Presentation on theme: "Characterization of Apoptosis Inducing Factor (AIF) in the Drosophila Visual System Zachary Lemmon and Joseph E. O’Tousa. Biological Sciences, University."— Presentation transcript:
Characterization of Apoptosis Inducing Factor (AIF) in the Drosophila Visual System Zachary Lemmon and Joseph E. O’Tousa. Biological Sciences, University of Notre Dame, Notre Dame, IN. Introduction We are investigating the localization and apparent activity of Apoptosis Inducing Factor (AIF) in both wild type photoreceptors and photoreceptors undergoing age dependent retinal degeneration. AIF is a phylogenically ancient flavoprotein that localizes to mitochondria with an amino terminal mitochondrial localization sequence (MLS) consisting of the first 162 amino acids of the protein. Mature protein is formed by proteolytic cleavage of the MLS in mitochondria (Cande et al. 2002). AIF has putative roles in electron transfer and apoptotic processes. Electron transport activities of AIF are conducted by FAD-binding and NADH-binding domains (aa 184-540). Upon cellular stress, AIF is released from the mitochondria and relocated to the nucleus, where it promotes chromatin alteration and cell death through an unknown mechanism that may involve direct DNA interaction or recruitment of various factors such as nucleases (Mate et al. 2002). The mechanism is speculated to involve exposed positively charged residues in the protein and an AIF specific insertion of 50 amino acids in the carboxy terminal domain (aa 572-622) (Cande et al. 2002). Previous work on AIF focuses on mammalian cell culture (Cande et al. 2002), biochemical (Mate et al. 2002), and mouse AIF models (Cheung et al. 2006). Several of these studies have shown AIF can be redirected to the nucleus and trigger apoptosis by removal of the amino MLS. To study AIF function in adult Drosophila photoreceptors, three mRFP-tagged AIF transgenic strains (full length AIF, AIF amino terminal domain, and AIF carboxy terminal domain) were generated. As predicted by previous studies, we show that the amino terminal domain and full length constructs specify mitochondrial localization in wild type Drosophila photoreceptors. In wild type photoreceptor cells, the carboxy terminal AIF construct failed to localize to mitochondria. We also used a recessive lethal AIF piggyback insertion allele (CG7263 e04281 ) in a genetic mosaic to study the effect of AIF loss of function on photoreceptor development and degeneration. Ommatidial Whole Mount Fluorescent Microscopy Investigation of AIF in Drosophila Photoreceptor Cells Figure 1: AIF Structure and Function. The 162 amino acid N-terminal mitochondrial localization sequence directs AIF to mitochondria. Cleavage of the N-terminal sequence results in the active form of AIF responsible for electron transport (FAD/NADH-Binding Domains) and apoptotic (C- terminus) functions. Upon apoptotic stress, permeabilization of the mitochondrial membrane occurs and causes AIF release and relocalization to the nucleus, where it takes part in an unknown apoptotic mechanism. Figure 2: AIF transgene constructs. Three AIF constructs were produced using standard PCR cloning methods and the Drosophila Gateway Cloning System. The constructs consisted of a UAS promoter, AIF specific sequence, and carboxy terminal mammalian RFP tag. A mitochondrial GFP tagged protein, mitoGFP (A. Pilling and B. Saxton), was used for the purpose of colocalization and was also under control of the UAS promoter. Expression of RFP and GFP tagged proteins was specifically driven in adult photoreceptor cells with GAL4 under control of the rhodopsin promoter (Rh1). Retinal Structure Disrupted by AIF Loss of Function Conclusions Works Cited The amino terminal domain of AIF is sufficient to target the protein into mitochondria. The AIF full length-RFP gene construct also is correctly targeted to mitochondria. This reagent now puts us in position to assess the movement of AIF during the retinal disease process. Carboxy terminal AIF does not localize to mitochondria, nor predominately to the nucleus as predicted by mammalian cell culture work. Currently we are investigating its role as a trigger of retinal degeneration. Altered eye morphology in AIF genetic mosaics indicates a cellular defect, likely impacting mitochondrial function, due to AIF mutation. Cande, Celine, F. Cecconi, P. Dessen, and G. Kroemer. 2002. Apoptosis-inducing factor (AIF): key to the conserved caspase-independent pathways of cell death? Journal of Cell Science, 115: 4727-4734. Cande, Celine. I. Cohen, E. Daugas, L. Ravagnan, N. Larochette, N. Zamzami, and G. Kroemer. 2002. Apoptosis-inducing factor (AIF): a novel caspase- independent death effector released from mitochondria. Biochimie, 84: 215-222. Mate, Maria. M. Ortiz-Lombardia, B. Boitel, A. Haouz, D. Tello, S. Susin, J. Penninger, G. Kroemer, and P. Alzari. 2002. The crystal structure of the mouse apoptosis-inducing factor AIF. Nature Structural Biology, 9 (6): 442-446. Cheung, Eric. N. Joza, N. Steenaart, K. McClellan, M. Neuspiel, S. McNamara, J. MacLaurin, P. Rippstein, D. Park, G. Shore, H. McBride, J. Penninger, and R. Slack. 2006. Dissociating the dual roles of apoptosis-inducing factor in maintaining mitochondrial structure and apoptosis. The EMBO Journal, 25 (17): 4061-4073. Figure 7: Eye phenotype observed in AIF mutant. CG7263 e04281 is a PBac element insertion in the second intron of AIF. This creates a loss of function homozygous lethal mutation. For this reason, FLP/FRT site somatic recombination was used to study the effect of AIF loss of function in the adult retina. Genetic mosaic flies (panel 7b) showed reduced eye size and a rough eye phenotype in comparison to an FRT CG7263 e04281 control (panel 7a). A GMR-Hid eye (panel 7c) is shown for comparison. Amino Terminal AIF-RFP Sufficient for Mitochondrial Localization Carboxy Terminal AIF-RFP does not localize to the mitochondria The Action of Apoptosis Inducing Factor (AIF) Full Length AIF-RFP Localizes to Mitochondria Figure 4: Amino terminal AIF specifies mitochondrial localization. The green fluorescent image shows mitochondria in the photoreceptor cells marked by a GFP tagged protein (panel 4a). A complementary red fluorescent image shows similar expression of the mRFP tagged AIF construct (panel 4b). A merged photograph combining the green, red, and blue (DAPI) fluorescent images shows strong colocalization of the amino terminal AIF construct and mitochondrial GFP marker (panel 4c). This shows that the amino terminal AIF construct, consisting of the MLS, is sufficient for mitochondrial localization. 4a 4b4c 7a 7b 7c 6a 6b 6c FRT CG7263 e04281 SM1, Cy FRT CG7263 e04281 GMR-Hid 40A FRT SM1, Cy GMR-Hid 40A FRT AIF insertion heterozygotes have normal eye structure. AIF insertion homozygotes (eyFLP retina mosaic) disrupt retinal development. GMR-Hid, in non- mosaic flies, eliminates eye tissues Figure 6: Localization of the Carboxy Terminal AIF-RFP construct. with a mitochondrial GFP marker. Localization of the carboxy terminal AIF construct was expected to be nuclear and cytosolic. Fluorescent pictures of the ommatidial bundles show the expected punctate green fluorescence (panel 6a) for the mitochondrial GFP marker. Red fluorescent pictures of the carboxy terminal AIF construct show a weak signal (panel 6b) that could be indicative of cytosolic localization, but there is no indication of nuclear localization. A merged image (panel 6c) of the green, red, and blue (DAPI) signal clearly shows carboxy AIF does not localize to the mitochondria. Figure 5: Full length AIF construct suggests mitochondrial localization. The AIF full length transgene shows a punctate expression pattern similar to amino terminal AIF, suggesting full length AIF also localizes to mitochondria. There are no green fluorescent images due to complications with the cross. Figure 3: Localization of cellular structures in photoreceptors. A single ommatidium composed of eight photoreceptors is prepared by manual dissection of the retina and mounted on standard microscope slides with 0.5% paraformaldehyde fix and DAPI mounting media. Fluorescent microscopy is then used to view the localization of various tagged proteins in different cellular structures. This type of preparation allows for colocalization studies using multiple proteins with GFP, RFP, or other fluorescent tags. DAPI (nuclei) DAPI (nuclei)
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