Speciální metody fyziologie živočichů Vítězslav BRYJA Ústav experimentální biologie, PřF MU & Oddělení cytokinetiky, Biofyzikální ústav AV ČR Metody studia buněčné signalizace •Wnt signalling •- eg. Wnt-1 or Wnt-3a frogs •Consequences of b-catenin activation in the brain: •Chenn & Walsh, 2002: Science;297:365-369. •Zechner et al., 2003: Dev. Biol.;258:406-418. •midbrain (Brn4-promotor) •cortex (nestin enhancer) exencephaly •Non-canonical/PCP (Planar cell polarity) pathway defects cause neural tube closure •phenotypes in mouse (and human) •Exencephaly •Hamblet et al., 2002, Development •Open neural tube antibody •Protilátka (imunoglobulin) Metoda 1: Western blotting Western wb-antibody •+ •- •Wnt-5a •ABC •Dvl3 •Dvl2 • • •+ •- •Wnt-3a • • • • • • •ABC – active b-catenin = b-catenin dephosphorylated on GSK3b target sites •Dvl – Dishevelled – activated by phosphorylation detected as phosphorylation dependent mobility shift •PS-Dvl • Compound Target Concn Activity PTX Galpha i/o 100 ng/ml No PDBu PKC activator 1 µM No Wortmannin PI3K 50 nM No LY294002 PI3K 50 µM No PD98059 MEK1/2 10 µM No UO126 MEK1/2 10 µM No SB203580 p38 10 µM No JNKII inhib JNK 6 µM No Genistein PKC 50 µM No chelerythrine PKC 10 µM No Ro-31 8220 PKC 1 µM No BIM I PKC 500 nM No KN93 CamKII 10 µM No I3M GSK-3 2 µM No Kenpaullone GSK-3 6 µM No H89 PKA 10 µM No 8-Br-cAMP cAMP pathway activator 10 µM No 8CPT-2Me-cAMP EPAC activator 30 µM No SQ22536 Adenylyl cyclase 100 µM No MDL12330 Adenylyl cyclase 10 µM No PP2 Src-like 10 µM No AG1276 EGFR 10 µM No ET-18-OCH3 PLC 10 µM No D4476 Casein kinase 1 100 µM Yes staurosporin Ser/Thr kinases, PKC 2 µM No •Bryja et al., J. Cell Sci., 2007 •D4476 (100 mM) •CHAPS •+ •- •+ •- •+ •- •Wnt-5a •Wnt-3a •Dvl2 • • •Both Wnt-3a and Wnt-5a activate Dvl2 and Dvl3 via casein kinase 1 (CK1) Metoda 2: Immunoprecipitace immunoprecipitation •WB: HA •TCL: •Flag-Dvl3 mutants •100 •75 •50 •37 •75 •37 •kDa •100 •50 •1 •2 •3 •4 •5 •6 •7 •DEP •WB: Flag •WB: Flag •Flag-Dvl3 constructs • •DEP •PDZ •DIX •FLAG • •DEP •PDZ •FLAG • •DEP •FLAG • •FLAG • •DIX •FLAG • •PDZ •DIX •FLAG • •DEP •PDZ •DIX •FLAG •1 •2 •3 •4 •5 •6 •7 •+ •+ •- •- •+ •+ •+ • •CK1 β-arrestin binds Dishevelled Metoda 3: Immunocytochemie ihc Fluorescenční proteiny gfpspectra •Dvl2-myc •barr2-GFP •merge β-arrestin co-localizes with Dvl in the cytoplasm Metoda 4: Reportérové eseje luciferase reporter •Is this relevant for Wnt signal transduction in vivo? •1. β-arrestin is required for β-catenin activation in vitro •Wnt-3a •- •+ •ABC •actin •- •+ •P-LRP6 •wt •barr1/2DKO •MEFs: •b-catenin •TopFlash reporter - b-catenin transcriptional activity •b-arr1 •b-arr2 •β-arrestin deficient MEFs Metoda č. 5: Analýza in vivo - drápatka MCBrevision_targeting injections •Is β-arrestin important for the Wnt/β-catenin signalling in vivo? •β-arrestin knockdown in Xenopus (axis duplication assay): embyrevisedembedded •Keller explants (Xenopus) control •CE •β-arrestin MO •control MO •β-arrestin regulates convergent extension movements in vivo keller%20explant Metody č. 6: Genetické modifikace myši Transgenní myš mouse transgenesis •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“ •WT •Lrp6 -/- •#1 •#2 Lrp6minusInt1plusminus •Lrp6 KO embryos display exencephaly.... •....which is rescued by loss of Wnt5a Metody č. 7: Afinitní purifikace a hmotnostní spektroskopie mso6078A mso2C273 •Dvl2 •Dvl3 •Afinitní purifikace Hmotnostní spektroskopie (Mass Spec) mass spec placka-south-park-postava [obrazky_4ever_sk]%20mys,%20past,%20spion,%20syr,%20mysion%20impossible%207410161 •Děkuji za pozornost! •Molekulární mechanismus •Celogenomové techniky •Celoproteomové techniky •Experimentální model • (in vitro - tkáňové kultury, in vivo - myš, C. elegans, Xenopus) •Pacient