Cryostat eye sections were cut at 10 m and examined using a Zeiss LSM 700 inverted confocal microscope with a plan-Apo 63x NA 1.4 oil-immersion objective. using Chlorprothixene a rabbit anti-OPA1 antibody (abcam ab42364) and revealed using a polyclonal goat anti-rabbit secondary antibody (Dako P0448) coupled to an ECL detection kit (WESTAR?; Supernova HRP Detection Substrate, Geneflow K1-0068) according to the manufacturer’s instructions and imaged with G:BOX (Syngene). Image_2.tif (180K) GUID:?0428E9E4-DC9E-4544-9412-4A1AC5C1F0BF Figure S3: Images from sections of brain and spleen from control, Chlorprothixene RedMIT/GFP-LC3, and OPA1Q285STOP/RedMIT/GFP-LC3. Brain (Bottom) and spleen (Top) from non-fluorescent (control), RedMIT/GFP-LC3 and OPA1Q285STOP/RedMIT/GFP-LC3 mice were sectioned (10 m) and imaged on a Zeiss LSM 700 inverted confocal microscope with a plan-Apo 63x NA 1.4 oil-immersion objective. The fluorescent signals, GFP-LC3 and mitochondrial mRFP, are visible but no clear colocalization between them could be visualized in the views shown. High throughput imaging identified infrequent colocalization that was more marked in OPA1Q285STOP/RedMIT/GFP-LC3 mice than RedMIT/GFP-LC3 mice (Figure ?(Figure5).5). Scale bars = 10 m. Image_3.jpg (355K) GUID:?A717EEB9-9988-488F-AEB8-CD28C37FEB07 Table S1: mRFP and GFP transgenes do not affect mice reproductive success. Table_1.pdf (37K) GUID:?68E221A7-91D8-42EB-86A4-DA1788DA2C5E Video S1: Movie of the RedMIT-GFP-LC3 MEFs. Live RedMIT-GFP-LC3 MEFs have been imaged using a custom Olympus IX81 inverted microscope equipped with temperature control (Solent scientific) every 30 s for 8 h. This can be used to quantify the early stages of mitophagy in real time. Video_1.MOV (1.6M) GUID:?1FDF42B7-0902-463C-9A60-743834B93C13 Abstract Background: Autosomal dominant optic atrophy (ADOA) is usually caused by mutations in the essential gene, OPA1. This encodes a ubiquitous protein involved in mitochondrial dynamics, hence tissue specificity is not understood. Dysregulated mitophagy (mitochondria recycling) is implicated in ADOA, being increased in OPA1 patient fibroblasts. Furthermore, autophagy may be increased in retinal ganglion cells Chlorprothixene (RGCs) of the OPA1Q285STOP mouse model. Aims: We developed a mouse model for studying mitochondrial dynamics in order to investigate mitophagy in ADOA. Methods: We crossed the OPA1Q285STOP mouse with our RedMIT/GFP-LC3 mouse, harboring red fluorescent mitochondria and green fluorescent autophagosomes. Colocalization between mitochondria and autophagosomes, the hallmark of mitophagy, was quantified in fluorescently labeled organelles in primary cell cultures, Chlorprothixene using two high throughput imaging methods Imagestream (Amnis) and IN Cell Analyzer 1000 (GE Healthcare Rabbit polyclonal to ZNF76.ZNF76, also known as ZNF523 or Zfp523, is a transcriptional repressor expressed in the testis. Itis the human homolog of the Xenopus Staf protein (selenocysteine tRNA genetranscription-activating factor) known to regulate the genes encoding small nuclear RNA andselenocysteine tRNA. ZNF76 localizes to the nucleus and exerts an inhibitory function onp53-mediated transactivation. ZNF76 specifically targets TFIID (TATA-binding protein). Theinteraction with TFIID occurs through both its N and C termini. The transcriptional repressionactivity of ZNF76 is predominantly regulated by lysine modifications, acetylation and sumoylation.ZNF76 is sumoylated by PIAS 1 and is acetylated by p300. Acetylation leads to the loss ofsumoylation and a weakened TFIID interaction. ZNF76 can be deacetylated by HDAC1. In additionto lysine modifications, ZNF76 activity is also controlled by splice variants. Two isoforms exist dueto alternative splicing. These isoforms vary in their ability to interact with TFIID Life Sciences). We studied colocalization between mitochondria and autophagosomes in fixed sections using confocal microscopy. Results: We validated our imaging methods for RedMIT/GFP-LC3 mouse cells, showing that colocalization of red fluorescent mitochondria and green fluorescent autophagosomes is a useful indicator of mitophagy. We showed that colocalization increases when lysosomal processing is impaired. Further, colocalization of mitochondrial fragments and autophagosomes is increased in cultures from the OPA1Q285STOP/RedMIT/GFP-LC3 mice compared to RedMIT/GFP-LC3 control mouse cells that were wild type for OPA1. This was apparent in both mouse embryonic fibroblasts (MEFs) using IN Cell 1000 and in splenocytes using ImageStream imaging flow cytometer (Amnis). We confirmed that this represents increased mitophagic flux using lysosomal inhibitors. We also used microscopy to investigate the level of mitophagy in the retina from the OPA1Q285STOP/RedMIT/GFP-LC3 mice and the RedMIT/GFP-LC3 control mice. However, the expression levels of fluorescent proteins and the image signal-to-background ratios precluded the detection of colocalization so we were unable to show any difference in colocalization between these mice. Conclusions: We show that colocalization of fluorescent mitochondria and autophagosomes in cell cultures, but not fixed tissues from the RedMIT/GFP-LC3, can be used to detect mitophagy. We used this model to confirm that mitophagy is increased in a mouse model of ADOA. It will be useful for cell based studies of diseases caused by impaired mitochondrial dynamics. and are present in this facility. Confocal microscopy MEFs were plated onto 0 thickness coverslips and treated as described in the main text. Four percent paraformaldehyde was used for fixation Chlorprothixene (10 min, room temperature). Cells were permeabilized and washed in 0.1% Triton-Tris buffered saline three times before mounting on slides using Vectashield (Vector Labs). Images were acquired on an upright Leica SP5 confocal microscope equipped with the appropriate filters and sequential 488, and 568 nm laser illumination. For mouse eyes, four samples (2x RedMIT-GFP-LC3 and 2x RedMIT-GFP-LC3-OPA1Q285STOP) were harvested from perfused-fixed mice and cryostat eye sections cut at 10 m, lightly counter-stained with DAPI.