Speciln metody fyziologie ivoich Metody studia Wnt signalizace

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

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

Consequences of -catenin activation in the brain: midbrain (Brn 4 -promotor) Zechner et al. , 2003: Dev. Biol. ; 258: 406 -418. cortex (nestin enhancer) Chenn & Walsh, 2002: Science; 297: 365 -369.

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

Protilátka (imunoglobulin)

Metoda 1: Western blotting

Wnt-5 a + Wnt-3 a + Dvl 2 Dvl 3 ABC – active -catenin = -catenin dephosphorylated on GSK 3 target sites Dvl – Dishevelled – activated by phosphorylation detected as phosphorylation dependent mobility shift PS-Dvl CHAPS D 4476 (100 M) Dvl 2 - + Wnt-3 a Wnt-5 a - - + + Compound Target Concn Activity PTX PDBu Wortmannin LY 294002 PD 98059 UO 126 SB 203580 JNKII inhib Genistein chelerythrine Ro-31 8220 BIM I KN 93 I 3 M Kenpaullone H 89 8 -Br-c. AMP 8 CPT-2 Me-c. AMP SQ 22536 MDL 12330 PP 2 AG 1276 ET-18 -OCH 3 D 4476 staurosporin Galpha i/o PKC activator PI 3 K MEK 1/2 p 38 JNK PKC PKC Cam. KII GSK-3 PKA c. AMP pathway activator EPAC activator Adenylyl cyclase Src-like EGFR PLC Casein kinase 1 Ser/Thr kinases, PKC 100 ng/ml 1 µM 50 n. M 50 µM 10 µM 6 µM 50 µM 1 µM 500 n. M 10 µM 2 µM 6 µM 10 µM 30 µM 10 µM 100 µM 2 µM No No No No No No Yes No Both Wnt-3 a and Wnt-5 a activate Dvl 2 and Dvl 3 via casein kinase 1 (CK 1) Bryja et al. , J. Cell Sci. , 2007

Metoda 2: Immunoprecipitace

Flag-Dvl 3 mutants 1 2 3 4 5 6 7 k. Da β-arrestin binds Dishevelled 100 TCL: 75 WB: Flag 50 37 Flag-Dvl 3 constructs FLAG DIX PDZ DEP FLAG DIX FLAG DIX PDZ CK 1 DEP + + + DEP 100 75 IP: HA- -arrestin 1 2 3 4 5 6 7 WB: Flag 50 37 WB: HA

Metoda 3: Immunocytochemie

Fluorescenční proteiny

β-arrestin co-localizes with Dvl in the cytoplasm arr 2 -GFP Dvl 2 -myc merge

Metoda 4: Reportérové eseje

1. β-arrestin is required for β-catenin activation in vitro -arr 1 -arr 2 Wnt-3 a - arr 1/2 DKO wt + - + Top. Flash reporter - -catenin transcriptional activity P-LRP 6 - ar r 1 - wt KO ar r 2 KO ar r 1 /2 d. K O β-arrestin deficient MEFs: ABC -catenin actin Is this relevant for Wnt signal transduction in vivo?

Metoda č. 5: Analýza in vivo - drápatka

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

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

Metody č. 6: Genetické modifikace myši

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“

Lrp 6 KO embryos display exencephaly. . WT Lrp 6 -/#1 #2 . . which is rescued by loss of Wnt 5 a

Metody č. 7: Afinitní purifikace a hmotnostní spektroskopie

Dvl 2 Dvl 3 p. Glue-Dvl 2 p. Glue Afinitní purifikace

Hmotnostní spektroskopie (Mass Spec)

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