Technical Reference #1653

1653.

Zyxin Mediates Actin Fiber Reorganization in Epithelial-Mesenchymal Transition and Contributes to Endocardial Morphogenesis Masaki Mori; Hironori Nakagami; Nobutaka Koibuchi; Yoichi Takami; Hiroshi Koritama; Hiroki Hayashi; Hisataka Sabe; Naoki Mochizuki; Ryuichi Morishita; Yasufumi Kaneda, Osaka University, Molecular Biology of the Cell, 20(1653), (2009)
Link To Paper

Abstract:
Epithelial-mesenchymal transition (EMT) confers destabilization of cell-cell adhesion and cellmotility required for morphogenesis or cancer metastasis. Here we report that Zyxin a focaladhesion-associated LIM protein is essential for actin reorganiz

Materials & Methods:
Cell culture NMuMG cells were obtained from ATCC. We cloned the cells by limiting dilution and obtained 13 different clones. Among them we used a cell line designated ‘C7’ that showed typical epithelial morphology and robust response to TGF-β1. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM Nacalai Tesque Japan) supplemented with 10% fetal bovine serum (FBS). TGF-β1-stimulation experiments were performed with recombinant human TGF-β1 (2 ng/ml; R&D systems Minneapolis MN). Deletion mutants and retroviral vector construction Zyxin deletion mutants were generated by polymerase chain reaction (PCR)-amplifying the amino acid 1-375 (ΔLIM) or aa 376-564 (LIM only) region and inserting into pCAGIP-EGFP vector. pCX4 puro vector was kindly provided by Tsuyoshi Akagi (Akagi et al. 2003). pCX4 puro-EGFP-Snail pCX4 puro-EGFP-Twist1 or pCX4 puro-HMGA2 were generated by inserting the full-length Snail cDNA from pCAGGS-EGFP-Snail Twist1 cDNA from pCAGIP-Twist1 (Hayashi et al. 2007) or HMGA2 cDNA from pENTR-HMGA2 into the pCX4 puro vector respectively. Each plasmid above was cotransfected with pGP and pE-eco (TAKARA BIO INC Japan) into BOSC to produce ecotropic retrovirus vector. Immunocytochemistry NMuMG-C7 cells were plated onto collagen-coated glass bottom dishes (MatTek Corporation Ashland MA). The cells were fixed in 4% paraformaldehyde (PFA) permeabilized in 0.1% Triton X-100 for 5 min and probed with mouse anti-Zyxin antibody (1:40 164D4 Synaptic Systems; 1:100 clone ZOL301 Sigma-Aldrich) mouse anti-E-cadherin antibody (1:50 clone 36) mouse 6 anti-N-cadherin antibody (1:50 clone 32 BD Bioscience) and rabbit anti-ZO-1-antibody (1:100 61-7300 Invitrogen). The primary antibody was detected by goat anti-mouse-Alexa 488 goat anti-mouse-Alexa 546 or goat anti-rabbit-Alexa 546 (1:1000 Invitrogen Carlsbad CA). Nuclei were stained with 4’-6-Diamidino-2-phenylindole (DAPI Invitrogen). F-actin was stained with Alexa Fluor® 546-phalloidin (Invitrogen). Fluorescence imaging NMuMG-C7 cells were stably transfected with pCAGIP-EGFP-Zyxin plasmid. Fluorescence images were recorded with a Nikon eclipse TE300 inverted microscope or with a confocal microscope with a Bio-Rad laser scanning system (Radiance 2100) coupled to a Nikon eclipse TE2000-U inverted microscope with a 60× oil immersion objective lens. For time-lapse recording of Zyxin images the cells were grown in 35-mm glass-bottom dishes (MatTek) and placed in a CO2 chamber (Model CZI-3; Zeiss) at 37 °C attached to the stage of an inverted microscope (Axiovert 200; Zeiss). Images were acquired every 3 min using a 40×1.3 NA objective lens (Plan-NEOFLUAR Zeiss) and a CCD camera (model HRm; Zeiss) and analysed using Axiovision software (version 4.6 Zeiss). SDS-PAGE and Western blot analysis Cells were harvested in a RIPA lysis buffer. Protein concentration was determined by the method of Lowry (Lowry et al. 1951). Protein samples (10 μg per lane) were separated by SDS-PAGE and transferred to a Hybond-P PVDF membrane (GE Healthcare Buckinghamshire England). Western blot was performed with the following antibodies: anti-Zyxin (1:1000 ZOL301) anti-vinculin (1:1000 V9264) anti-β-actin (1:5000 AC-15 Sigma-Aldrich) anti-E-cadherin (1:5000 clone 36) anti-N-cadherin (1:5000 clone 32) anti-Paxillin (1:1000 clone 165) anti-FAK (1:200 clone 77) anti-p130Cas (1:1000 clone 21) anti-Hic-5 (1:200 clone 34 BD biosciences) anti-GFP (1:1000 598 MBL) anti-HMGA2 (1:200 ab41878) and anti-GAPDH (1:5000 6C5 Abcam Inc.). All 7 secondary antibodies were horseradish peroxidase conjugated (GE Healthcare) and were used at 1:1000 dilution. Immunoreactive bands were detected with Chemi-LumiOne L (Nacalai Tesque). Quantification was done by densitometry using ImageJ software. Modified Boyden’s chamber assay Modified Boyden’s chamber assay was carried out as previously described (Saito et al. 2006). Briefly 27 μl DMEM with 10% FBS was added to the lower chamber and 50 μl of cell suspension (5 × 105 cells/mL) in DMEM with 10% FBS was added to the upper chamber. After 4 hr of incubation the cells on the lower side of the membrane were stained with Diff-Quick (Sysmex Hyogo Japan) to facilitate visualization and counting of cells. The count was done in eight randomly chosen fields. RNAi experiments Zyxin or Twist1 Stealth siRNA oligonucleotides were purchased from Invitrogen. The dsRNA oligonucleotides against Zyxin were as follows: (sense) 5'-AACAAAUGGAGUGGCAACUGGUG GG-3' and (antisense) 5'-CCCACCAGUUGCCACUCCAUUUGUU-3'. The dsRNA oligonucleotides against Twist1 were: (sense) 5'-UUGAGGGUCUGAAUCUUGCUCAGCU-3' and (antisense) 5'-AGCUGAGCAAGAUUCAGACCCUCAA-3'. The scrambled siRNA oligonucleotides were: (sense) 5'-UUCCUCAAUAGAUGCGUGUTT-3' and (antisense) 5'-ACACGCAUCUAUUGAGGAATT-3'. These sequences did not correspond to any sequence in the mouse genome when subjected to a BLAST search. Transfections were carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. RT-PCR analysis and real-time PCR quantification Total RNA was isolated using an RNeasy RNA isolation kit (QIAGEN Valencia CA). cDNA was 8 synthesized using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems Foster City CA). The sequences of PCR primers are shown in the Supplementary Table І. The reactions were performed as follows: 98°C for 30 s; 25 cycles of 98°C for 10 s 52°C for 30 s and 72°C for 60 s. PCR products were separated in a 1.5% agarose gel. Real-time PCR was performed using an Applied Biosystems 7900 HT Fast Real-Time PCR system with TaqMan Probe for Zyxin (Mm00496120_m1) and GAPDH (Mm99999915_g1) and TaqMan Universal PCR Master Mix (Applied Biosystems). The reactions were incubated at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The relative expression level was calculated from a standard curve obtained by using log dilutions of cDNA containing the gene of interest. The average of two independent analyses for each gene and sample was calculated and was normalized to the endogenous reference control gene GAPDH. In situ hybridization In situ hybridization was performed as previously described (Koibuchi and Chin 2007). The cDNAs used for generation of Dig-labeled mouse riboprobes were Zyxin (nucleotides 2002 to 2404; GenBank no. NM_011777) and Twist1 (nucleotides 644 to 1524; GenBank no. NM_011658). Lentivirus vector pcDNA6.2-shZyxin and pcDNA6.2-shLacZ were generated by inserting the oligonucleotide containing the specific siRNA target sequence into pcDNA6.2 vector (Invitrogen). pLenti6-shZyxin and pLenti6-shLacZ were generated from pcDNA6.2 constructs. Lentivirus was generated by cotransfection of the above construct with packaging plasmids into 293FT cells according to the manufacturer’s instruction (Invitrogen). The sequences of shZyxin DNA oligo and shLacZ control oligo are available in the Supplementary Table ІІ. 9 Ex vivo EMT assay The experiments were approved by the Ethics Committee for animal experiments of Osaka University Graduate School of Medicine. C57BL/6 mice were purchased from Charles River Breeding Laboratories. The ex vivo EMT assay was performed as described (Camenisch et al. 2002) with some modifications. After putting E9.5 AVC explants on collagen gels lentivirus-containing medium was added and incubated for 24 hr. Then the explants were incubated with M199 media (Gibco/BRL Rockville MD) supplemented with 0.01% insulin transferrin and selenium (ITS Gibco/BRL) for 48 hr prior to determining the extent of outgrowth and matrix invasion. The criteria for ‘Migratory’ were the appearance of the stellate cells migrating outward frequently invading the collagen gel matrix. The criteria for ‘Nonmigratory’ were the appearance of polygonal cells that form cobblestone-like colonies on the gel surface (Camenisch et al. 2002). XZ confocal images were acquired to visualize collagen gel invasion. Cells that migrated into the collagen gel were stained with DAPI and counted in randomly collected areas. Phalloidin staining of the explants was done after the fixation with 4% PFA for 20 min on the collagen gel.

Microscopic Technique
Confocal Microscopy

Cell Type(s)
NMuMG