Fig. 8

Increased cytoplasmic Ca²⁺ load induces mitochondrial fragmentation. a, b Representative traces and quantitative analysis of the average cytoplasmic Ca²⁺ peak showing enhanced cytoplasmic Ca²⁺ load in Mul1-deficient neurons. Cortical neurons were co-transfected at DIV7-8 with pcDNA-D3cpv and Flag or Flag-MUL1ΔRing, followed by live imaging at DIV12-13 just before mitochondrial fragmentation. The emission spectra of the ratiometric probe (525 nm/458 nm) were obtained by exciting 405 nm following stimulation with 10 μM histamine. Note that Mul1-deficient neurons display an increased cytoplasmic Ca²⁺ peak (p = 0.01) following histamine-induced Ca²⁺ release from the ER. Data were analyzed from 2 to 3 live neurons per field with 3 to 4 fields per experiment and the number of trials is indicated in the bars. c, d The role of calcineurin activity in mitochondrial fragmentation in Mul1-depleted neurons. Cortical neurons, co-transfected with DsRed-Mito and Mul1shRNA at DIV7-8, were treated with CsA (0.5 μM) or FK506 (0.6 μM) for 48 h starting at DIV12. Note that blocking calcineurin activity by CsA or FK506 abolishes mitochondrial fragmentation in Mul1-depleted neurons at DIV14. All data were analyzed from the total number of neurons indicated in the bars in three experiments and are expressed as mean ± s.e.m. Unpaired Student’s t-test (b) or Ordinary one-way ANOVA with Dunnett’s post hoc test (d). Scale Bars: 10 μm