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Immunofluorescence Study of Endogenous YAP in Mammalian Cells

  • Valentina Rausch
  • Carsten G. HansenEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1893)

Abstract

Immunocytochemistry enables determination of cellular localization and relative abundance of proteins. This protocol describes a rapid and cost-effective approach to study the cellular localization of YAP (and TAZ), the transcriptional co activators of the Hippo pathway, in mammalian cells. Cells are seeded onto coated cover slips, cultivated and treated as required. Subsequently, they are chemically fixed, and cellular proteins are fluorescently labeled by means of specific antibodies. Multiplexing antibodies enables ascertaining the subcellular localization of YAP and TAZ and thereby also the activation state of the Hippo pathway in various cell types.

Key words

Immunofluorescence Immunocytochemistry Cellular localization YAP Hippo pathway 

Notes

Acknowledgments

Members of the Gram Hansen lab are thanked for their comments as well as for helping taking the photos for Figs. 1, 2, 3, 4, 5, and 6. Work ongoing in the Gram Hansen lab is supported by a Chancellor’s Fellowship start-up fund and by the Wellcome Trust-University of Edinburgh Institutional Strategic Support Fund. We acknowledge the technical support and guidance provided by the Centre for Reproductive Health SuRF Histology and imaging as well as the Centre for Inflammation Research imaging staff.

References

  1. 1.
    Hansen CG, Moroishi T, Guan KL (2015) YAP and TAZ: a nexus for Hippo signaling and beyond. Trends Cell Biol 25(9):499–513.  https://doi.org/10.1016/j.tcb.2015.05.002 CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Gomez M, Gomez V, Hergovich A (2014) The Hippo pathway in disease and therapy: cancer and beyond. Clin Transl Med 3(22):1–12.  https://doi.org/10.1186/2001-1326-3-22 CrossRefGoogle Scholar
  3. 3.
    Gaspar P, Tapon N (2014) Sensing the local environment: actin architecture and Hippo signalling. Curr Opin Cell Biol 31:74–83.  https://doi.org/10.1016/j.ceb.2014.09.003 CrossRefPubMedGoogle Scholar
  4. 4.
    Wada K, Itoga K, Okano T, Yonemura S, Sasaki H (2011) Hippo pathway regulation by cell morphology and stress fibers. Development 138(18):3907–3914.  https://doi.org/10.1242/dev.070987 CrossRefPubMedGoogle Scholar
  5. 5.
    Zhao B, Li L, Wang L, Wang CY, Yu J, Guan KL (2012) Cell detachment activates the Hippo pathway via cytoskeleton reorganization to induce anoikis. Genes Dev 26(1):54–68.  https://doi.org/10.1101/gad.173435.111 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Yu FX, Zhao B, Panupinthu N, Jewell JL, Lian I, Wang LH, Zhao J, Yuan H, Tumaneng K, Li H, Fu XD, Mills GB, Guan KL (2012) Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling. Cell 150(4):780–791.  https://doi.org/10.1016/j.cell.2012.06.037 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Park HW, Kim YC, Yu B, Moroishi T, Mo JS, Plouffe SW, Meng Z, Lin KC, Yu FX, Alexander CM, Wang CY, Guan KL (2015) Alternative Wnt signaling activates YAP/TAZ. Cell 162(4):780–794.  https://doi.org/10.1016/j.cell.2015.07.013 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Kim NG, Gumbiner BM (2015) Adhesion to fibronectin regulates Hippo signaling via the FAK-Src-PI3K pathway. J Cell Biol 210(3):503–515.  https://doi.org/10.1083/jcb.201501025 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Dupont S, Morsut L, Aragona M, Enzo E, Giulitti S, Cordenonsi M, Zanconato F, Le Digabel J, Forcato M, Bicciato S, Elvassore N, Piccolo S (2011) Role of YAP/TAZ in mechanotransduction. Nature 474(7350):179–183.  https://doi.org/10.1038/nature10137 CrossRefPubMedGoogle Scholar
  10. 10.
    Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126(4):677–689.  https://doi.org/10.1016/j.cell.2006.06.044 CrossRefPubMedGoogle Scholar
  11. 11.
    Panciera T, Azzolin L, Cordenonsi M, Piccolo S (2017) Mechanobiology of YAP and TAZ in physiology and disease. Nat Rev Mol Cell Biol 18(12):758–770.  https://doi.org/10.1038/nrm.2017.87 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Koontz LM, Liu-Chittenden Y, Yin F, Zheng Y, Yu J, Huang B, Chen Q, Wu S, Pan D (2013) The Hippo effector Yorkie controls normal tissue growth by antagonizing scalloped-mediated default repression. Dev Cell 25(4):388–401.  https://doi.org/10.1016/j.devcel.2013.04.021 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Jiao S, Wang H, Shi Z, Dong A, Zhang W, Song X, He F, Wang Y, Zhang Z, Wang W, Wang X, Guo T, Li P, Zhao Y, Ji H, Zhang L, Zhou Z (2014) A peptide mimicking VGLL4 function acts as a YAP antagonist therapy against gastric cancer. Cancer Cell 25(2):166–180.  https://doi.org/10.1016/j.ccr.2014.01.010 CrossRefPubMedGoogle Scholar
  14. 14.
    Zhang W, Gao Y, Li P, Shi Z, Guo T, Li F, Han X, Feng Y, Zheng C, Wang Z, Li F, Chen H, Zhou Z, Zhang L, Ji H (2014) VGLL4 functions as a new tumor suppressor in lung cancer by negatively regulating the YAP-TEAD transcriptional complex. Cell Res 24(3):331–343.  https://doi.org/10.1038/cr.2014.10 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Hansen CG, Ng YL, Lam WL, Plouffe SW, Guan KL (2015) The Hippo pathway effectors YAP and TAZ promote cell growth by modulating amino acid signaling to mTORC1. Cell Res 25(12):1299–1313.  https://doi.org/10.1038/cr.2015.140 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.University of Edinburgh Centre for Inflammation ResearchQueen’s Medical Research InstituteEdinburghUK

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