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• W2761946181 abstract "•APC controls microtubule organization and NFAT-driven cytokine gene expression•APC silencing impairs NFAT nuclear localization and activation•NFAT associates with microtubules that control its localization and its activation•ApcMin/+ Tregs have intrinsically reduced differentiation and IL-10 production Adenomatous polyposis coli (APC) is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer development. Although extensively studied in epithelial transformation, the effect of APC on T lymphocyte activation remains poorly defined. We found that APC ensures T cell receptor-triggered activation through Nuclear Factor of Activated T cells (NFAT), since APC is necessary for NFAT’s nuclear localization in a microtubule-dependent fashion and for NFAT-driven transcription leading to cytokine gene expression. Interestingly, NFAT forms clusters juxtaposed with microtubules. Ultimately, mouse Apc deficiency reduces the presence of NFAT in the nucleus of intestinal regulatory T cells (Tregs) and impairs Treg differentiation and the acquisition of a suppressive phenotype, which is characterized by the production of the anti-inflammatory cytokine IL-10. These findings suggest a dual role for APC mutations in colorectal cancer development, where mutations drive the initiation of epithelial neoplasms and also reduce Treg-mediated suppression of the detrimental inflammation that enhances cancer growth. Adenomatous polyposis coli (APC) is a polarity regulator and tumor suppressor associated with familial adenomatous polyposis and colorectal cancer development. Although extensively studied in epithelial transformation, the effect of APC on T lymphocyte activation remains poorly defined. We found that APC ensures T cell receptor-triggered activation through Nuclear Factor of Activated T cells (NFAT), since APC is necessary for NFAT’s nuclear localization in a microtubule-dependent fashion and for NFAT-driven transcription leading to cytokine gene expression. Interestingly, NFAT forms clusters juxtaposed with microtubules. Ultimately, mouse Apc deficiency reduces the presence of NFAT in the nucleus of intestinal regulatory T cells (Tregs) and impairs Treg differentiation and the acquisition of a suppressive phenotype, which is characterized by the production of the anti-inflammatory cytokine IL-10. These findings suggest a dual role for APC mutations in colorectal cancer development, where mutations drive the initiation of epithelial neoplasms and also reduce Treg-mediated suppression of the detrimental inflammation that enhances cancer growth. T lymphocytes recognize peptide antigens associated with major histocompatibility complex molecules (MHCs) on antigen-presenting cells. Antigen recognition induces T cell polarization toward the antigen-presenting cell. This forms an organized interface, the immunological synapse that regulates T cell activation leading to T cell growth, differentiation and cytokine production. T cell receptor (TCR) and signaling molecules dynamically concentrate at the immunological synapse to optimally control T cell activation. This depends on the orchestrated action of the actin and microtubule cytoskeleton, and intracellular vesicle traffic (Agüera-Gonzalez et al., 2015Agüera-Gonzalez S. Bouchet J. Alcover A. Immunological Synapse. John Wiley & Sons, 2015https://doi.org/10.1002/9780470015902.a0004027.pub2http://onlinelibrary.wiley.com/doi/10.1002/9780470015902.a0004027.pub2/fullCrossref Google Scholar, Soares et al., 2013Soares H. Lasserre R. Alcover A. Orchestrating cytoskeleton and intracellular vesicle traffic to build functional immunological synapses.Immunol. Rev. 2013; 256: 118-132Crossref PubMed Scopus (66) Google Scholar). Cell polarity is regulated by an array of evolutionary conserved polarity complexes crucial for stably polarized epithelial cells (Rodriguez-Boulan and Macara, 2014Rodriguez-Boulan E. Macara I.G. Organization and execution of the epithelial polarity programme.Nat. Rev. Mol. Cell Biol. 2014; 15: 225-242Crossref PubMed Scopus (459) Google Scholar) or for induced polarization in migrating cells (Elric and Etienne-Manneville, 2014Elric J. Etienne-Manneville S. Centrosome positioning in polarized cells: common themes and variations.Exp. Cell Res. 2014; 328: 240-248Crossref PubMed Scopus (41) Google Scholar). Scribble, Dlg1, and PKCζ polarity regulators were shown to control lymphocyte migration, immunological synapse formation, and T cell activation (Bertrand et al., 2010Bertrand F. Esquerré M. Petit A.E. Rodrigues M. Duchez S. Delon J. Valitutti S. Activation of the ancestral polarity regulator protein kinase C zeta at the immunological synapse drives polarization of Th cell secretory machinery toward APCs.J. Immunol. 2010; 185: 2887-2894Crossref PubMed Scopus (49) Google Scholar, Lasserre et al., 2010Lasserre R. Charrin S. Cuche C. Danckaert A. Thoulouze M.I. de Chaumont F. Duong T. Perrault N. Varin-Blank N. Olivo-Marin J.C. et al.Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse.EMBO J. 2010; 29: 2301-2314Crossref PubMed Scopus (94) Google Scholar, Ludford-Menting et al., 2005Ludford-Menting M.J. Oliaro J. Sacirbegovic F. Cheah E.T. Pedersen N. Thomas S.J. Pasam A. Iazzolino R. Dow L.E. Waterhouse N.J. et al.A network of PDZ-containing proteins regulates T cell polarity and morphology during migration and immunological synapse formation.Immunity. 2005; 22: 737-748Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar, Real et al., 2007Real E. Faure S. Donnadieu E. Delon J. Cutting edge: atypical PKCs regulate T lymphocyte polarity and scanning behavior.J. Immunol. 2007; 179: 5649-5652Crossref PubMed Scopus (49) Google Scholar, Round et al., 2007Round J.L. Humphries L.A. Tomassian T. Mittelstadt P. Zhang M. Miceli M.C. Scaffold protein Dlgh1 coordinates alternative p38 kinase activation, directing T cell receptor signals toward NFAT but not NF-kappaB transcription factors.Nat. Immunol. 2007; 8: 154-161Crossref PubMed Scopus (96) Google Scholar, Xavier et al., 2004Xavier R. Rabizadeh S. Ishiguro K. Andre N. Ortiz J.B. Wachtel H. Morris D.G. Lopez-Ilasaca M. Shaw A.C. Swat W. Seed B. Discs large (Dlg1) complexes in lymphocyte activation.J. Cell Biol. 2004; 166: 173-178Crossref PubMed Scopus (88) Google Scholar). The polarity regulator and tumor suppressor adenomatous polyposis coli (APC) is known for its association with familial adenomatous polyposis (FAP), large numbers of sporadic human colorectal tumors, and intestinal carcinomas in mice (McCartney and Näthke, 2008McCartney B.M. Näthke I.S. Cell regulation by the Apc protein Apc as master regulator of epithelia.Curr. Opin. Cell Biol. 2008; 20: 186-193Crossref PubMed Scopus (117) Google Scholar, Moser et al., 1990Moser A.R. Pitot H.C. Dove W.F. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse.Science. 1990; 247: 322-324Crossref PubMed Scopus (1309) Google Scholar, Su et al., 1992Su L.K. Kinzler K.W. Vogelstein B. Preisinger A.C. Moser A.R. Luongo C. Gould K.A. Dove W.F. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene.Science. 1992; 256: 668-670Crossref PubMed Scopus (1347) Google Scholar, Zeineldin and Neufeld, 2013Zeineldin M. Neufeld K.L. More than two decades of Apc modeling in rodents.Biochim. Biophys. Acta. 2013; 1836: 80-89PubMed Google Scholar). APC contains several protein-protein interaction domains (Figure 1A), permitting its involvement in various cellular processes including proliferation, differentiation, migration, and death. APC-interacting proteins include β-catenin, the polarity regulators Dlg1 or Scribble, cytoskeleton regulators, nuclear pore and nuclear transport proteins, and apoptosis- or mitosis-related proteins (Etienne-Manneville, 2009Etienne-Manneville S. APC in cell migration.Adv. Exp. Med. Biol. 2009; 656: 30-40Crossref PubMed Scopus (45) Google Scholar, Nelson and Näthke, 2013Nelson S. Näthke I.S. Interactions and functions of the adenomatous polyposis coli (APC) protein at a glance.J. Cell Sci. 2013; 126: 873-877Crossref PubMed Scopus (59) Google Scholar). The effect of APC mutations on intestinal epithelium differentiation and tumor progression has been widely investigated in colorectal cancer patients and in mouse models (Béroud and Soussi, 1996Béroud C. Soussi T. APC gene: database of germline and somatic mutations in human tumors and cell lines.Nucleic Acids Res. 1996; 24: 121-124Crossref PubMed Scopus (170) Google Scholar, McCartney and Näthke, 2008McCartney B.M. Näthke I.S. Cell regulation by the Apc protein Apc as master regulator of epithelia.Curr. Opin. Cell Biol. 2008; 20: 186-193Crossref PubMed Scopus (117) Google Scholar, Moser et al., 1990Moser A.R. Pitot H.C. Dove W.F. A dominant mutation that predisposes to multiple intestinal neoplasia in the mouse.Science. 1990; 247: 322-324Crossref PubMed Scopus (1309) Google Scholar, Nelson and Näthke, 2013Nelson S. Näthke I.S. Interactions and functions of the adenomatous polyposis coli (APC) protein at a glance.J. Cell Sci. 2013; 126: 873-877Crossref PubMed Scopus (59) Google Scholar, Su et al., 1992Su L.K. Kinzler K.W. Vogelstein B. Preisinger A.C. Moser A.R. Luongo C. Gould K.A. Dove W.F. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene.Science. 1992; 256: 668-670Crossref PubMed Scopus (1347) Google Scholar, Zeineldin and Neufeld, 2013Zeineldin M. Neufeld K.L. More than two decades of Apc modeling in rodents.Biochim. Biophys. Acta. 2013; 1836: 80-89PubMed Google Scholar). Altered intestinal immune homeostasis was found in Apc mutant mice, together with the impaired control of inflammation by regulatory T lymphocytes (Tregs) (Akeus et al., 2014Akeus P. Langenes V. von Mentzer A. Yrlid U. Sjöling Å. Saksena P. Raghavan S. Quiding-Järbrink M. Altered chemokine production and accumulation of regulatory T cells in intestinal adenomas of APC(Min/+) mice.Cancer Immunol. Immunother. 2014; 63: 807-819Crossref PubMed Scopus (24) Google Scholar, Chae and Bothwell, 2015Chae W.J. Bothwell A.L. Spontaneous intestinal tumorigenesis in Apc (/Min+) mice requires altered T cell development with IL-17A.J. Immunol. Res. 2015; 2015: 860106Crossref PubMed Scopus (17) Google Scholar, Gounaris et al., 2009Gounaris E. Blatner N.R. Dennis K. Magnusson F. Gurish M.F. Strom T.B. Beckhove P. Gounari F. Khazaie K. T-regulatory cells shift from a protective anti-inflammatory to a cancer-promoting proinflammatory phenotype in polyposis.Cancer Res. 2009; 69: 5490-5497Crossref PubMed Scopus (155) Google Scholar). However, whether Apc defects in T cells contribute to this loss of anti-inflammatory functions remains only vaguely explored (Tanner et al., 2016Tanner S.M. Daft J.G. Hill S.A. Martin C.A. Lorenz R.G. Altered T-cell balance in lymphoid organs of a mouse model of colorectal cancer.J. Histochem. Cytochem. 2016; 64: 753-767Crossref PubMed Scopus (9) Google Scholar). Our data unveil the requirement for APC in T cell receptor-dependent nuclear factor of activated T cells (NFAT) activation. APC permits NFAT nuclear localization in a microtubule-dependent fashion. Moreover, ApcMin/+ mice display modestly reduced levels of NFAT expression and nuclear localization in intestinal regulatory T cells (Tregs). Heteroinsufficiency of Apc leads to induced Treg (iTreg) populations with impaired capacity to differentiate and produce NFAT-regulated cytokines, particularly interleukin-10 (IL-10), which is essential to control intestinal inflammation and adenocarcinoma progression (Rubtsov et al., 2008Rubtsov Y.P. Rasmussen J.P. Chi E.Y. Fontenot J. Castelli L. Ye X. Treuting P. Siewe L. Roers A. Henderson Jr., W.R. et al.Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces.Immunity. 2008; 28: 546-558Abstract Full Text Full Text PDF PubMed Scopus (1121) Google Scholar). We first investigated the expression of APC in human T cells. Jurkat cells and peripheral blood T cells express apparent full-length APC (311 kDa), as compared with HCT-116 and DLD-1 colon cancer cell lines that express full-length and truncated APC forms, respectively (Béroud and Soussi, 1996Béroud C. Soussi T. APC gene: database of germline and somatic mutations in human tumors and cell lines.Nucleic Acids Res. 1996; 24: 121-124Crossref PubMed Scopus (170) Google Scholar) (Figure 1B). We then localized APC in T cells. As we have previously shown (Lasserre et al., 2010Lasserre R. Charrin S. Cuche C. Danckaert A. Thoulouze M.I. de Chaumont F. Duong T. Perrault N. Varin-Blank N. Olivo-Marin J.C. et al.Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse.EMBO J. 2010; 29: 2301-2314Crossref PubMed Scopus (94) Google Scholar), T cells on stimulatory anti-CD3-coated coverslips (pseudo synapses) display radially organized microtubules, with the centrosome apposed to the contact site (Figures 1C–1E). Similar to other cell types (Etienne-Manneville et al., 2005Etienne-Manneville S. Manneville J.B. Nicholls S. Ferenczi M.A. Hall A. Cdc42 and Par6-PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization.J. Cell Biol. 2005; 170: 895-901Crossref PubMed Scopus (244) Google Scholar, Näthke et al., 1996Näthke I.S. Adams C.L. Polakis P. Sellin J.H. Nelson W.J. The adenomatous polyposis coli tumor suppressor protein localizes to plasma membrane sites involved in active cell migration.J. Cell Biol. 1996; 134: 165-179Crossref PubMed Scopus (448) Google Scholar), APC appeared as discrete puncta apposed to microtubules (Figure 1C; Movie S1), frequently localized at the synapse periphery, likely corresponding to microtubule tips, as shown at the leading edge of migrating cells (Etienne-Manneville et al., 2005Etienne-Manneville S. Manneville J.B. Nicholls S. Ferenczi M.A. Hall A. Cdc42 and Par6-PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization.J. Cell Biol. 2005; 170: 895-901Crossref PubMed Scopus (244) Google Scholar) (Figure 1C, right, arrowheads; Movie S1). APC regulates microtubule stability and organization in polarized cells (Etienne-Manneville et al., 2005Etienne-Manneville S. Manneville J.B. Nicholls S. Ferenczi M.A. Hall A. Cdc42 and Par6-PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization.J. Cell Biol. 2005; 170: 895-901Crossref PubMed Scopus (244) Google Scholar, Kroboth et al., 2007Kroboth K. Newton I.P. Kita K. Dikovskaya D. Zumbrunn J. Waterman-Storer C.M. Näthke I.S. Lack of adenomatous polyposis coli protein correlates with a decrease in cell migration and overall changes in microtubule stability.Mol. Biol. Cell. 2007; 18: 910-918Crossref PubMed Scopus (100) Google Scholar, Mogensen et al., 2002Mogensen M.M. Tucker J.B. Mackie J.B. Prescott A.R. Näthke I.S. The adenomatous polyposis coli protein unambiguously localizes to microtubule plus ends and is involved in establishing parallel arrays of microtubule bundles in highly polarized epithelial cells.J. Cell Biol. 2002; 157: 1041-1048Crossref PubMed Scopus (135) Google Scholar). We therefore investigated the role of APC in microtubule network organization at the immunological synapse. We assessed microtubule patterns at pseudo synapses of control and APC-silenced T cells. Control T cells mostly displayed radial microtubule patterns, whereas APC-silenced cells frequently displayed disorganized microtubule patterns in both Jurkat and primary T cells (Figures 1D and 1E). Moreover, centrosome polarization to the immunological synapse was less efficient in APC-silenced cells, as assessed by the centrosome distance to the contact site (Figure 1F). Tubulin acetylation correlates with microtubule stability and is controlled by APC (Kroboth et al., 2007Kroboth K. Newton I.P. Kita K. Dikovskaya D. Zumbrunn J. Waterman-Storer C.M. Näthke I.S. Lack of adenomatous polyposis coli protein correlates with a decrease in cell migration and overall changes in microtubule stability.Mol. Biol. Cell. 2007; 18: 910-918Crossref PubMed Scopus (100) Google Scholar). Consistently, APC-silenced T cells had lower levels of acetylated tubulin in unstimulated and CD3+CD28-stimulated cells (Figures 1G and 1H). Lower tubulin acetylation was also observed in DLD-1 epithelial carcinoma cells expressing truncated APC (Figure 1I) and in CD4+ and Tregs from ApcMin/+ heterozygous mutant mice (Figures S2B and S2H). In sum, APC depletion results in impaired microtubule acetylation, microtubule network organization, and centrosome polarization at the immunological synapse. Immunological synapses direct T cell activation through the actin- and microtubule-dependent spatial and temporal organization of TCR signaling complexes (Bunnell et al., 2002Bunnell S.C. Hong D.I. Kardon J.R. Yamazaki T. McGlade C.J. Barr V.A. Samelson L.E. T cell receptor ligation induces the formation of dynamically regulated signaling assemblies.J. Cell Biol. 2002; 158: 1263-1275Crossref PubMed Scopus (504) Google Scholar, Campi et al., 2005Campi G. Varma R. Dustin M.L. Actin and agonist MHC-peptide complex-dependent T cell receptor microclusters as scaffolds for signaling.J. Exp. Med. 2005; 202: 1031-1036Crossref PubMed Scopus (452) Google Scholar, Lasserre et al., 2010Lasserre R. Charrin S. Cuche C. Danckaert A. Thoulouze M.I. de Chaumont F. Duong T. Perrault N. Varin-Blank N. Olivo-Marin J.C. et al.Ezrin tunes T-cell activation by controlling Dlg1 and microtubule positioning at the immunological synapse.EMBO J. 2010; 29: 2301-2314Crossref PubMed Scopus (94) Google Scholar, Yokosuka et al., 2005Yokosuka T. Sakata-Sogawa K. Kobayashi W. Hiroshima M. Hashimoto-Tane A. Tokunaga M. Dustin M.L. Saito T. Newly generated T cell receptor microclusters initiate and sustain T cell activation by recruitment of Zap70 and SLP-76.Nat. Immunol. 2005; 6: 1253-1262Crossref PubMed Scopus (561) Google Scholar). APC silencing affected the generation and dynamics of signaling complexes at the immunological synapse, as monitored by the number, intensity, and trajectories of YFP-SLP76 microclusters (Figures S1A and S1B, arrowheads, and S1C and S1D). However, APC-silenced cells did not have altered tyrosine phosphorylation of the proximal TCR signaling molecules ZAP70 and PLCγ1, or calcium flux (Figures S1E–S1H; Movies S6 and S7). Therefore, APC is necessary for the generation and dynamics of signaling microcluster at the immunological synapse, without significantly affecting early signaling. The transcription factors NFAT, nuclear factor κB (NF-κB), and AP1 (Fos and Jun) are crucial for antigen-triggered T cell growth, differentiation, and cytokine gene regulation. We investigated the effect of APC silencing on NFAT-, NF-κB-, and AP1-driven transcription using luciferase expression vectors. Despite normal TCR-induced phosphorylation and Ca2+ flux in APC-silenced cells (Figures S1E–S1H), NFAT-driven luciferase expression was significantly lower (Figure 2A). In contrast, NF-κB- or AP1-driven luciferase was not affected (Figure 2B and 2C). Similar effects were observed when T cells were stimulated with calcium ionophore (Iono) and phorbol myristate acetate (PMA) that activate calcineurin and protein kinase C (PKC), respectively, bypassing TCR proximal signal events (Figures 2D–2F). Moreover, APC silencing decreased IL2 gene expression in Jurkat and primary T cells, as assessed by qRT-PCR (Figures 2G and 2H). Similarly, T cells from mice with heteroinsufficiency for Apc (ApcMin/+) exhibited reduced proliferative, cytokine production and Treg lineage commitment responses to TCR stimulation compared to their wild-type counterparts (Figures S2A and S2C–S2G). Moreover, the early response gene c-Myc was also inhibited (Figures S2B and S2H). NFAT shuttles between the cytoplasm and the nucleus in a phosphorylation-dependent manner. Cytoplasmic NFAT is phosphorylated on several serine residues whose dephosphorylation by the calcium-dependent phosphatase calcineurin drives NFAT nuclear translocation and gene transcription (Beals et al., 1997aBeals C.R. Clipstone N.A. Ho S.N. Crabtree G.R. Nuclear localization of NF-ATc by a calcineurin-dependent, cyclosporin-sensitive intramolecular interaction.Genes Dev. 1997; 11: 824-834Crossref PubMed Scopus (342) Google Scholar, Okamura et al., 2000Okamura H. Aramburu J. García-Rodríguez C. Viola J.P. Raghavan A. Tahiliani M. Zhang X. Qin J. Hogan P.G. Rao A. Concerted dephosphorylation of the transcription factor NFAT1 induces a conformational switch that regulates transcriptional activity.Mol. Cell. 2000; 6: 539-550Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Conversely, phosphorylation by the serine kinases GSK-3β facilitates NFAT nuclear export and cytoplasmic retention (Beals et al., 1997bBeals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3.Science. 1997; 275: 1930-1934Crossref PubMed Scopus (634) Google Scholar, Shibasaki et al., 1996Shibasaki F. Price E.R. Milan D. McKeon F. Role of kinases and the phosphatase calcineurin in the nuclear shuttling of transcription factor NF-AT4.Nature. 1996; 382: 370-373Crossref PubMed Scopus (434) Google Scholar). We investigated the effect of APC silencing on NFATC2 (NFAT1), an isoform constitutively expressed in T cells, which concentrates in the nucleus in response to TCR-CD28 stimulation or PMA-Iono. Nuclear NFATC2 in APC-silenced activated Jurkat or primary T cells was lower compared to controls. (Figures 2J–2L, S3A, and S3B). Similarly, DLD-1 carcinoma cells expressing truncated APC were less efficient at translocating NFATC2 and producing NFAT-driven luciferase in response to PMA-Iono than HCT-116 controls (Figures S3C and S3D). Moreover, we observed that NFATC2 was less expressed at the mRNA and protein level in APC-silenced Jurkat cells (Figures 2I and 2M), whereas NFATC1 (NFAT2) isoform expression was not significantly affected (Figure 2N). Since APC and GSK-3 form a complex (Stamos and Weis, 2013Stamos J.L. Weis W.I. The β-catenin destruction complex.Cold Spring Harb. Perspect. Biol. 2013; 5: a007898Crossref Scopus (675) Google Scholar), and GSK-3 phosphorylates NFAT and regulates its nuclear-to-cytoplasmic cycling (Beals et al., 1997bBeals C.R. Sheridan C.M. Turck C.W. Gardner P. Crabtree G.R. Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3.Science. 1997; 275: 1930-1934Crossref PubMed Scopus (634) Google Scholar), we investigated whether APC silencing affects NFATC2 phosphorylation. We therefore analyzed NFATC2 phosphorylation status by comparing its electrophoretic mobility. Bands with slower mobility represent more phosphorylated protein species. Apc silencing did not significantly affect the ratio of higher to lower mobility NFAT electrophoretic bands, suggesting that phosphorylation was not altered by APC depletion (Figures S4A–S4H). In sum, APC is necessary for NFATC2 expression and nuclear localization, as well as NFAT-driven gene transcription with functional consequences for proliferation, differentiation, and cytokine expression. Microtubule disorganization in APC-silenced cells (Figures 1D–1F) prompted us to hypothesize that APC regulation of microtubules may influence NFAT activation, including nuclear localization and transcriptional activity. Therefore, we first analyzed NFATC2 localization with respect to microtubules. We observed discrete puncta of endogenous NFATC2 juxtaposed to microtubules (Figure 3A, arrowheads). We then measured the distance of NFATC2 microclusters to the closest microtubule at different activation times, using 3D segmentations and quantitative image analysis (Figures 3B and 3C; Movies S2, S3, S4, and S5). NFATC2-microtubule distance was ≈0.2 μm in non-stimulated cells and significantly increased upon TCR stimulation (Figures 3C and 3D). NFATC2 microclusters approached to the synapse surface at early activation times and then progressively moved away to a central, peri-nuclear region of the cell as the cell retracted from the coverslip (Z-position; Figure 3E). The number of NFAT microclusters was conserved during the course of T cell activation (Figure 3F). These data are consistent with NFATC2 forming microclusters associated with microtubules in resting and early activated T cells and then separating at later times as NFAT translocates to the nucleus. It was reported that NFAT and APC form cytoplasmic complexes that share common partners, like the Ser/Thr kinase GSK-3β, and the cytoskeleton regulator IQGAP (Sharma et al., 2011Sharma S. Findlay G.M. Bandukwala H.S. Oberdoerffer S. Baust B. Li Z. Schmidt V. Hogan P.G. Sacks D.B. Rao A. Dephosphorylation of the nuclear factor of activated T cells (NFAT) transcription factor is regulated by an RNA-protein scaffold complex.Proc. Natl. Acad. Sci. USA. 2011; 108: 11381-11386Crossref PubMed Scopus (203) Google Scholar, Stamos and Weis, 2013Stamos J.L. Weis W.I. The β-catenin destruction complex.Cold Spring Harb. Perspect. Biol. 2013; 5: a007898Crossref Scopus (675) Google Scholar). Moreover, both APC and NFAT associate with microtubules displaying spotted patterns (Figures 1C and 3A). Therefore, we investigated the relative localization of APC and NFAT. APC and NFAT microclusters did not fully overlap, although they were often found in close proximity (Figure 3G, arrowheads). Next, we investigated whether microtubule integrity was required for NFATC2 localization and transcriptional activity. Treatment of T cells with the microtubule polymerization inhibitor colchicine significantly reduced the number of NFAT clusters at the immunological synapse, providing further evidence of the close relationship between NFAT and the microtubule network (Figure 4A). Furthermore, colchicine significantly inhibited NFAT-driven luciferase gene expression under both CD3-CD28 and PMA-Iono T cell stimulations (Figures 4B and 4C). Consistently, IL2 mRNA levels were reduced in colchicine-treated cells (Figure 4D). In addition, NFAT nuclear detection was significantly reduced in colchicine-treated T cells (Figures 4E–4G), suggesting that the defect of NFAT-dependent transcription could be related to impaired NFAT nuclear localization. In contrast with APC-silenced cells, colchicine-treated T cells displayed comparable amounts of total NFAT protein and mRNA (Figures 4H and 4I), suggesting that NFAT nuclear localization could be microtubule-dependent. Finally, overexpression of two APC truncated molecules containing the C-terminal APC microtubule and EB1 interacting regions (Etienne-Manneville et al., 2005Etienne-Manneville S. Manneville J.B. Nicholls S. Ferenczi M.A. Hall A. Cdc42 and Par6-PKCzeta regulate the spatially localized association of Dlg1 and APC to control cell polarization.J. Cell Biol. 2005; 170: 895-901Crossref PubMed Scopus (244) Google Scholar) significantly inhibited NFAT-driven luciferase expression in Jurkat cells, although less efficiently than APC silencing (Figure 4J). Altogether, our data indicate that APC controls NFAT-driven gene transcription by regulating NFAT nuclear localization in a microtubule-dependent manner. APC also affects NFAT mRNA and protein levels. APC silencing inhibits NFAT, without altering NF-κB or AP1, leading to reduced IL2 gene expression. Unlike conventional T cells, Tregs constitutively retain some NFAT in the nucleus, even in the absence of TCR stimulation (Li et al., 2012Li Q. Shakya A. Guo X. Zhang H. Tantin D. Jensen P.E. Chen X. Constitutive nuclear localization of NFAT in Foxp3+ regulatory T cells independent of calcineurin activity.J. Immunol. 2012; 188: 4268-4277Crossref PubMed Scopus (30) Google Scholar, Vaeth et al., 2012Vaeth M. Schliesser U. Müller G. Reissig S. Satoh K. Tuettenberg A. Jonuleit H. Waisman A. Müller M.R. Serfling E. et al.Dependence on nuclear factor of activated T-cells (NFAT) levels discriminates conventional T cells from Foxp3+ regulatory T cells.Proc. Natl. Acad. Sci. USA. 2012; 109: 16258-16263Crossref PubMed Scopus (103) Google Scholar). This nuclear NFAT fraction is required to maintain a suppressive phenotype, as it cooperates with Foxp3 to drive the Treg transcriptional profile and enhances Foxp3 expression (Tone et al., 2008Tone Y. Furuuchi K. Kojima Y. Tykocinski M.L. Greene M.I. Tone M. Smad3 and NFAT cooperate to induce Foxp3 expression through its enhancer.Nat. Immunol. 2008; 9: 194-202Crossref PubMed Scopus (619) Google Scholar, van der Veeken et al., 2013van der Veeken J. Arvey A. Rudensky A. Transcriptional control of regulatory T-cell differentiation.Cold Spring Harb. Symp. Quant. Biol. 2013; 78: 215-222Crossref PubMed Scopus (20) Google Scholar, Wu et al., 2006Wu Y. Borde M. Heissmeyer V. Feuerer M. Lapan A.D. Stroud J.C. Bates D.L. Guo L. Han A. Ziegler S.F. et al.FOXP3 controls regulatory T cell function through cooperation with NFAT.Cell. 2006; 126: 375-387Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar). Because we showed that APC was necessary for optimal accumulation of NFAT in the nucleus of human T cells activated in vitro, we examined the impact of Apc mutation on NFAT localization in Tregs isolated from the intestinal lamina propria of pre-cancerous (11-week-old) ApcMin/+ mice. Using high-throughput image analysis with imaging flow cytometry, we assessed the nuclear localization of NFAT in Tregs from wild-type (WT) and ApcMin/+ mice, finding a small but significant change in the cellular distribution of NFAT in mutant Tregs, with a shift away from the nucleus (Figure 5A). Moreover, conventional flow cytometric analysis confirmed a significant reduction in Foxp3 protein levels within ApcMin/+ Treg (Figure 5B). Further investigation into Treg phenotypes revealed a dramatic shift in Treg subtypes populating the in" @default.
• W2761946181 created "2017-10-20" @default.
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• W2761946181 date "2017-10-01" @default.
• W2761946181 modified "2023-10-18" @default.
• W2761946181 title "Adenomatous Polyposis Coli Defines Treg Differentiation and Anti-inflammatory Function through Microtubule-Mediated NFAT Localization" @default.
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