Tyrosine Phosphorylation of an Actin-Binding Protein Girdin Specifically Marks Tuft Cells in Human and Mouse Gut

JHCXXX10.1369/0022155417702586Girdin phosphorylation marks tuft cellsKuga et al. 702586research-article2017 Article Journal of Histochemistry & Cytochemistry 1 –20 © The Author(s) 2017 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI:https://doi.org/10.1369/0022155417702586 10.1369/0022155417702586 journals.sagepub.com/home/jhc Tyrosine Phosphorylation of an Actin-Binding Protein Girdin Specifically Marks Tuft Cells in Human and Mouse Gut Daisuke Kuga, Kaori Ushida, Shinji Mii, Atsushi Enomoto, Naoya Asai, Masato Nagino, Masahide Takahashi, and Masato Asai Department of Pathology (DK, KU, SM, AE, NA, MT, MA), Division of Surgical Oncology, Department of Surgery (DK, MN), Division of Molecular Pathology, Center for Neurological Disease and Cancer (NA, MT), and Department of Endocrinology and Diabetes (MA), Nagoya University Graduate School of Medicine, Nagoya, Japan Summary Tuft cells (TCs) are minor components of gastrointestinal epithelia, characterized by apical tufts and spool-shaped somas. The lack of reliable TC-markers has hindered the elucidation of its role. We developed site-specific and phosphorylation- status–specific antibodies against Girdin at tyrosine-1798 (pY1798) and found pY1798 immunostaining of mouse jejunum clearly depicted epithelial cells closely resembling TCs. This study aimed to validate pY1798 as a TC-marker. Double- fluorescence staining of intestines was performed with pY1798 and known TC-markers, for example, hematopoietic- prostaglandin-D-synthase (HPGDS), or doublecortin-like kinase 1 (DCLK1). Odds ratios (ORs) were calculated from cell counts to determine whether two markers were attracting (OR<1) or repelling (OR>1). In consequence, pY1798 signals strongly attracted those of known TC-markers. ORs for HPGDS in mouse stomach, small intestine, and colon were 0 for all, and 0.08 for DCLK1 in human small intestine. pY1798-positive cells in jejunum were distinct from other minor epithelial cells, including goblet, Paneth, and neuroendocrine cells. Thus, pY1798 was validated as a TC-marker. Interestingly, apoptosis inducers significantly increased relative TC frequencies despite the absence of proliferation at baseline. In conclusion, pY1798 is a novel TC-marker. Selective tyrosine phosphorylation and possible resistance to apoptosis inducers implied the activation of certain kinase(s) in TCs, which may become a clue to elucidate the enigmatic roles of TCs. (J Histochem Cytochem XX:XXX–XXX, XXXX) Journal of Histochemistry & Cytochemistry Keywords brush cell, Girdin, tuft cell marker, tyrosine phosphorylation Introduction long and thick microvilli extending to the supra-nuclear region to form rootlets without forming a terminal web Tuft cells (TCs) are minor scattered components in the mammalian gastrointestinal tract, originally described in 19561 as spool-shaped epithelial cells with “tuft”-like Received for publication November 6, 2016; accepted March 7, 2017. bristles. The definition of TCs (or brush cells) is largely dependent on their morphology.2 Regardless of their Corresponding Author: resident organ (digestive tract or respiratory tract),1,3,4 Masato Asai, Department of Pathology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, TCs share common characteristics, including (1) spo- Japan. 5 radic distribution, (2) spool-shaped somas, and (3) E-mail: [email protected] 2 Kuga et al. at the lumenal surface, unlike enterocytes in the small Materials and Methods intestine. Little is currently known, however, about the primary causes of such morphological characteristics. Ethics Statement Several molecular markers of TCs are also known, The animal experiments were approved by the Animal for example, villin, F-actin–reactive phalloidin, glycoca- Care and Use Committee (26323) and by the lyx-reactive lectins, cyclooxygenase-1/2 (Cox1/2), Recombinant DNA Safety Committee (09-73) of the doublecortin-like kinase 1 (DCLK1), and hematopoi- Nagoya University Graduate School of Medicine. 6,7 etic-prostaglandin-D-synthase (HPGDS). However, Paraffin blocks of human tissues were obtained under these known TC-markers are less than optimal. For the approval of ethical committee of Nagoya University example, some TC-markers (e.g., villin, phalloidin) Graduate School of Medicine. This article does not only stain limited subcellular regions of TCs, whereas contain any individual person’s data. other markers (e.g., lectin Ulex europaeus agglutinin-1 [UEA-1]) also show reactivity in epithelial cells other than TCs, such as neuroendocrine cells, goblet cells, Mouse Strains 7 and Paneth cells. To the best of our knowledge, there All mice were housed in cages with woodchip-bedding is no TC-marker, which can universally mark TCs in at 23C on a 12-hr light/dark cycle. For apoptosis induc- multiple organs of multiple species with high contrast. tion tests and age-dependent expression analysis, Given this limitation, it has been difficult to elucidate DDY mice were obtained from Japan SLC (Shizuoka, the enigmatic role of TCs in multiple tissues. Even Japan). The construction of the gene-targeting vector though TCs account for a considerable proportion of for global Girdin knockout mice has been previously epithelial cells in certain organs (7% of rat salivary described.14 The following PCR primers were used: 2 8 glands and 30% of rat common bile duct ), until 5′-GCCGGAATGAACTTTCATTGATTCCGTC-3′ and recently there have been very few functional studies 5′-CAGCCAGAGGTCCAAACGTTTTAACC-3′ to detect 6 on TCs. A series of reports has recently identified a wild-type alleles (amplicon 293-bp) and 5′-CGCGG possible role for TCs in protection against parasitic ATTGGCCTGAACTGC-3′ and 5′-CCAGGAGTCGTC 9,10 infection in the gut. However, these reports did not GCCACCAA-3′ to detect LacZ alleles (352-bp). identify a broader role for TCs within organs other than the small intestine. In 2005, our group identified the Girdin/CCDC88A Human Tissues gene product as an F-actin–binding protein containing a Normal regions of surgically resected or endoscopi- coiled-coil domain, which has putative roles in cell cally obtained tissues were collected in Nagoya 11 migration via the modulation of the actin cytoskeleton. University Hospital. Other groups also identified that Girdin interacts with several key cellular elements and proteins, including microtubules, heterotrimeric G proteins, and early endo- Paraffin Sections and Free-Floating Cryosections 12,13 somes. Global Girdin knockout mice exhibited com- Anesthetized mice were perfused with a 10% buffered plete preweaning lethality due to malnutrition, as well as neutral formalin solution. Collected tissues were 14–17 18 the developmental abnormalities in brain. Lin et al. immersed in ethanol, xylene, and molten paraffin. discovered that on ligand stimulation of various recep- Paraffin-embedded tissues were sectioned at 4 µm, tors, Girdin is phosphorylated at tyrosine-1798 of Girdin and were mounted on MAS-coated glass slides (Y1798), eventually triggering cell migration. As the (S9441; Matsunami, Osaka, Japan). For free-floating amino-acid sequence of human Girdin around Y1798 sections, tissues were cryoprotected with 15% (KDSNPYATLPRAS) is highly conserved among mam- sucrose in formalin solution overnight, and were snap- malian species, we generated site-specific and phos- frozen in liquid isopentane cooled with liquid nitrogen. phorylation-status–specific antibodies againstFrozen tissues were sectioned at 30 µm at −20C phosphorylated tyrosine-1798 (pY1798) in human using a cryostat (CM3050S; Leica Microsystems, 19 Girdin. We applied this antibody for immunohisto- Wetzlar, Germany). chemical analysis of mammalian gut, and observed specific staining of epithelial cells closely resembling TCs. The aims of this study were to validate pY1798 as Antibodies an authentic TC-marker, and to open up a new field in The sources of antibodies and staining reagents are TC research using pY1798 antibodies as a novel tool. listed in Supplemental Fig. 6. Site-specific and Girdin Phosphorylation Marks Tuft Cells 3 phosphorylation-status–specific anti-Girdin rabbit with excitation wavelengths of 405, 488, and 555 nm polyclonal antibodies were collectively developed with using a confocal laser-scanning microscope (LSM700; IBL, Co., Ltd. (Gunma, Japan).20 Zeiss, Jena, Germany). Immunohistochemistry Apoptosis Induction Test by Cisplatin Deparaffinized sections were boiled for target retrieval Cisplatin [cis-diamminedichloroplatinum (CDDP), at each optimal pH (6, 7, or 9) at 98C for 30 min. Briplatin, 10 mg/20 mL] was purchased from Bristol Sections were serially treated with 3% hydrogen per- Myers Squibb (New York, NY). Seven-week-old male oxide in methanol, protein block serum-free (X0909; DDY mice (n=32) were divided into four groups: (1) Dako, Glostrup, Denmark), and primary antibodies at control (n=8), (2) low-dose (CDDP 0.2 mg/body, n=8), 4C overnight. After washing in phosphate buffered (3) middle-dose (0.4 mg/body, n=8), and (4) high-dose saline (PBS), sections were incubated with secondary (0.8 mg/body, n=8). The indicated dose of cisplatin antibodies. When the host species of primary antibod- was administered in a single intraperitoneal injection. ies were rabbit or mouse, EnVision System HRP- Mice were euthanized for tissue collection 24 or 48 hr labeled polymer (rabbit, K4003, or mouse, K4001; after administration. Dako) was used. For primary antibodies from goat, donkey polyclonal anti-MTR antibody (705-065-147; Jackson-Immunoresearch,

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