Speciální metody fyziologie živočichů, 4.5.2011 Vítězslav BRYJA Ústav experimentální biologie, PřF MU & Oddělení cytokinetiky, Biofyzikální ústav AV ČR Metody studia Wnt signalizace Wnts (Wingless/Int) - family of ligands - 19 members in human and mouse - glycosylated and palmitoylated extracellular proteins - short range of action, bind to extracellular matrix - only in multicellular animals canonical (eg. Wnt-1 or Wnt-3a) non-canonical (eg. Wnt-5a) Canonical Wnt signalling - eg. Wnt-1 or Wnt-3a - induce axis duplication in Xenopus - induce transformation of mammary cell line C57mg -signals via nuclear translocation of b-catenin - frogs Canonical Wnt signalling and tumours according to Beachy et al., Nature 2004 Reya & Clevers 2005, Nature Lo Celso, C. L. et al. Development 2004;131:1787-1799 Consequences of b-catenin activation in the epidermis (keratin 14 promotor): 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) Non-canonical Wnt signalling - e.g. Wnt5a - do not induce axis duplication in Xenopus - do not induce transformation of mammary cell line C57mg - do not signal via nuclear translocation of b-catenin - frogs-gastrulation Drosophila – PCP (planar cell polarity) PCP scheme drosophila wing 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 Vincan, 2004, Frontiers in Bioscience DISHEVELLED (Dvl – mouse, XDsh – Xenopus, Dsh – Drosophila) -phosphoprotein – phosphorylated by numerous kinases, significance often unknow - acts between Frizzled and downstream signalling components - required for signal transduction of all Wnt signalling pathways Fundamental question of Wnt signalling: How the specificity is achieved? 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: Immunocytochemistry ihc Fluorescenční proteiny gfpspectra Dvl2-myc barr2-GFP merge β-arrestin co-localizes with Dvl in the cytoplasm Metoda 4: Reporter assays 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: In vivo analysis in Xenopus MCBrevision_targeting injections Metoda č. 5: In vivo analysis in Xenopus 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 Genotype Wnt5a -/- Lrp6+/+ Wnt5a -/- Lrp6+/- Wnt5a+/+ Lrp6 -/- Wnt-5a +/- Lrp6-/- others outflow tract defects/total number of embryos 7/7 8/8 6/6 2/4 0/6 Fig4B2 Fig4A2 Lrp6 and Wnt5a KO show similar heart abnormalities associated with non-canonical Wnt signalling defects; heart abnormalities in Lrp6 KO are 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