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• W2809358976 abstract "•Photobleaching coupled with mathematical modeling identifies YAP1 dynamics•Regulation of nuclear export is key determinant of YAP1 localization•Serine phosphorylation is required for YAP1 nuclear export through XPO1•Nuclear YAP1 remains sensitive to actin and Src-family kinase regulation The transcriptional regulator YAP1 is critical for the pathological activation of fibroblasts. In normal fibroblasts, YAP1 is located in the cytoplasm, while in activated cancer-associated fibroblasts, it is nuclear and promotes the expression of genes required for pro-tumorigenic functions. Here, we investigate the dynamics of YAP1 shuttling in normal and activated fibroblasts, using EYFP-YAP1, quantitative photobleaching methods, and mathematical modeling. Imaging of migrating fibroblasts reveals the tight temporal coupling of cell shape change and altered YAP1 localization. Both 14-3-3 and TEAD binding modulate YAP1 shuttling, but neither affects nuclear import. Instead, we find that YAP1 nuclear accumulation in activated fibroblasts results from Src and actomyosin-dependent suppression of phosphorylated YAP1 export. Finally, we show that nuclear-constrained YAP1, upon XPO1 depletion, remains sensitive to blockade of actomyosin function. Together, these data place nuclear export at the center of YAP1 regulation and indicate that the cytoskeleton can regulate YAP1 within the nucleus. The transcriptional regulator YAP1 is critical for the pathological activation of fibroblasts. In normal fibroblasts, YAP1 is located in the cytoplasm, while in activated cancer-associated fibroblasts, it is nuclear and promotes the expression of genes required for pro-tumorigenic functions. Here, we investigate the dynamics of YAP1 shuttling in normal and activated fibroblasts, using EYFP-YAP1, quantitative photobleaching methods, and mathematical modeling. Imaging of migrating fibroblasts reveals the tight temporal coupling of cell shape change and altered YAP1 localization. Both 14-3-3 and TEAD binding modulate YAP1 shuttling, but neither affects nuclear import. Instead, we find that YAP1 nuclear accumulation in activated fibroblasts results from Src and actomyosin-dependent suppression of phosphorylated YAP1 export. Finally, we show that nuclear-constrained YAP1, upon XPO1 depletion, remains sensitive to blockade of actomyosin function. Together, these data place nuclear export at the center of YAP1 regulation and indicate that the cytoskeleton can regulate YAP1 within the nucleus. The transmission of signals from the cytoplasm to the transcriptional machinery in the nucleus can occur in many ways. Signal transducing kinases can enter the nucleus and modulate transcription factor activity (Taagepera et al., 1998Taagepera S. McDonald D. Loeb J.E. Whitaker L.L. McElroy A.K. Wang J.Y.J. Hope T.J. Nuclear-cytoplasmic shuttling of C-ABL tyrosine kinase.Proc. Natl. Acad. Sci. USA. 1998; 95: 7457-7462Crossref PubMed Scopus (264) Google Scholar). Alternatively, DNA binding transcription factors can shuttle between the cytoplasm and the nucleus (Nicolás et al., 2004Nicolás F.J. De Bosscher K. Schmierer B. Hill C.S. Analysis of Smad nucleocytoplasmic shuttling in living cells.J. Cell Sci. 2004; 117: 4113-4125Crossref PubMed Scopus (106) Google Scholar, Vartiainen et al., 2007Vartiainen M.K. Guettler S. Larijani B. Treisman R. Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL.Science. 2007; 316: 1749-1752Crossref PubMed Scopus (480) Google Scholar, Xu and Massague, 2004Xu L. Massague J. Nucleocytoplasmic shuttling of signal transducers.Nat. Rev. Mol. Cell Biol. 2004; 5: 209-219Crossref PubMed Scopus (217) Google Scholar). YAP1 and TAZ (WWTR1) are transcriptional regulators that are believed to be sequestered in the cytoplasm via interaction with 14-3-3 proteins when phosphorylated. In the absence of phosphorylation, YAP1 and TAZ are released and can interact with transcription factors, such as TEADs, in the nucleus (Piccolo et al., 2014Piccolo S. Dupont S. Cordenonsi M. The biology of YAP/TAZ: hippo signaling and beyond.Physiol. Rev. 2014; 94: 1287-1312Crossref PubMed Scopus (995) Google Scholar, Zhao et al., 2011Zhao B. Tumaneng K. Guan K.L. The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal.Nat. Cell Biol. 2011; 13: 877-883Crossref PubMed Scopus (861) Google Scholar). Structural studies have shown that the YAP1/TEADs interaction is critically dependent on serine 94 in YAP1 (Chen et al., 2010Chen L. Chan S.W. Zhang X. Walsh M. Lim C.J. Hong W. Song H. Structural basis of YAP recognition by TEAD4 in the hippo pathway.Genes Dev. 2010; 24: 290-300Crossref PubMed Scopus (173) Google Scholar, Li et al., 2010Li Z. Zhao B. Wang P. Chen F. Dong Z. Yang H. Guan K.-L. Xu Y. Structural insights into the YAP and TEAD complex.Genes Dev. 2010; 24: 235-240Crossref PubMed Scopus (263) Google Scholar, Zhao et al., 2008Zhao B. Ye X. Yu J. Li L. Li W. Li S. Yu J. Lin J.D. Wang C.Y. Chinnaiyan A.M. et al.TEAD mediates YAP-dependent gene induction and growth control.Genes Dev. 2008; 22: 1962-1971Crossref PubMed Scopus (1650) Google Scholar). YAP1 and TAZ are negatively regulated by serine phosphorylation by the LATS1/2 kinases, which are themselves regulated by the MST1/2 kinases (Chan et al., 2005Chan E.H.Y. Nousiainen M. Chalamalasetty R.B. Schafer A. Nigg E.A. Sillje H.H.W. The Ste20-like kinase Mst2 activates the human large tumor suppressor kinase Lats1.Oncogene. 2005; 24: 2076-2086Crossref PubMed Scopus (436) Google Scholar, Dong et al., 2007Dong J. Feldmann G. Huang J. Wu S. Zhang N. Comerford S.A. Gayyed M.F. Anders R.A. Maitra A. Pan D. Elucidation of a universal size-control mechanism in Drosophila and mammals.Cell. 2007; 130: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (1762) Google Scholar, Hao et al., 2008Hao Y. Chun A. Cheung K. Rashidi B. Yang X. Tumor suppressor LATS1 is a negative regulator of oncogene YAP.J. Biol. Chem. 2008; 283: 5496-5509Crossref PubMed Scopus (609) Google Scholar, Lei et al., 2008Lei Q.-Y. Zhang H. Zhao B. Zha Z.-Y. Bai F. Pei X.-H. Zhao S. Xiong Y. Guan K.-L. TAZ promotes cell proliferation and epithelial-mesenchymal transition and is inhibited by the hippo pathway.Mol. Cell. Biol. 2008; 28: 2426-2436Crossref PubMed Scopus (708) Google Scholar, Oka et al., 2008Oka T. Mazack V. Sudol M. Mst2 and Lats kinases regulate apoptotic function of Yes kinase-associated protein (YAP).J. Biol. Chem. 2008; 283: 27534-27546Crossref PubMed Scopus (276) Google Scholar, Zhao et al., 2007Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. et al.Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (2095) Google Scholar). This pathway, often called the Hippo pathway, is critical for controlling the extent of tissue growth and organ size (Dong et al., 2007Dong J. Feldmann G. Huang J. Wu S. Zhang N. Comerford S.A. Gayyed M.F. Anders R.A. Maitra A. Pan D. Elucidation of a universal size-control mechanism in Drosophila and mammals.Cell. 2007; 130: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (1762) Google Scholar, Halder and Johnson, 2011Halder G. Johnson R.L. Hippo signaling: growth control and beyond.Development. 2011; 138: 9-22Crossref PubMed Scopus (797) Google Scholar, Pan, 2010Pan D. The hippo signaling pathway in development and cancer.Dev. Cell. 2010; 19: 491-505Abstract Full Text Full Text PDF PubMed Scopus (1693) Google Scholar, Zhao et al., 2011Zhao B. Tumaneng K. Guan K.L. The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal.Nat. Cell Biol. 2011; 13: 877-883Crossref PubMed Scopus (861) Google Scholar). It is regulated by a network of epithelial junctional molecules that transmit information about tissue integrity. Further, regulation by glucagon and other soluble factors couples tissue growth to nutrient availability (Enzo et al., 2015Enzo E. Santinon G. Pocaterra A. Aragona M. Bresolin S. Forcato M. Grifoni D. Pession A. Zanconato F. Guzzo G. et al.Aerobic glycolysis tunes YAP/TAZ transcriptional activity.EMBO J. 2015; 34: 1349-1370Crossref PubMed Scopus (247) Google Scholar, Santinon et al., 2016Santinon G. Pocaterra A. Dupont S. Control of YAP/TAZ activity by metabolic and nutrient-sensing pathways.Trends Cell Biol. 2016; 26: 289-299Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, Yu et al., 2012Yu F.-X. Zhao B. Panupinthu N. Jewell J.L. Lian I. Wang L.H. Zhao J. Yuan H. Tumaneng K. Li H. et al.Regulation of the Hippo-YAP pathway by G-protein-coupled receptor signaling.Cell. 2012; 150: 780-791Abstract Full Text Full Text PDF PubMed Scopus (1101) Google Scholar). In all these cases, the activity of YAP1 and TAZ is negatively regulated by direct LATS1/2-mediated serine phosphorylation on several serine residues, including serine 127 in YAP1 (Zhao et al., 2007Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. et al.Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (2095) Google Scholar). Low levels of YAP1 and TAZ phosphorylation are linked to nuclear accumulation, leading to cell proliferation, wound healing, or tissue regeneration (Camargo et al., 2007Camargo F.D. Gokhale S. Johnnidis J.B. Fu D. Bell G.W. Jaenisch R. Brummelkamp T.R. YAP1 increases organ size and expands undifferentiated progenitor cells.Curr. Biol. 2007; 17: 2054-2060Abstract Full Text Full Text PDF PubMed Scopus (936) Google Scholar, Dong et al., 2007Dong J. Feldmann G. Huang J. Wu S. Zhang N. Comerford S.A. Gayyed M.F. Anders R.A. Maitra A. Pan D. Elucidation of a universal size-control mechanism in Drosophila and mammals.Cell. 2007; 130: 1120-1133Abstract Full Text Full Text PDF PubMed Scopus (1762) Google Scholar, Gao et al., 2013Gao T. Zhou D. Yang C. Singh T. Penzo-Mendez A. Maddipati R. Tzatsos A. Bardeesy N. Avruch J. Stanger B.Z. Hippo signaling regulates differentiation and maintenance in the exocrine pancreas.Gastroenterology. 2013; 144 (1553.e1): 1543-1553Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, Lavado et al., 2013Lavado A. He Y. Pare J. Neale G. Olson E.N. Giovannini M. Cao X. Tumor suppressor Nf2 limits expansion of the neural progenitor pool by inhibiting Yap/Taz transcriptional coactivators.Development. 2013; 140: 3323-3334Crossref PubMed Scopus (79) Google Scholar, Schlegelmilch et al., 2011Schlegelmilch K. Mohseni M. Kirak O. Pruszak J. Rodriguez J.R. Zhou D. Kreger B.T. Vasioukhin V. Avruch J. Brummelkamp T.R. et al.Yap1 acts downstream of α-catenin to control epidermal proliferation.Cell. 2011; 144: 782-795Abstract Full Text Full Text PDF PubMed Scopus (772) Google Scholar, Zhao et al., 2011Zhao B. Tumaneng K. Guan K.L. The Hippo pathway in organ size control, tissue regeneration and stem cell self-renewal.Nat. Cell Biol. 2011; 13: 877-883Crossref PubMed Scopus (861) Google Scholar). High levels of phosphorylation are linked to cell quiescence via promotion of complexes with 14-3-3 proteins in the cytoplasm (Aitken, 2006Aitken A. 14-3-3 proteins: a historic overview.Semin. Cancer Biol. 2006; 16: 162-172Crossref PubMed Scopus (640) Google Scholar, Muslin and Xing, 2000Muslin A.J. Xing H. 14-3-3 proteins: regulation of subcellular localization by molecular interference.Cell Signal. 2000; 12: 703-709Crossref PubMed Scopus (348) Google Scholar). Mechanical cues and tyrosine phosphorylation can modulate YAP1 function (Dupont et al., 2011Dupont S. Morsut L. Aragona M. Enzo E. Giulitti S. Cordenonsi M. Zanconato F. Le Digabel J. Forcato M. Bicciato S. et al.Role of YAP/TAZ in mechanotransduction.Nature. 2011; 474: 179-183Crossref PubMed Scopus (3277) Google Scholar, Li et al., 2016Li P. Silvis M.R. Honaker Y. Lien W.H. Arron S.T. Vasioukhin V. alphaE-catenin inhibits a Src-YAP1 oncogenic module that couples tyrosine kinases and the effector of Hippo signaling pathway.Genes Dev. 2016; 30: 798-811Crossref PubMed Scopus (119) Google Scholar) and are proposed to enable epithelial cells to monitor organ size (Benham-Pyle et al., 2015Benham-Pyle B.W. Pruitt B.L. Nelson W.J. Cell adhesion. Mechanical strain induces E-cadherin-dependent Yap1 and beta-catenin activation to drive cell cycle entry.Science. 2015; 348: 1024-1027Crossref PubMed Scopus (334) Google Scholar, Fernandez et al., 2011Fernandez B.G. Gaspar P. Bras-Pereira C. Jezowska B. Rebelo S.R. Janody F. Actin-Capping Protein and the Hippo pathway regulate F-actin and tissue growth in Drosophila.Development. 2011; 138: 2337-2346Crossref PubMed Scopus (235) Google Scholar, Porazinski et al., 2015Porazinski S. Wang H. Asaoka Y. Behrndt M. Miyamoto T. Morita H. Hata S. Sasaki T. Krens S.F.G. Osada Y. et al.YAP is essential for tissue tension to ensure vertebrate 3D body shape.Nature. 2015; 521: 217-221Crossref PubMed Scopus (164) Google Scholar, Sansores-Garcia et al., 2011Sansores-Garcia L. Bossuyt W. Wada K.-I. Yonemura S. Tao C. Sasaki H. Halder G. Modulating F-actin organization induces organ growth by affecting the Hippo pathway.EMBO J. 2011; 30: 2325-2335Crossref PubMed Scopus (331) Google Scholar). This may depend on Src-mediated phosphorylation of tyrosine 357; however, the full details of how mechanical cues regulate YAP1 are not determined. YAP1 activation in fibroblasts within tumors depends on the actin cytoskeleton and is correlated with increased nuclear YAP1 and Y357 phosphorylation, but S127 phosphorylation and LATS1/2 activity are not changed (Calvo et al., 2013Calvo F. Ege N. Grande-Garcia A. Hooper S. Jenkins R.P. Chaudhry S.I. Harrington K. Williamson P. Moeendarbary E. Charras G. et al.Mechanotransduction and YAP-dependent matrix remodelling is required for the generation and maintenance of cancer-associated fibroblasts.Nat. Cell Biol. 2013; 15: 637-646Crossref PubMed Scopus (826) Google Scholar). Little is known about the dynamics of YAP1 shuttling in and out of the nucleus (Zhao et al., 2007Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. et al.Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (2095) Google Scholar). Many binding partners have been identified, including the TEADs, 14-3-3, and cytoplasmic proteins localized at cell junctions (Couzens et al., 2013Couzens A.L. Knight J.D.R. Kean M.J. Teo G. Weiss A. Dunham W.H. Lin Z.-Y. Bagshaw R.D. Sicheri F. Pawson T. et al.Protein interaction network of the mammalian Hippo pathway reveals mechanisms of kinase-phosphatase interactions.Sci. Signal. 2013; 6: rs15Crossref PubMed Scopus (313) Google Scholar, Moya and Halder, 2014Moya I.M. Halder G. Discovering the Hippo pathway protein-protein interactome.Cell Res. 2014; 24: 137-138Crossref PubMed Scopus (22) Google Scholar), yet it remains unclear if YAP1 is stably sequestered at these sites in either the cytoplasm or the nucleus. The rate of YAP1 shuttling between the cytoplasm and nucleus is not known, and apart from the implication of XPO1 (also called Exportin1 or Crm1) in YAP1 nuclear export (Dupont et al., 2011Dupont S. Morsut L. Aragona M. Enzo E. Giulitti S. Cordenonsi M. Zanconato F. Le Digabel J. Forcato M. Bicciato S. et al.Role of YAP/TAZ in mechanotransduction.Nature. 2011; 474: 179-183Crossref PubMed Scopus (3277) Google Scholar, Ren et al., 2010Ren F. Zhang L. Jiang J. Hippo signaling regulates Yorkie nuclear localization and activity through 14-3-3 dependent and independent mechanisms.Dev. Biol. 2010; 337: 303-312Crossref PubMed Scopus (144) Google Scholar, Wei et al., 2015Wei S.C. Fattet L. Tsai J.H. Guo Y. Pai V.H. Majeski H.E. Chen A.C. Sah R.L. Taylor S.S. Engler A.J. et al.Matrix stiffness drives epithelial-mesenchymal transition and tumour metastasis through a TWIST1-G3BP2 mechanotransduction pathway.Nat. Cell Biol. 2015; 17: 678-688Crossref PubMed Scopus (535) Google Scholar), the machinery regulating YAP1 entry and exit from the nucleus is not known. We answer these questions by using a variety of live imaging methods and mathematical analysis (Nicolás et al., 2004Nicolás F.J. De Bosscher K. Schmierer B. Hill C.S. Analysis of Smad nucleocytoplasmic shuttling in living cells.J. Cell Sci. 2004; 117: 4113-4125Crossref PubMed Scopus (106) Google Scholar, Vartiainen et al., 2007Vartiainen M.K. Guettler S. Larijani B. Treisman R. Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL.Science. 2007; 316: 1749-1752Crossref PubMed Scopus (480) Google Scholar). Fluorescence recovery after photobleaching (FRAP) is used to provide information about sequestration, diffusion, and the rate of dissociation from TEAD transcription factors. Fluorescence loss in photobleaching (FLIP) is used to assess nuclear import and export rates and the rate of TEAD association. By combining these methods with YAP1 point mutations, actomyosin manipulations, and a screen for regulators of YAP1 nuclear import/export, we are able to derive a detailed model of YAP1 dynamics in normal fibroblasts (NFs) and pathologically activated fibroblasts (CAFs). To image the localization and dynamics of YAP1 in normal mammary fibroblasts (NF1) and mammary carcinoma-associated fibroblasts (CAF1), we fused EYFP to the N terminus of the protein and generated NF1 and CAF1 stably expressing levels of EYFP-YAP1 similar to the level of the endogenous YAP1 (Figures 1A and S1A), estimated at ∼130,000 EYFP-YAP1 molecules/cell (Figure S1B). There was no correlation between the level of EYFP-YAP1 and its subcellular distribution, indicating that the system is not sensitive to modest variation in the level of YAP1 (Figure S1H). Functional matrix contraction assays demonstrated that the expression of EYFP-YAP1 in both cells did not erroneously activate them (Figure S1C). We confirmed that EYFP-YAP1 is regulated in a similar manner to endogenous YAP1. Figure 1B shows that EYFP-YAP1 is more nuclear in CAF1 than NF1, mirroring the difference in endogenous protein localization (Figure S1D). EYFP-YAP1 also showed a similar cytoplasmic shift upon actomyosin blockade using blebbistatin (Figure 1C), and this was accompanied by reduced YAP1-dependent transcription (Figures 1D and 1E; note elevated YAP1 transcriptional activity in CAF1 versus NF1). We further probed the behavior of EYFP-YAP1 by introducing well-characterized mutations at serine 94 (S94A) (Zhao et al., 2008Zhao B. Ye X. Yu J. Li L. Li W. Li S. Yu J. Lin J.D. Wang C.Y. Chinnaiyan A.M. et al.TEAD mediates YAP-dependent gene induction and growth control.Genes Dev. 2008; 22: 1962-1971Crossref PubMed Scopus (1650) Google Scholar) and serines 61, 109, 127, 164, and 381 (termed 5SA) (Zhao et al., 2007Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. et al.Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (2095) Google Scholar), blocking the interaction of YAP1 with TEAD and 14-3-3, respectively (Figures 1A and S1E). Both mutants showed the expected cytoplasmic and nuclear localization, respectively (Figures 1F and 1G); this pattern was not altered by depletion of the endogenous YAP1 (Figures S1G and S1I). We confirmed that the altered localization of EYFP-YAP1_S94A was due to defective TEAD binding with additional R89A, L91A, and F95A mutations in the TEAD binding domain (Li et al., 2010Li Z. Zhao B. Wang P. Chen F. Dong Z. Yang H. Guan K.-L. Xu Y. Structural insights into the YAP and TEAD complex.Genes Dev. 2010; 24: 235-240Crossref PubMed Scopus (263) Google Scholar) and TEAD1-4 siRNA (Figures S1J–S1M). Depletion of LATS1/2 promoted the nuclear accumulation of EYFP-YAP1 (Figures S1L and S1M). These data confirm that EYFP-YAP1 recapitulates key features of YAP1 regulation. The functionality of the EYFP-YAP1 was also evidenced by the increased TEAD reporter activity in cells expressing EYFP-YAP1_5SA (Figure 1H). Cells expressing EYFP-YAP1_S94A showed reduced TEAD reporter and matrix contraction activity, indicating that this construct acts as a dominant negative (Figures 1H and S1F). Having shown that EYFP-YAP1 is a valid tool to probe YAP1 function, we embarked on FRAP experiments combined with mathematical modeling in order to assess protein diffusion (DC and DN) and dissociation rates (k-0 and k-1) in the cytoplasm and nucleus, respectively (Figure 2A) (Fritzsche and Charras, 2015Fritzsche M. Charras G. Dissecting protein reaction dynamics in living cells by fluorescence recovery after photobleaching.Nat. Protoc. 2015; 10: 660-680Crossref PubMed Scopus (44) Google Scholar, Sprague et al., 2004Sprague B.L. Pego R.L. Stavreva D.A. McNally J.G. Analysis of binding reactions by fluorescence recovery after photobleaching.Biophys. J. 2004; 86: 3473-3495Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). During FRAP, a region of interest was bleached and the time for fluorescence recovery compared with an adjacent non-bleached region was assessed. An incomplete recovery would indicate an “immobile fraction” sequestered in the compartment of interest. FRAP analyses in both the cytoplasm and the nucleus of NF1 and CAF1 were performed. Figure 2B shows that the bleached area has a fluorescent intensity equivalent to a non-bleached region within ∼15 s (see also Figure S2A). This indicates that there is no measurable “immobile” fraction of EYFP-YAP1 on the timescale of our experiments and thus that the molecule does not engage in permanent binding to a fixed component in the nucleus or in the cytoplasm. We confirmed the validity of our FRAP experiments on fast-diffusing EGFP and stably chromatin-bound H2B-GFP (Figures S2B and S2C; Video S1). The H2B-GFP analysis also demonstrates that chromatin can be considered immobile in the time frame of our experiments. Both diffusion of EYFP-YAP1 and its release from a short-lasting interaction could influence the observed rate of fluorescence recovery (half-time), the effective radius (re) and the depth (K) of the postbleach profile (Figure S2D; Mathematical Methods section of Mathematical Modeling and Model Validation) (Fritzsche and Charras, 2015Fritzsche M. Charras G. Dissecting protein reaction dynamics in living cells by fluorescence recovery after photobleaching.Nat. Protoc. 2015; 10: 660-680Crossref PubMed Scopus (44) Google Scholar, Kang et al., 2009Kang M. Day C.A. Drake K. Kenworthy A.K. DiBenedetto E. A generalization of theory for two-dimensional fluorescence recovery after photobleaching applicable to confocal laser scanning microscopes.Biophys. J. 2009; 97: 1501-1511Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, Kang et al., 2010Kang M. Day C.A. DiBenedetto E. Kenworthy A.K. A quantitative approach to analyze binding diffusion kinetics by confocal FRAP.Biophys. J. 2010; 99: 2737-2747Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). If diffusion is slow enough, these parameters would change according to the size of the bleached region, as diffusion into a larger region will take longer (Figure S2D, Situation 1). We therefore repeated FRAP analyses with different-sized bleached regions in the nucleus and the cytoplasm. The intensity recoveries (Figure 2B) and the postbleach profile (Figure S3A) showed no differences among the three bleached regions. Furthermore, the half-time, the effective radius, and the bleach depth did not change significantly (Figures 2C and S3). Taken together, these results indicate that diffusion is rapid relative to the imaging rate of 16.7 Hz, suggesting that the recovery observed reflects unbinding/binding reactions (Figure S2D, Situation 2). Consistent with this, Figure 2D shows that a pure diffusion model did not fit the experimental recovery curves well (orange versus blue curves). Both reaction-diffusion (red) and reaction models (green and magenta) fit the data well. Akaike Information Criterion (AIC) analysis indicated that in the majority of cases, reaction-based models fit best (Figure 2G; Table S1; Mathematical Methods section of Mathematical Modeling and Model Validation). In the cases where reaction-diffusion models fit well, the diffusion value was in the range of 25–40 μm2s−1, which is in the range reported for multimerized GFP with similar molecular mass to EYFP-YAP1 (Baum et al., 2014Baum M. Erdel F. Wachsmuth M. Rippe K. Retrieving the intracellular topology from multi-scale protein mobility mapping in living cells.Nat. Commun. 2014; 5: 4494Crossref PubMed Scopus (101) Google Scholar). Therefore, diffusion is so rapid that it has largely occurred by the first postbleach image acquisition and the subsequent recovery captured in our analyses mainly reflects the reaction component, such as the dissociation of molecules from their bound immobile state (Figures 2E and 2F). In the majority of cells, a one-reaction rate model gave the best fit (Figure 2G). When the two-reactions model was better, one of the rates was always similar to the dissociation rates generated in the single rate analysis and the other rate was much faster with very wide confidence intervals. Overall the two-reactions approach did not improve the match to the experimental data enough to justify the increase in number of parameters (Table S1). We therefore used a one-reaction model to extract YAP1 dissociation rate. Intriguingly, this rate in the nucleus was significantly lower in CAF1 (0.39 s−1) than in NF1 (0.56 s−1) (Figure 2H; Video S2). This suggests that YAP1 associates more stably with a nuclear partner in CAFs, likely a chromatin-bound factor. Depletion of endogenous YAP1 did not significantly alter these dissociation rates, excluding the possibility of saturation of the chromatin by endogenous YAP1 (data not shown). Similarly, FRAP analyses in the cytoplasm determined that the observed fluorescence recoveries could be explained by dissociation rates of 0.67 s−1 in NF1 and 0.65 s−1 in CAF1 (Figure 2H; Video S3). This means that there is relatively little difference between normal and activated fibroblasts and that YAP1 does not have a long-lived site of sequestration in the cytoplasm. Having established that YAP1 has differential binding in the nucleus in CAF1 compared with NF1, we sought to determine what the binding partner was. The most likely partners of YAP1 in the nucleus are TEAD transcription factors (Zanconato et al., 2015Zanconato F. Forcato M. Battilana G. Azzolin L. Quaranta E. Bodega B. Rosato A. Bicciato S. Cordenonsi M. Piccolo S. Genome-wide association between YAP/TAZ/TEAD and AP-1 at enhancers drives oncogenic growth.Nat. Cell Biol. 2015; 17: 1218-1227Crossref PubMed Scopus (626) Google Scholar, Zhao et al., 2008Zhao B. Ye X. Yu J. Li L. Li W. Li S. Yu J. Lin J.D. Wang C.Y. Chinnaiyan A.M. et al.TEAD mediates YAP-dependent gene induction and growth control.Genes Dev. 2008; 22: 1962-1971Crossref PubMed Scopus (1650) Google Scholar). We therefore repeated the FRAP analyses with S94A mutation, which is known to abrogate TEAD interaction. Figures 3A and 3B show that S94A mutation did indeed affect fluorescence recovery (see also Video S4). In some cases, it became so rapid that it was not possible to reliably determine a dissociation rate. In the CAFs in which a rate could be measured, it was 0.97 s−1, while the median rate of the whole dataset including the rapid recovery cells was 1.29 s−1 (Figure 3E). These data confirm that the altered dynamics of EYFP-YAP1 in the nucleus of CAF1 is due to TEAD binding. In contrast, the active 5SA mutant, unable to bind 14-3-3 proteins, exhibited a slower dissociation rate, consistent with increased TEAD binding and transcriptional activation (Figures 3C–3E; Video S4). To determine if the dissociation rate measured in CAF1 (0.21 s−1) represents the dissociation rate of an intact YAP1-TEAD complex from chromatin or the dissociation rate of YAP1 from TEAD that remains bound to chromatin, we performed FRAP of TEAD1-mCherry. Figures 3F and 3G show that TEAD1-mCherry had a slower recovery than YAP1 with a dissociation rate of 0.05/0.06 s−1 (see also Video S5). This suggests that in CAFs, TEAD1 typically spends 30 s bound to chromatin, whereas YAP1 typically spends ∼2.5 s bound to TEADs, indicating that the rate we measured primarily represents the dissociation of YAP1 from TEAD transcription factors. Next, we turned our attention to determining the rate of nuclear import and export. We employed FLIP analysis to continually bleach EYFP-YAP1 in the nucleus and assess import rates (k−2), export rates (k2), as well as the nuclear association rates (k1) (Figure 4A). Figure 4B shows the different loss of signal at the bleached point (black) and “reporting” points in the nucleus (green) and the cytoplasm (orange) that were selected for being a similar distance from the bleached point. FLIP analyses on fast-diffusing EGFP revealed no differences in the loss of signals between the bleached and the reporting point in the nucleus and a fast drop of the cytoplasmic intensity (Figure S4A). The greater difference in curves of the nuclear and cytoplasmic “reporting” points (green and orange) in CAF1 compared with NF1 provide a qualitative indication of reduced exchange between the nucleus and the cytoplasm in these cells (Figure 2B). However, we sought quantitative determination of the rates (Ungricht et al., 2015Ungricht R. Klann M. Horvath P. Kutay U. Diffusion and retention are major determinants of protein targeting to the inner nuclear membrane.J. Cell Biol. 2015; 209: 687-703Crossref PubMed Scopus (76) Google Scholar, Wustner et al., 2012Wustner D. Solanko L.M. Lund F.W. Sage D. Schroll H.J. Lomholt M.A. Quantitative f" @default.
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• W2809358976 title "Quantitative Analysis Reveals that Actin and Src-Family Kinases Regulate Nuclear YAP1 and Its Export" @default.
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