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Immunofluorescence Microscopy to Study Endogenous TAZ in Mammalian Cells

  • Nathan M. Kingston
  • Andrew M. Tilston-Lunel
  • Julia Hicks-Berthet
  • Xaralabos VarelasEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1893)

Abstract

The transcriptional coactivator with PDZ-binding motif (TAZ), which is encoded by the WWTR1 gene, is a key transcriptional effector of the Hippo signaling pathway. TAZ function has been implicated in a variety of developmental processes and diseases, most notably in driving oncogenesis. Given that nuclear-cytoplasmic localization dynamics dictate TAZ activity, techniques for visualizing TAZ localization are critical for its study. Here we describe an immunofluorescence microscopy protocol that allows for the visualization of TAZ subcellular localization in mammalian cells, offering an approach that can aid in the analysis of TAZ regulation and function.

Key words

TAZ WWTR1 Hippo pathway Immunofluorescence microscopy 

Notes

Acknowledgments

We would like to thank Dr. Jeffrey Wrana (Lunenfeld-Tanenbaum Research Institute, Toronto, Canada) for providing the Taz-loxP/loxP mice. X.V. is supported by NIH R01HL124392, the March of Dimes Foundation Grant no. 1-FY17-375, and an American Cancer Society Ellison New England Research Scholar Grant (RSG-17-138-01-CSM). N. M. K. is supported by NIH T32HL007035-40. J.H.B. is supported by NIH F31HL13250601.

References

  1. 1.
    Fu V, Plouffe SW, Guan KL (2018) The Hippo pathway in organ development, homeostasis, and regeneration. Curr Opin Cell Biol 49:99–107.  https://doi.org/10.1016/j.ceb.2017.12.012 CrossRefGoogle Scholar
  2. 2.
    Varelas X (2014) The Hippo pathway effectors TAZ and YAP in development, homeostasis and disease. Development 141(8):1614–1626.  https://doi.org/10.1242/dev.102376 CrossRefPubMedGoogle Scholar
  3. 3.
    Mahoney WM Jr, Hong JH, Yaffe MB, Farrance IK (2005) The transcriptional co-activator TAZ interacts differentially with transcriptional enhancer factor-1 (TEF-1) family members. Biochem J 388(Pt 1):217–225.  https://doi.org/10.1042/BJ20041434 CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Zhang H, Liu CY, Zha ZY, Zhao B, Yao J, Zhao S, Xiong Y, Lei QY, Guan KL (2009) TEAD transcription factors mediate the function of TAZ in cell growth and epithelial-mesenchymal transition. J Biol Chem 284(20):13355–13362.  https://doi.org/10.1074/jbc.M900843200 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Hiemer SE, Szymaniak AD, Varelas X (2014) The transcriptional regulators TAZ and YAP direct transforming growth factor beta-induced tumorigenic phenotypes in breast cancer cells. J Biol Chem 289(19):13461–13474.  https://doi.org/10.1074/jbc.M113.529115 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Levasseur A, St-Jean G, Paquet M, Boerboom D, Boyer A (2017) Targeted disruption of YAP and TAZ impairs the maintenance of the adrenal cortex. Endocrinology 158(11):3738–3753.  https://doi.org/10.1210/en.2017-00098 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Nishioka N, Inoue K, Adachi K, Kiyonari H, Ota M, Ralston A, Yabuta N, Hirahara S, Stephenson RO, Ogonuki N, Makita R, Kurihara H, Morin-Kensicki EM, Nojima H, Rossant J, Nakao K, Niwa H, Sasaki H (2009) The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass. Dev Cell 16(3):398–410.  https://doi.org/10.1016/j.devcel.2009.02.003 CrossRefPubMedGoogle Scholar
  8. 8.
    Poitelon Y, Lopez-Anido C, Catignas K, Berti C, Palmisano M, Williamson C, Ameroso D, Abiko K, Hwang Y, Gregorieff A, Wrana JL, Asmani M, Zhao R, Sim FJ, Wrabetz L, Svaren J, Feltri ML (2016) YAP and TAZ control peripheral myelination and the expression of laminin receptors in Schwann cells. Nat Neurosci 19(7):879–887.  https://doi.org/10.1038/nn.4316 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Xin M, Kim Y, Sutherland LB, Murakami M, Qi X, McAnally J, Porrello ER, Mahmoud AI, Tan W, Shelton JM, Richardson JA, Sadek HA, Bassel-Duby R, Olson EN (2013) Hippo pathway effector Yap promotes cardiac regeneration. Proc Natl Acad Sci U S A 110(34):13839–13844.  https://doi.org/10.1073/pnas.1313192110 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Reginensi A, Hoshi M, Boualia SK, Bouchard M, Jain S, McNeill H (2015) Yap and Taz are required for Ret-dependent urinary tract morphogenesis. Development 142(15):2696–2703.  https://doi.org/10.1242/dev.122044 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Meng Z, Moroishi T, Guan KL (2016) Mechanisms of Hippo pathway regulation. Genes Dev 30(1):1–17.  https://doi.org/10.1101/gad.274027.115 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kanai F, Marignani PA, Sarbassova D, Yagi R, Hall RA, Donowitz M, Hisaminato A, Fujiwara T, Ito Y, Cantley LC, Yaffe MB (2000) TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins. EMBO J 19(24):6778–6791.  https://doi.org/10.1093/emboj/19.24.6778 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Lei QY, Zhang H, Zhao B, Zha ZY, Bai F, Pei XH, Zhao S, Xiong Y, Guan KL (2008) TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway. Mol Cell Biol 28(7):2426–2436.  https://doi.org/10.1128/MCB.01874-07 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Liu CY, Zha ZY, Zhou X, Zhang H, Huang W, Zhao D, Li T, Chan SW, Lim CJ, Hong W, Zhao S, Xiong Y, Lei QY, Guan KL (2010) The hippo tumor pathway promotes TAZ degradation by phosphorylating a phosphodegron and recruiting the SCF{beta}-TrCP E3 ligase. J Biol Chem 285(48):37159–37169.  https://doi.org/10.1074/jbc.M110.152942 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Moroishi T, Hansen CG, Guan KL (2015) The emerging roles of YAP and TAZ in cancer. Nat Rev Cancer 15(2):73–79.  https://doi.org/10.1038/nrc3876 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Zanconato F, Cordenonsi M, Piccolo S (2016) YAP/TAZ at the roots of cancer. Cancer Cell 29(6):783–803.  https://doi.org/10.1016/j.ccell.2016.05.005 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Beyer TA, Weiss A, Khomchuk Y, Huang K, Ogunjimi AA, Varelas X, Wrana JL (2013) Switch enhancers interpret TGF-beta and Hippo signaling to control cell fate in human embryonic stem cells. Cell Rep 5(6):1611–1624.  https://doi.org/10.1016/j.celrep.2013.11.021 CrossRefPubMedGoogle Scholar
  18. 18.
    Varelas X, Samavarchi-Tehrani P, Narimatsu M, Weiss A, Cockburn K, Larsen BG, Rossant J, Wrana JL (2010) The Crumbs complex couples cell density sensing to Hippo-dependent control of the TGF-beta-SMAD pathway. Dev Cell 19(6):831–844.  https://doi.org/10.1016/j.devcel.2010.11.012 CrossRefPubMedGoogle Scholar
  19. 19.
    Reginensi A, Scott RP, Gregorieff A, Bagherie-Lachidan M, Chung C, Lim DS, Pawson T, Wrana J, McNeill H (2013) Yap- and Cdc42-dependent nephrogenesis and morphogenesis during mouse kidney development. PLoS Genet 9(3):e1003380.  https://doi.org/10.1371/journal.pgen.1003380 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J, Golland P, Sabatini DM (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7(10):R100.  https://doi.org/10.1186/gb-2006-7-10-r100 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Nathan M. Kingston
    • 1
  • Andrew M. Tilston-Lunel
    • 1
  • Julia Hicks-Berthet
    • 1
  • Xaralabos Varelas
    • 1
    Email author
  1. 1.Department of BiochemistryBoston University School of MedicineBostonUSA

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