loading control, TOMM20, by western blot in lysates from WT and KO cortical tissue under non-reducing conditions. Representative membranes are shown. (E) [Ca 2+ ] c signals measured by Cal520 and induced by electrical field stimulation indicated on the graph in intact DIV11–15 WT and KO primary cortical neurons with Cal520 and infected with mtRCaMP; n = 4 (time pregnancies for each group). (F) [Ca 2+ ] m signals measured simultaneously with (E) as mtRCaMP fluorescence intensity and normalized to the baseline; n = 4 (time pregnancies for each group). (G) [Ca 2+ ] m signals measured simultaneously with (E) as mtRCaMP fluorescence lifetime; n = 4 (time pregnancies for each group). (H) Resting [Ca 2+ ] m in DIV11–15 WT and KO primary cortical neurons transfected with mtGEM-GECO1. Statistical analysis between the groups was determined using Mann-Whitney test; n (WT) = 10 and n (KO) = 13 (embryos). (I) Resting [Ca 2+ ] m in DIV11–15 WT and KO primary cortical neurons infected with mtRCaMP and measured as fluorescence lifetime. Statistical analysis between the groups was determined using Student’s t test; n = 4 (time pregnancies for each group). (J) Quantification of the peak for (D). (K) Quantification of the peak for (E). (L) Quantification of the peak for (F). (M) [Ca 2+ ] c signals measured by fura-2 and induced by electrical field stimulation indicated on the graph in intact DIV11–15 WT and KO primary cortical neurons with fura-2 and transfected with 4mtGCaMP6f; n = 5 (time pregnancies for each group). (N) Quantification of AUC for the first stimulation in (M). (O) Quantification of total AUC in (M). (P) [Ca 2+ ] m signals measured simultaneously with (M) as 4mtGCaMP6f fluorescence intensity; n = 5 (time pregnancies for each group). (Q) Quantification of AUC for the first stimulation in (P). (R) Quantification of total AUC in (P). Statistical analysis between the groups was determined using one-tail Welch’s t test in (J)–(L), (N), (O), (Q), and (R). p values are indicated in the graphs. Data are represented as the mean ± SEM. See also and .
Article Snippet: The cells were subjected to electrical field stimulation at 20 and 40 V/cm achieved by passing 2-ms current pulses in a biphasic mode at different frequencies using a platinum field stimulation electrode (RC-49MFSH) and High-Power Bi-Phase Stimulator 701C (Aurora Scientific) under the control of Optogenetic TTL pulse generator with corresponding software (Doric lenses).
Techniques: Western Blot, Control, Immunodetection, Infection, Fluorescence, Transfection, MANN-WHITNEY
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![(A) Generation of Micu2 −/− (KO) mice. The Cre transgene under the EIIa promoter was used to target exon 3 of the floxed Micu2 (WT) gene for removal on the C57BL/6J background, as described previously. (B) Micu2 −/− mice showing Mendelian distribution. (C) Abundance of different mtCU subunits (MICU1, MICU2, MICU3, MCU, and EMRE) quantified by western blot in lysates from DIV11–15 WT and KO cortical neurons under reducing conditions normalized to the mitochondrial mass loading control, TOMM20, and WT. Significant differences ( p < 0.05) between conditions are highlighted in bold. Statistical analysis between the groups was determined using Student’s t test or Welch’s t test if criteria for equal variances are not met; n = 8 (embryos) for each group. (D) Immunodetection of MICU dimers and loading control, TOMM20, by western blot in lysates from WT and KO cortical tissue under non-reducing conditions. Representative membranes are shown. (E) [Ca 2+ ] c signals measured by Cal520 and induced by electrical field <t>stimulation</t> indicated on the graph in intact DIV11–15 WT and KO primary cortical neurons with Cal520 and infected with mtRCaMP; n = 4 (time pregnancies for each group). (F) [Ca 2+ ] m signals measured simultaneously with (E) as mtRCaMP fluorescence intensity and normalized to the baseline; n = 4 (time pregnancies for each group). (G) [Ca 2+ ] m signals measured simultaneously with (E) as mtRCaMP fluorescence lifetime; n = 4 (time pregnancies for each group). (H) Resting [Ca 2+ ] m in DIV11–15 WT and KO primary cortical neurons transfected with mtGEM-GECO1. Statistical analysis between the groups was determined using Mann-Whitney test; n (WT) = 10 and n (KO) = 13 (embryos). (I) Resting [Ca 2+ ] m in DIV11–15 WT and KO primary cortical neurons infected with mtRCaMP and measured as fluorescence lifetime. Statistical analysis between the groups was determined using Student’s t test; n = 4 (time pregnancies for each group). (J) Quantification of the peak for (D). (K) Quantification of the peak for (E). (L) Quantification of the peak for (F). (M) [Ca 2+ ] c signals measured by fura-2 and induced by electrical field stimulation indicated on the graph in intact DIV11–15 WT and KO primary cortical neurons with fura-2 and transfected with 4mtGCaMP6f; n = 5 (time pregnancies for each group). (N) Quantification of AUC for the first stimulation in (M). (O) Quantification of total AUC in (M). (P) [Ca 2+ ] m signals measured simultaneously with (M) as 4mtGCaMP6f fluorescence intensity; n = 5 (time pregnancies for each group). (Q) Quantification of AUC for the first stimulation in (P). (R) Quantification of total AUC in (P). Statistical analysis between the groups was determined using one-tail Welch’s t test in (J)–(L), (N), (O), (Q), and (R). p values are indicated in the graphs. Data are represented as the mean ± SEM. See also and .](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6669/pmc12906669/pmc12906669__nihms-2132728-f0004.jpg)