After washing, the cells were incubated with Alexa Fluor 488 or 555-conjugated secondary antibodies at space temperature (RT) for 1?h. part in sensory understanding and signal transduction. Mitochondria, the powerhouse of the cell, control cell survival, and death. The cellular ability to remove dysfunctional CGP60474 mitochondria through mitophagy is definitely important for cell survival. We show here that mitochondrial stress, caused by respiratory complex inhibitors and excessive fission, robustly stimulates ciliogenesis in different types of cells including neuronal cells. Mitochondrial stress-induced ciliogenesis is definitely mediated by mitochondrial reactive oxygen species generation, subsequent activation of AMP-activated protein kinase and autophagy. Conversely, abrogation of ciliogenesis compromises mitochondrial stress-induced autophagy, leading to enhanced cell death. In mice, treatment with mitochondrial toxin, MPTP elicits ciliary elongation and autophagy in the substantia nigra dopamine neurons. Blockade of cilia formation in these neurons attenuates MPTP-induced autophagy but facilitates dopamine neuronal loss and motor disability. Our findings demonstrate the important role of main cilia in cellular pro-survival responses during mitochondrial stress. siRNA treatment in SH-SY5Y and RPE cells. Successful induction of mitochondrial fusion and fission was confirmed by examining the mitochondrial morphology using Mito Tracker staining and the expression CGP60474 of OPA1 and Drp1 (Fig. ?(Fig.2a2a and Supplementary Fig. 1). Mitochondrial fission via OPA1 depletion robustly increased ciliogenesis, whereas mitochondrial fusion following Drp1 depletion experienced a minimal effect on ciliary frequency and lengths in either cell type (Fig. ?(Fig.2a2a and Supplementary Fig. 2). We further evaluated changes in the primary cilia of siRNA and the mitochondrial fission inhibitor Mdivi-1 significantly suppressed ciliogenesis as well as mitochondrial fragmentation (Fig. ?(Fig.2c).2c). These data show that mitochondrial fusion and fission control main ciliogenesis in reverse directions. Open in a separate windows Fig. 2 Mitochondrial fission induces RCBTB2 ciliogenesis.a Effects of mitochondrial fusion induced by siRNA (sisiRNA (sior siwere stained with a MitoTracker (white), ARL13B (green), and Hoechst 33342 dye (blue). b transfection or Mdivi-1 (10?M). Representative cilia images are offered. Cilia measurement data were obtained from about 200 cells per group and the experiments were repeated at least three times. Data are the mean??SEM. *siRNA treatment. The measurement of mtROS via the expression of a mitochondrial hydrogen peroxide sensor (MT-HyPer) revealed that NAC completely blocks mtROS overproduction caused by rotenone, MPP+, and the knockdown of OPA1 (Fig. ?(Fig.3a).3a). A striking loss of rotenone- and OPA1 knockdown-induced ciliogenesis was observed in NAC-treated SH-SY5Y and RPE cells (Fig. ?(Fig.3b3b and Supplementary Fig. 4). NAC treatment also suppressed mitochondrial fission induced by OPA1 knockdown or rotenone in SH-SY5Y cells (Supplementary Fig. 5). These results suggest that mtROS critically mediates mitochondrial stress-induced ciliogenesis. Open in a separate window Fig. 3 Mitochondrial ROS and AMPK mediate mitochondrial stress-induced ciliogenesis.a, b SH-SY5Y cells were transfected with scrambled control siRNA (Sc) or siRNA against (sior rotenone in SH-SY5Y cells. Main cilia were immunostained with ARL13B antibody CGP60474 (green) and the nucleus was counterstained with Hoechst 33342 dye (blue). c SH-SY5Y cells transfected with sifor 3 days or treated with rotenone (200?nM) were analyzed by Western blotting with a phosphorylated-AMPK (p-AMPK) (T172) antibody. d SH-SY5Y cells transfected with Sc or siRNA for (sidouble-knockout (DKO) MEFs were treated with rotenone or MPP+. After 24?h, the cells were stained with ARL13B (green), Hoechst 33342 dye (blue). Level bar, 5?m. Experiments were repeated at least three times. Data are the mean??SEM of about 200 cells per group. *siRNA or rotenone treatment increased AMPK -subunit (T172) phosphorylation, a marker of AMPK activation, in CGP60474 SH-SY5Y cells (Fig. ?(Fig.3c).3c). We thus tested whether AMPK is an important downstream mediator of mitochondria stress-related ciliogenesis. The downregulation of AMPK completely inhibited rotenone- or MPP+-induced ciliary changes in SH-SY5Y cells (Fig. ?(Fig.3d3d and Supplementary Fig. 6). Consistently, double-knockout (DKO) MEF cells failed to increase cilia formation or growth in response to OPA1 depletion, rotenone, or MPP+ treatment (Fig. ?(Fig.3e3e and Supplementary Fig..