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Speciální metody fyziologie živočichů

Speciální metody fyziologie živočichů. Metody studia Wnt signalizace. Vítězslav BRYJA. Ústav experimentální biologie, PřF MU & Odd ělení cytokinetiky, Biofyzikální ústav AV ČR. Wnt signalling. - eg. Wnt-1 or Wnt-3a. Chenn & Walsh, 2002: Science;297:365-369.

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Speciální metody fyziologie živočichů

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  1. Speciální metody fyziologie živočichů Metody studia Wnt signalizace Vítězslav BRYJA Ústav experimentální biologie, PřF MU&Oddělení cytokinetiky, Biofyzikální ústav AV ČR

  2. Wnt signalling - eg. Wnt-1 or Wnt-3a

  3. Chenn & Walsh, 2002: Science;297:365-369. Zechner et al., 2003: Dev. Biol.;258:406-418. Consequences of -catenin activation in the brain: midbrain (Brn4-promotor) cortex (nestin enhancer)

  4. Non-canonical/PCP (Planar cell polarity) pathway defects cause neural tube closure phenotypes in mouse (and human) Exencephaly Open neural tube Hamblet et al., 2002, Development

  5. Protilátka (imunoglobulin)

  6. Metoda 1: Western blotting

  7. Wnt-5a Wnt-3a - - + + Dvl2 Dvl3 ABC ABC – active -catenin = -catenin dephosphorylated on GSK3 target sites Dvl – Dishevelled – activated by phosphorylation detected as phosphorylation dependent mobility shift CHAPS Wnt-3a Wnt-5a - - D4476 (100 M) - + + + PS-Dvl Dvl2 Both Wnt-3a and Wnt-5a activate Dvl2 and Dvl3 via casein kinase 1 (CK1) Bryja et al., J. Cell Sci., 2007

  8. Metoda 2: Immunoprecipitace

  9. Flag-Dvl3 mutants Flag-Dvl3 constructs kDa + 100 1 DIX PDZ DEP FLAG + 2 TCL: PDZ DEP - FLAG 75 3 1 2 3 4 5 6 7 DEP - FLAG 4 WB: Flag FLAG + 5 DIX FLAG + 6 DIX PDZ FLAG + CK1 50 7 DIX PDZ DEP FLAG 37 DEP 100 75 WB: Flag 50 IP: HA- -arrestin 37 WB: HA β-arrestin binds Dishevelled

  10. Metoda 3: Immunocytochemie

  11. Fluorescenční proteiny

  12. barr2-GFP Dvl2-myc merge β-arrestin co-localizes with Dvl in the cytoplasm

  13. Metoda 4: Reportérové eseje

  14. MEFs: wt barr1/2DKO Wnt-3a β-arrestin deficient MEFs P-LRP6 ABC b-arr1/2dKO b-arr2KO b-arr1KO 1. β-arrestin is required for β-catenin activation in vitro b-catenin wt b-arr1 actin b-arr2 - - + + TopFlash reporter - -catenin transcriptional activity Is this relevant for Wnt signal transduction in vivo?

  15. Metoda č. 5: Analýza in vivo - drápatka

  16. Is β-arrestin important for the Wnt/β-catenin signalling in vivo? β-arrestin knockdown in Xenopus (axis duplication assay):

  17. β-arrestin regulates convergent extension movements in vivo control MO β-arrestin MO Keller explants (Xenopus) CE

  18. Metody č. 6: Genetické modifikace myši

  19. Transgenní myš Nobelova cena 2007 Mario R. Capecchi, Martin J. Evans and Oliver Smithies za „principles for introducing specific gene modifications in mice by the use of embryonic stem cells“

  20. Lrp6 KO embryos display exencephaly.... WT Lrp6 -/- #1 #2 ....which is rescued by loss of Wnt5a

  21. Metody č. 7: Afinitní purifikace a hmotnostní spektroskopie

  22. Afinitní purifikace pGlue pGlue-Dvl2 pGlue-Dvl3 Dvl2 Dvl3

  23. Hmotnostní spektroskopie(Mass Spec)

  24. Děkuji za pozornost! Celogenomové techniky Experimentální model (in vitro - tkáňové kultury, in vivo - myš, C. elegans, Xenopus) Molekulární mechanismus Pacient Celoproteomové techniky

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