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. 2017 Jun;65(6):347-366.
doi: 10.1369/0022155417702586. Epub 2017 Apr 4.

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

Daisuke Kuga  1   2 , Kaori Ushida  1 , Shinji Mii  1 , Atsushi Enomoto  1 , Naoya Asai  1   3 , Masato Nagino  2 , Masahide Takahashi  1   3 , Masato Asai  1   4
Affiliations

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

Daisuke Kuga et al. J Histochem Cytochem. 2017 Jun.

Abstract

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. .

Keywords: Girdin; brush cell; tuft cell marker; tyrosine phosphorylation.

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Figures

Figure 1.
Figure 1.
pY1798 reveals epithelial cells closely resembling tuft cells (TCs) in the mammalian gut. (A) Immunohistochemistry of mouse jejunum with anti-Girdin phospho-Y1798 (pY1798) antibodies or with cyclooxygenase-2 (Cox2) antibodies. The boxed areas in the low-magnification images (scale bar, 100 µm) were magnified, rotated, and shown on each right side. Red dots represent positive epithelial cells. Immunohistochemistry of mouse jejunum with pY1798 antibodies pre-absorption (B, upper), or post-absorption (B, lower) using unphosphorylated Y1798 peptides (scale bars, 10 µm). Immunohistochemistry of mouse (C) and human (D) gastrointestinal tracts with pY1798 antibodies (scale bars, 10 µm).
Figure 2.
Figure 2.
Validation of pY1798 as a tuft cell (TC)-marker by transmission electron microscopy (TEM). TEM of pY1798-immunostained jejunum (A and B). Boxed regions are shown magnified in the right panels. (A) A positive epithelial cell (arrowhead) protrudes beyond the brush border. (B) The signal condensation at the lumenal tip (asterisk) of a pY1798-positive epithelial cell comprises thick microvilli accompanied by rootlets (open arrowheads; scale bars in A, left, 10 µm; A, right, 5 µm; B, left, 1 µm; and B, right, 0.5 µm).
Figure 3.
Figure 3.
Validation of pY1798 by co-staining with known tuft cells (TC)–markers. (A) The resultant counts of immunofluorescent cells were arranged in a 2 × 2 contingency table. Odds ratios (ORs; bc/ad) were calculated from these cell counts. Representative results of immunofluorescence using a reference marker (pY1798) and one or two target markers including hematopoietic-prostaglandin-D-synthase (HPGDS; B and E), phalloidin (C and D), cyclooxygenase-2 (Cox2; D), lectin UEA-I (D), and villin (F–H), in tissue sections from multiple organs, including stomach (B and F), small intestine (C, D, and G), and colon (E and H) from mouse (B–E) and human (F–H). The columnar side of the squamocolumnar junction (SCJ) of mouse stomach frequently contained pY1798–HPGDS double-positive TCs (B). Focal thickenings of the brush border (white arrowheads) consistently colocalized with pY1798 (C; scale bars, upper panels, 20 µm; lower panels, 5 µm in B and E–H). In Panel C, the boxed region in the left panel (scale bar, 20 µm) is shown magnified and rotated in the four panels at right (scale bar, 10 µm; Panel D, 10 µm). Abbreviation: DAPI, 4,6-diamidino-2-phenylindole.
Figure 4.
Figure 4.
pY1798-positive cells are distinct from other minor epithelial cells. Immunohistochemistry of mouse jejunum with pY1798 antibodies, counterstained with alcian blue (A, left, low magnification, scale bar, 50 µm; right, high magnification of the boxed region, scale bar, 20 µm) or with hematoxylin–eosin (B, left, low magnification, scale bar, 100 µm; right, high magnification of the boxed region, scale bar, 20 µm). Neither goblet cells (black arrowheads) nor Paneth cells (red dotted circle) were pY1798 positive (open arrowheads). (C) Immunofluorescence of mouse jejunum with pY1798 antibodies and Insm1 (pan-neuroendocrine markers). pY1798-positive cells (white open arrowheads) and Insm1-positive cells (white closed arrowheads) were mutually exclusive (scale bars, 20 µm). (D) Immunohistochemistry (left) and immunofluorescence (right) of mouse jejunum with sodium glucose transporter 1 (SGLT1) antibodies. SGLT1 was found at the apical surfaces (black arrows) except for at the surface of goblet cells (red arrowhead). One cell (yellow arrowhead) is shown with a pY1798-positive signal in the cytoplasm (left). Cytoplasmic SGLT consistently colocalized with pY1798, whereas surface SGLT1 (white arrows) never did (right; scale bars, 10 µm). (E) Immunofluorescence of mouse jejunum with pY1798 antibodies and proliferation markers [Ki67 and proliferating cell nuclear antigen (PCNA)]. From the left are shown pY1798 antibodies, proliferation marker (upper, Ki67, and lower, PCNA), and digitally merged images with DAPI (4,6-diamidino-2-phenylindole; scale bars, 20 µm).
Figure 5.
Figure 5.
Lack of apoptosis in tuft cells (TCs). (A) Double-fluorescence staining of mouse jejunum with TdT-mediated dUTP nick end labeling (TUNEL) and pY1798. TUNEL-positive cells (white dotted circles) at the villus tip and TCs (white closed arrowheads) were mutually exclusive (scale bars, 20 µm). (B) Immunofluorescence of mouse jejunum for pY1798 (left) and cleaved caspase 3 (C-caspase 3; right). C-Caspase 3–positive epithelial cells (white open arrowheads) and TCs (white closed arrowheads) were mutually exclusive (scale bars, 20 µm). (C) Apoptosis induction test using cisplatin (cis-diamminedichloroplatinum or CDDP). Mice were divided into four groups (n=8 each): control, low-dose (0.2 mg/body), middle-dose (0.4 mg/body), and high-dose (0.8 mg/body), and euthanized for 24 or 48 hr after administration. Jejunum sections were stained for C-caspase 3, pY1798, or cyclooxygenase-2 (Cox2). The percentages of positive epithelial cells (red arrowheads) are summarized in bar graphs (24 hr, filled bars; 48 hr, open bars). *p<0.05 (scale bars, 50 µm). (D) Immunofluorescence of mouse jejunum for pY1798 and C-caspase 3 from the high-dose group. Boxed regions in the upper row (scale bars, 20 µm) are shown magnified below (scale bars, 5 µm). C-Caspase 3–positive epithelial cells (white open arrowheads) and TCs (white closed arrowheads) were mutually exclusive. Abbreviation: DAPI, 4,6-diamidino-2-phenylindole.
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