Supplementary MaterialsTransparent reporting form. as a ratio of red to green

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Supplementary MaterialsTransparent reporting form. as a ratio of red to green fluorescence intensity. Compared to age-matched WT/Het siblings, mutants exhibited significantly lower JC-1 fluorescence ratios, indicating that the mitochondria are depolarized (Figure 2C) (WT/Het: 0.25??0.24 n?=?8 fish, Mutant: 0.05??0.07 n?=?8 fish, Mann-Whitney U test, p? ?0.01, mean ratio??SD). These results suggest that mitochondrial activity is reduced in the absence of MET. Open in a separate window Figure 2. Acute mitochondrial activity is reduced in the absence of MET.(A, B) Maximum projections of hair cells from WT/Het and mutant siblings incubated in JC-1 dye. Hair cells were imaged from a dorsal view, as indicated in the schematic shown in Figure 1figure supplement 1B. (C) Mean JC-1 fluorescence plotted as a ratio of red:green. WT/Het: 0.25 0.24 n?=?8 fish; Mutant: 0.05? 0.07 n?=?8 fish; mean ratio??SD. Mann-Whitney U Suvorexant manufacturer test was used to assess significance. Value for each fish represents the mean of 3 neuromasts. Scale bar?=?5 m. Measuring mitochondrial aging and oxidation with mitoTimer The waterjet and calcium imaging studies reveal that mitochondria respond to hair cell MET activity. Ca2+ flux can have multiple effects on mitochondrial function, including regulation of electron transport during oxidative phosphorylation (OXPHOS) and generation of ROS?(Brookes et al., 2004). We next wanted to examine Suvorexant manufacturer whether acute MET activity causes persistent effects on the state of mitochondria. To look at cumulative mitochondrial activity over time, we used a transgenic zebrafish expressing the reporter mitoTimer in all hair cells (Tg[mutant siblings. WTHet: 13.3??0.92; Mut: 0.33??0.27; mean?SD; n?=?7 fish per group; Mann-Whitney U test p?=?0006. Values for each fish represent the mean of 3 neuromasts. Hair cells Suvorexant manufacturer were imaged at 5dpf, just following Hoechst treatment. The lack of labeling in mutant hair cell suggests that Hoechst nuclear labeling is MET-dependent. Figure 4figure supplement 2. Open in a separate window Hoechst incubation does not affect mitoTimer ratio compared to control.Mean mitoTimer fluorescence ratio with and without Hoechst incubation. Incubation: 1.51 . 093, n?=?105 cells; No incubation: 1.48??1.08, n?=?119 cells; mean ratio?SD; Mann-Whitney U test p?=?0.34; five fish, three neuromasts per fish. Blocking mechanotransduction has long-term effects on mitochondria We next tested whether long-term changes in MET activity would result in alterations in mitochondrial activity, reflected by changes in mitoTimer fluorescence. We first assayed this in the absence of hair cell stimulation by measuring mitoTimer fluorescence in mutants. Compared to age-matched WT/Het siblings, the mitoTimer fluorescence ratio was significantly decreased in mutants, with a difference of 66.3% (Figure 5ACC; Mann-Whitney U test, p? ?0.001; n?=?14 WT/Het fish Rabbit Polyclonal to RAB38 and 15 mutant fish; data combined from three experiments). Similar results were obtained when embryos were incubated in the MET-blocking drug benzamil (200 M) for 48 hr (3-5dpf; Figure 5DCF)?(Hailey et al., 2017). Suvorexant manufacturer We observed a reduction of 43.4% relative to 0.5% DMSO control (Mann-Whitney U test, p? ?0.001; n?=?17 fish per group; data combined from two experiments). Open in a separate window Figure 5. Mitochondrial activity depends on hair cell mechanotransduction.(A, B) Maximum projections of hair cells from WT/Het and mutant siblings crossed to Tg[larvae. WT/Het: 100??18.7, n?=?14 fish; Mutant: 34??13.1,15 mutant fish; mean (% WT/Het)SD. Value for each fish represents the mean of 2C4 neuromasts. (D, E) Maximum projections of mitoTimer-expressing hair cells from control larvae and larvae incubated in 200 M benzamil. (F) mitoTimer mean fluorescence ratio for larvae incubated in benzamil compared to DMSO control. Control: 100??28.7; Treated: 56.6??22.7, n?=?17 fish per group; mean (% Control)SD. Value for each fish represents the mean of 2C4 neuromasts. Mann-Whitney U test was used to assess significance. All larvae imaged at 5dpf. Scale bar?=?5 m. Figure 5figure supplement 1. Open in a separate window Reduced hair cell activity does not influence hair cell turnover.Maximum projects of (A) DMSO control and (B) benzamil-treated fish expressing hair cell-specific membrane GFP (Tg[locus (Tg[mutants. Mitochondrially-localized Eos in hair cells was photoconverted at 5dpf and then monitored to measure loss of red fluorescence as an estimate of mitochondrial or mitochondrial protein turnover. Mitochondrial fluorescence was measured at 8 and 16.