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. 2017 Nov 15;5(2):145-156.
doi: 10.1016/j.jcmgh.2017.11.001. eCollection 2018.

The Endosomal Protein Endotubin Is Required for Enterocyte Differentiation

Christopher M Cox  1 , Ruifeng Lu  1   2 , Kaan Salcin  1   3 , Jean M Wilson  1
Affiliations

The Endosomal Protein Endotubin Is Required for Enterocyte Differentiation

Christopher M Cox et al. Cell Mol Gastroenterol Hepatol. .

Abstract

Background & aims: During late embryonic development and through weaning, enterocytes of the ileum are highly endocytic. Defects in endocytosis and trafficking are implicated in neonatal disease, however, the mechanisms regulating trafficking during the developmental period are incompletely understood. The apical endosomal protein endotubin (EDTB) is highly expressed in the late embryonic and neonatal ileum. In epithelial cells in vitro, EDTB regulates both trafficking of tight junction proteins and proliferation through modulation of YAP activity. However, EDTB function during the endocytic stage of development of the intestine is unknown.

Methods: By using Villin-CreERT2, we induced knockout of EDTB during late gestation and analyzed the impact on endocytic compartments and enterocyte structure in neonates using immunofluorescence, immunocytochemistry, and transmission electron microscopy.

Results: Deletion of the apical endosomal protein EDTB in the small intestine during development impairs enterocyte morphogenesis, including loss of the apical endocytic complex, defective formation of the lysosomal compartment, and some cells had large microvillus-rich inclusions similar to those observed in microvillus inclusion disease. There also was a decrease in apical endocytosis and mislocalization of proteins involved in apical trafficking.

Conclusions: Our results show that EDTB-mediated trafficking within the epithelial cells of the developing ileum is important for maintenance of endocytic compartments and enterocyte integrity during early stages of gut development.

Keywords: AEC, apical endocytic complex; AP, alkaline phosphatase; CRISPR/Cas9, clustered regularly interspaced short palindromic repeats/cas9 endonuclease; EDTB, endotubin; EEA1, early endosomal antigen 1; Endosomes; Endotubin; G, guide; GFP, green fluorescent protein; GTPase, guanosine triphosphatase; KO, knockout; LAMP1, lysosome-associated membrane protein 1; MAMDC4, MAM domain containing 4; MVID, microvillus inclusion disease; P, postnatal day; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; Rab; SDS, sodium dodecyl sulfate; TBST, tris-buffered saline with 0.1% tween-20; TEM, transmission electron microscopic; TJ, tight junction; Tight Junction; Trafficking.

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Figures

None
Graphical abstract
Figure 1
Figure 1
EDTB is present in apical endosomes and is deleted upon tamoxifen induction of the Cre-recombinase. (A) P3 ileum labeled with antibodies against NHE3 (green) and EDTB (red). EDTB labeling is adjacent to NHE3 and localized in the apical cytoplasm (boxed area is magnified). Nuclei (4′,6-diamidino-2-phenylindole, blue). (B) PCR genotyping of P3 pups shows the presence of the floxed alleles and CreERT2 in KO animals. (C) Lysates from the proximal ileum were analyzed by Western blot and confirm loss of EDTB protein in KO intestine. (D) Immunofluorescence of distal ileum shows apical localization of EDTB (red) in control animals and loss of expression in KO animals. Nuclei (4′,6-diamidino-2-phenylindole, blue). Scale bars: 25 μm.
Figure 2
Figure 2
Disruption of enterocyte morphology after EDTB KO. (A) H&E staining of control and EDTB KO ileum. In control animals, the enterocytes have basal nuclei. In contrast, in knockout animals the nuclei are displaced, with many nuclei close to the brush border (arrows). Scale bar: 25 μm. (B) Whisker plot representation showing fold change in cell height from the nuclei to the apical border of enterocytes. ***P < .005. (C) TEM of enterocytes of control and EDTB KO animals. In both cases the enterocytes are a simple epithelium with a well-developed brush border. However, in KO animals the cells are shorter and lack the giant lysosome. Dashed line marks the basal border of the enterocytes. Scale bar: 5 μm. GL, giant lysosome; N, nucleus.
Figure 3
Figure 3
Disruption of apical endocytic compartment after EDTB KO. (A) TEM of the apical domain of control and EDTB KO enterocytes at P3. Control animals have a well-developed AEC. EDTB KO animals largely lack apical endosomal tubules and vesicles. Scale bar: 0.5 μm. (B) CRISPR/Cas9 genome editing tool was used to disrupt EDTB in Caco2BBE cells followed by pooling of cells. Loss of EDTB expression was verified by Western blot. The presence of some EDTB likely is owing to the mixed population. (C) Uptake of 4 kilodaltons of fluorescent dextran for 30 minutes followed by lysis and fluorometric analysis. Dextran uptake was decreased after EDTB KO in Caco2BBE cells. *P < .05, **P < .01.
Figure 4
Figure 4
EDTB KO disrupts lysosomes. Immunofluorescent labeling of ileum using the lysosome marker LAMP1 (green). In control animals, LAMP1 labeling is present in large structures above the nucleus but distinct from the EDTB-positive AEC (red). After EDTB KO, LAMP1 labeling is more compact and displaced apically. Nuclei (blue). Scale bars: 20 μm (6 upper panels) and 5 μm (2 lower panels).
Figure 5
Figure 5
EDTB KO results in intracellular accumulation of apical proteins in some enterocytes. (A) Alkaline phosphatase histochemistry of P3 ileum of control and EDTB KO enterocytes. Alkaline phosphatase is present on the apical membrane and AP-positive inclusions are present in EDTB KO enterocytes (arrows). Scale bar: 25 μm. (B) NHE3 labeling of P3 ileum of control and EDTB KO enterocytes. NHE3 inclusions are present in EDTB KO enterocytes (arrow). Scale bar: 25 μm. (C and D) TEM of enterocytes shows large invaginations and inclusions containing microvilli. Scale bar: 5 μm. (E) Labeling with antibodies against EEA1, Rab11a, and Rab14. EEA1 is localized to the apical cytoplasm in control and EDTB KO ileum. Both Rab11 and Rab14 are enriched in the apical cytoplasm of control enterocytes. After EDTB KO, the labeling is diffuse within the enterocytes. Scale bar: 50 μm. (F) Lysates of proximal ileum were analyzed by Western blot to examine levels of Rab protein. The levels of both Rab11 and Rab14 appear unchanged.
Figure 6
Figure 6
EDTB KO disrupts TJ protein localization. (A) Claudin (cldn) 1 and claudin 2 labeling of control and EDTB KO ileum. Claudin 1 is present at the lateral membrane of control cells. However, in EDTB KO ileum the labeling is diffuse, with limited labeling at the lateral membrane. Claudin 2 is localized at the apical membrane in both control and EDTB KO ileum. Scale bar: 25 μm. (B) Lysates of the proximal ileum were analyzed by Western blot to examine levels of TJ proteins. The levels of claudin 1 and claudin 2 are unchanged.
Figure 7
Figure 7
Reduced growth after endotubin KO. Control and KO mice were weighed on P3. Mice with EDTB KO are lighter than controls. *P < .05.
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