Myosin 5b is required for proper localization of the intermicrovillar adhesion complex in the intestinal brush border

Abstract

Intestinal enterocytes have an elaborate apical membrane of actin-rich protrusions known as microvilli. The organization of microvilli is orchestrated by the intermicrovillar adhesion complex (IMAC), which connects the distal tips of adjacent microvilli. The IMAC is composed of CDHR2 and CDHR5 as well as the scaffolding proteins USH1C, ANKS4B, and Myosin 7b (MYO7B). To create an IMAC, cells must transport the proteins to the apical membrane. Myosin 5b (MYO5B) is a molecular motor that traffics ion transporters to the apical membrane of enterocytes, and we hypothesized that MYO5B may also be responsible for the localization of IMAC proteins. To address this question, we used two different mouse models: 1) neonatal germline MYO5B knockout (MYO5B KO) mice and 2) adult intestinal-specific tamoxifen-inducible VillinCre^ERT2;MYO5B^flox/flox mice. In control mice, immunostaining revealed that CDHR2, CDHR5, USH1C, and MYO7B were highly enriched at the tips of the microvilli. In contrast, neonatal germline and adult MYO5B-deficient mice showed loss of apical CDHR2, CDHR5, and MYO7B in the brush border and accumulation in a subapical compartment. Colocalization analysis revealed decreased Mander's coefficients in adult inducible MYO5B-deficient mice compared with control mice for CDHR2, CDHR5, USH1C, and MYO7B. Scanning electron microscopy images further demonstrated aberrant microvilli packing in adult inducible MYO5B-deficient mouse small intestine. These data indicate that MYO5B is responsible for the delivery of IMAC components to the apical membrane.NEW & NOTEWORTHY The intestinal epithelium absorbs nutrients and water through an elaborate apical membrane of highly organized microvilli. Microvilli organization is regulated by the intermicrovillar adhesion complexes, which create links between neighboring microvilli and control microvilli packing and density. In this study, we report a new trafficking partner of the IMAC, Myosin 5b. Loss of Myosin 5b results in a disorganized brush border and failure of IMAC proteins to reach the distal tips of microvilli.

Keywords: enterocyte; intermicrovillar adhesion complex (IMAC); intestine; microvilli; myosin.

Graphical abstract

Figure 1.

Neonatal germline MYO5B KO mice exhibit altered IMAC gene expression and protein localization in the small intestine. Immunostaining of the small intestine for: the IMAC components (A) CDHR2 (magenta) and (B) CDHR5 (magenta), apical membrane marker phosphorylated-ezrin-radixin-moesin (P-ERM; green) and nuclei (blue) in neonatal control mice and germline MYO5B KO mice. Arrows indicate inclusions containing CDHR5 in the merged image. C: small intestinal tissue stained for the IMAC protein USH1C (magenta), the apical membrane protein γ actin (green) and nuclei (blue) in neonatal control and germline MYO5B KO mice. D: immunofluorescence images of the IMAC protein MYO7B (magenta), the apical membrane marker Villin (green) and nuclei (blue) in control and MYO5B KO mice. n = 4–6 mice/group. Scale bars = 50 µm. IMAC, intermicrovillar adhesion complex; KO, knockout.

Figure 2.

Adult tamoxifen-induced VillinCre^ERT2;MYO5B^flox/flox mice have aberrant localization of IMAC proteins. Small intestinal tissue stained for (A) CDHR2 (magenta), γ-actin (green) and nuclei (blue) in littermate control and tamoxifen-induced VillinCre^ERT2;MYO5B^flox/flox mice. Colocalization analysis of confocal micrographs for CDHR2 and γ-actin overlap. B: immunostaining of small intestinal tissue showing CDHR5 (magenta), Villin (green) and nuclei (blue) in control and VillinCre^ERT2;MYO5B^flox/flox mice. Graph shows colocalization analysis of CDHR5 and Villin in control and VillinCre^ERT2;MYO5B^flox/flox mice. C: staining for USH1C (magenta), γ-actin (green), and nuclei (blue) in control and VillinCre^ERT2;MYO5B^flox/flox mice along with colocalization analysis of USH1C and γ-actin. D: immunostaining for the IMAC protein MYO7B (magenta), γ-actin (green), and nuclei in the small intestine of control and VillinCre^ERT2;MYO5B^flox/flox mice. Mean fluorescence intensity measurements from immunofluorescence staining of adult control and VillinCre^ERT2;MYO5B^flox/flox mice for CDHR2 (E) and MYO7B (F). Colocalization analysis of MYO7B and γ-actin coexpression. *P < 0.05, n = 3 or 4 mice/group. Scale bars = 50 µm. IMAC, intermicrovillar adhesion complex.

Figure 3.

Loss of MYO5B results in increased localization of CDHR2 and CDHR5 to Lamp1⁺ lysosomes. Adult control and VillinCre^ERT2;MYO5B^flox/flox mice were immunostained for the IMAC components (A) CDHR2 (magenta), (B) CDHR5 (magenta), (C) USH1C (magenta), (D) MYO7B (magenta), and Lamp1 (green) to identify lysosomes. Colocalization analysis of the degree of overlap between IMAC protein and Lamp1⁺ lysosomes was performed and plotted. **P < 0.01, n = 3 or 4 mice/group. Scale bars = 50 µm. IMAC, intermicrovillar adhesion complex.

Figure 4.

Loss of MYO5B in vitro impacts the expression and localization of CDHR2. Intestinal organoids derived from uninduced VillinCre^ERT2;MYO5B^flox/flox mice and treated with either ethanol (control) or 4-hydroxytamoxifen (4-OHT) to deplete MYO5B in vitro. Differentiated intestinal organoids were immunostained for CDHR2 (magenta), γ actin (green), and nuclei (blue). Three uninduced VillinCre^ERT2;MYO5B^flox/flox mice were used to generate organoids and multiple wells of each treatment group (i.e., ethanol or 4-OHT) were pooled for paraffin embedding and staining. Scale bars = 50 µm. IMAC, intermicrovillar adhesion complex.

Figure 5.

Adult mice lacking MYO5B have decreased brush-border height and altered microvilli packing. A: confocal microscopy of small intestinal enterocytes from littermate control and VillinCre^ERT2;MYO5B^flox/flox mice stained for the terminal web marker α-actinin 4 (magenta), the microvilli label villin (green), and nuclei (blue). Scale bars = 10 µm. Graphs showing measurements of (B) α-actinin 4, (C) villin, and (D) total brush-border height in control and VillinCre^ERT2;MYO5B^flox/flox mice. **P < 0.01, ***P < 0.001, n = 4 mice/group. Scanning electron micrographs of the apical membrane of (E) control and (F) VillinCre^ERT2;MYO5B^flox/flox mice. Scale bars = 5 µm.