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The functional role of Optineurin

The functional role of Optineurin. Formation. Theory Experiments Perspective. Optineurin (OPTN). a cytoplasmic protein ubiquitously expressed in brain , heart , skeletal muscle , placenta and kidney

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The functional role of Optineurin

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  1. The functionalroleofOptineurin Institut der Anatomie Fakultät für Humananatomie und Embryologie Universität Regensburg Biochemisches Praktikum im Jahr 2013 Betreuerin: Christiane Sippl 16.01.2014 Riedlberger Felix

  2. Formation • Theory • Experiments • Perspective

  3. Optineurin (OPTN) • a cytoplasmicproteinubiquitouslyexpressed in brain, heart, skeletalmuscle, placentaandkidney • expressed in severalocularcelltypes, e.g. retinalganglioncells, retinalpigmentepithelium, etc. • itsgenespans a ~37 kbgenomicregionandconsists 13 activeexonscodingfor a 577 aminoacidprotein (human)

  4. Optineurin (OPTN) • Optineurin was foundtobelocalized at Golgi apparatuswithinteractingproteins: Rab8, Huntingtinand Myosin VI • stabilizesmorphologyof Golgi apparatus, whereitisinvolved in vesiculartrafficking • Itisinvolved in a lotofprocesses, e.g. regulationofinterferon

  5. Optineurin (OPTN) • Mutations in OPTN can: • causeglaucoma • Causeamytrophic lateral sclerosis (ALS) • (motorneurondiseasecausedbythedegeneration • ofneurons)

  6. Glaucoma • Progressive opticneuropathywithlossofretinalganglioncells (RGCs) • Results in blindness, ifisleftuntreated • secondleadingcauseofblindness • Most common form (90%): Primary open-angelglaucoma (POAG)

  7. Primary open-angle glaucoma (POAG) • Genes associatedwith POAG: Myocilin, optineurinand WDR36 • OPTN isparticularlylinkedto normal tensionglaucoma • normal visionvisionasitmightbeaffectedbyglaucoma

  8. Study ofOptineurin • through "knockout mice“ (KO-Type) • The mouseorganismoffersparticularadvantages: • themouseis a mammalanditsdevelopment, body plan, physiology, behavioranddiseaseshavemuch in commonwiththoseofhumans • almost all (99%) mouse genes haveallogenics in human • Targetedmutagenesisbyhomologousrecombinatoinpossible

  9. ConditionalOptineurin Knock-Out Mouse ES cells = embryonicstemcells

  10. ConditionalOptineurin Knock-Out Mouse ES cells = embryonicstemcells normal OPTN-sequence

  11. ConditionalOptineurin Knock-Out Mouse + + =

  12. ConditionalOptineurin Knock-Out Mouse + + = “gefloxtes“ OPTN

  13. ConditionalOptineurin Knock-Out Mouse “gefloxtes“ OPTN

  14. ConditionalOptineurin Knock-Out Mouse

  15. ConditionalOptineurin Knock-Out Mouse

  16. OptineurinKnock-Out Mouse 3000 1000 500 400 300 200 100 due to PCR youcan find thegenotypeofnewbornmice control Flox-PCR Only KO ifthisgeneishomozygous!

  17. Knock-Out Mouse 3000 1000 500 400 300 200 100 another type of KO: CTGF-PCR (SV40) control This PCR shows, ifthemice will overexpressthe Protein CTGF CTGF will cause a higherintraocularpressure

  18. Experiments – Arrestin/Tranducin • ArrestinandTransducinhaveimportant • role in convertingthe light stimulus • eitherifitisdarkorbright, Arrestin • orTransducinislocated at OS/IS • transportmechanismneededtomove • theseproteinstotheothersegment • Photoreceptor • Question: does OPTN participate in thistransportmechanism?

  19. Experiments – Arrestin/Tranducin • Execution: • Mice (3 months)weredarkadaptedovernight • 4 KO mice, 4 WT mice • Next day different exposure time at 120 Lux (0, 5, 10, 30 min) • after killing: fix proteins in bodieswith PFA • stainingArrestinandTransducin

  20. Experiments – Arrestin/Tranducin Result:

  21. Experiments – Sperm • watch, if KO spermareaffected in anyway, causeofOPTN • isolationofsperm in theepididymides • watchspermwithphasecontrastmicroscopeandcellculture medium (medium = DMEM: high glucose, 37°C, pH = 7.4) • Result: nodifferences, bothtypesofspermsare mobile and fertil

  22. Experiments – CTGF • Pre-experiment: CTGF-KO mice (4 weeksold) • Assumption: retinabecomesthinner due to CTGF overexpresion • But: retina not thinner! • → oldermice! (6 monthsold)

  23. Experiments – CTGF • 6 monthsold CTGF-KO micegetkilledandfixedwith PFA • watch at numberofaxonsandthicknessofretinawithelectronmicroscope

  24. Experiments – CTGF • → • Retina didn‘tbecomethinner!

  25. Experiments – CTGF • TUNEL-staining • blue: DAPI-staining • (normal nucleus) • green: dieingcell • Onlyifcellsaredieing, theretinabecomesthinner.

  26. Perspective • Try tofigure out, if OPTN participates in thetransportmechanismofarrestin/tranducin • analyze different KO-miceforretinathickness

  27. thankyoufor yourattention!

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