Recruitment of Polarity Complexes and Tight Junction Proteins to the Site of Apical Bulk Endocytosis

Figures

• 

  Figure 1. Myo5b KO mice have microvilli-lined inclusions associated with α-actinin and Rab11a. (A) Immunofluorescence staining for the basolateral protein p120 (magenta) and the plasma membrane and actin cytoskeleton linker Ezrin (green) shows the presence of numerous intracellular inclusions in Myo5b KO mice. (B) Micrograph of α-actinin-4 (magenta), which delineates the terminal web immediately below the apical brush border and the lateral membrane of intestinal epithelial cells and γ-actin (green), which identifies the actin-rich apical membrane immunostaining. No intracellular inclusions were observed in the proximal small intestine of neonatal control mice. Myo5b KO mice had numerous γ-actin–positive inclusions that also were ringed by α-actinin–4. (C) The small intestine of littermate control and Myo5b KO mice were stained for Rab11a (magenta) and γ-actin (green), which showed altered Rab11a localization in Myo5b-deficient enterocytes. Moreover, Rab11a surrounded numerous inclusions at the apical membrane and inclusions that were fully internalized. (D) Z-stack projections of the proximal small intestine of control and Myo5b KO mice to visualize the 3-dimensional nature of inclusions…

• 

  Figure 2. The Par polarity complex associates with inclusions in Myo5b KO enterocytes. (A) aPKC (magenta), a component of the Par polarity complex, is concentrated in inclusions in Myo5b KO mice, whereas in control mice, aPKC was localized to the apical brush border and the lateral membrane. (B) In control mice, Par3 (magenta) was present at the apical tight junctions. In the small intestine of Myo5b KO mice, cytoplasmic Par3 was evident and Par3 was enriched at the site of inclusions forming from endocytosis of the apical membrane. (C) Similar to Par3, Par6 (magenta) was observed at the apical tight junction in control mice. Enterocytes from Myo5b KO mice showed an accumulation of Par6 at the apical membrane of forming inclusions. (D) The small guanosine triphosphatase, Cdc42 (magenta) was present at the brush border and basolateral membrane of control mice. Myo5b KO mice had decreased apical Cdc42 compared with littermate control neonatal mice. n = 4–6 mice per group. Scale bars: 50 μm.

• 

  Figure 3. Micrographs of components of the Crumbs3 polarity complex in control and Myo5b KO proximal small intestine. (A) Crumbs3 (magenta) and γ-actin (green) staining showed Crumbs3 localized to the apical membrane and lateral membrane in control mice. In Myo5b KO mice Crumbs3 was concentrated in both internalized and forming inclusions. (B) The Crumbs3 binding partner Pals1 (magenta) was present at the apical junctional complex of enterocytes in control mice. In Myo5b KO mice, Pals1 was associated with inclusions. (C) Patj (magenta), another Crumbs3 polarity complex protein, was localized to the apical junctional complex in the proximal small intestine of control mice. In Myo5b KO mice, Patj was enriched at the site of inclusions forming from internalization of the apical membrane. n = 4–6 mice per group. Scale bars: 50 μm.

• 

  Figure 4. Apical tight junction proteins accumulate at the sites of inclusion formation in Myo5b KO enterocytes. In control mice, the apical tight junction proteins (A) claudin-2, (B) ZO-1, (C) ZO-2, and (D) occludin (all in magenta) were distributed normally at the site of apical cell–cell contact in control mice. (A) In Myo5b KO mice, claudin-2 was found to be dispersed within the cytoplasm in enterocytes at the base of the villi and was associated with inclusions at the apical membrane. (B) ZO-1, (C) ZO-2, and (D) occludin also accumulated at the site of brush-border endocytosis of forming inclusions in Myo5b-deficient enterocytes. n = 4–6 mice per group. Scale bars: 50 μm.

• 

  Figure 5. Cytoplasmic inclusions lack ZO-1, ZO-2, and occludin, and infrequently associate with claudin-2. (A) Immunostaining for tight junction proteins in Myo5b KO mice showed internalized inclusions occasionally stained positive for claudin-2. In Myo5b KO, small intestine inclusions rarely contained (B) ZO-1, (C) ZO-2, or (D) occludin. n = 4–6 mice per group. Scale bars: 50 μm.

• 

  Figure 6. Three-dimensional imaging of inclusions in Myo5b KO proximal small intestine show a cap of claudin-2 and ZO-1 formed over inclusions contiguous with the apical membrane. Imaging of 25-μm frozen sections of Myo5b KO proximal small intestine showed the dense accumulation of (A) claudin-2 (magenta) and (B) ZO-1 (magenta) immediately above forming inclusions identified by F-actin (green). Claudin-2 and ZO-1 appear to form a cap over inclusions where F-actin is absent. (C) Quantification of total inclusions that were present at the apical membrane and inclusions that were contiguous with the apical membrane and positive for Claudin-2 or ZO-1. ∗P < .05, n = 4–5 mice per group. TJ, tight junction.

• 

  Figure 7. Inclusions at the apical membrane have enrichment of claudin-2 in a pig model of MVID. Small intestinal tissue from aged matched wild-type (WT) pigs and pigs that expressed a P663L mutation in MYO5B that resulted in MVID were immunostained for claudin-2 (magenta) and γ-actin (green) to determine whether a physiologically relevant model of MVID formed inclusions in a similar manner as Myo5b KO mice. Inclusions in MVID pigs showed claudin-2 accumulated at the site of inclusion formation. n = 2 MVID pigs and 3 WT pigs. Scale bars: 50 μm.

• 

  Figure 8. Cortactin associates with ZO-1 during inclusion formation in Myo5b KO mice. (A) Immunostaining for cortactin (magenta), ZO-1 (green), P-ERM (cyan), and nuclei (white) in control and Myo5b KO mice. Magenta arrows indicate cortactin at sites of inclusion formation in Myo5b KO mice. (B) Maximum-intensity Z-stack projection of a representative inclusion in a Myo5b KO enterocyte. n = 4 mice per group. Scale bars: 50 μm.

• 

  Figure 9. Intestinal organoids generated from Myo5b KO mice have inclusions that are associated with claudin-2 and ZO-1. (A) Intestinal organoids derived from the proximal small intestine of Myo5b KO mice were treated with DMSO vehicle or ζ-Stat, an aPKC inhibitor to determine whether inhibition of aPKC would prevent inclusion formation. Both DMSO- and ζ-Stat–treated intestinal organoids from Myo5b KO mice developed numerous inclusions. (B) Immunostaining for Crumbs3 in DMSO-and ζ-Stat–treated Myo5b KO intestinal organoids showed no alteration in the localization of Crumbs3. However, a decreased intensity of Crumbs3 in intestinal organoids treated with ζ-Stat was observed compared with DMSO-treated organoids. Intestinal organoids from littermate control and Myo5b KO mice were immunostained for γ-actin and (C) claudin-2 (magenta) or (D) ZO-1 (green). Control-derived organoids showed a normal distribution of claudin-2 and ZO-1, while Myo5b KO mouse–derived organoids showed inclusions that were associated with accumulations of claudin-2 and ZO-1. n = 3 mice per group were used to generate organoids. Scale bars: (C and D) 10 μm, and (A and B) 50 μm.

• 

  Figure 10. Diagram of proposed model of inclusion formation in Myo5b KO enterocytes. Based on our findings, we propose that during apical bulk endocytosis, tight junction proteins are recruited to cover the forming bulk endosome/inclusion. These tight junction proteins form a dense cap over inclusions that are contiguous with the apical membrane, implying a functional role during apical bulk endocytosis excision from the membrane. In addition, apical polarity complexes (the Crumbs and Par complexes) are present at the apical membrane and in internalized inclusions.