Myosin Vb Traffics P-glycoprotein to the Apical Membrane of Intestinal Epithelial Cells.

BACKGROUND & AIMS: The xenobiotic efflux pump P-glycoprotein is highly expressed on the apical membrane of the gastrointestinal tract where it regulates the levels of intracellular substrates. P-glycoprotein is altered in disease, but the mechanisms which regulate the levels of P-glycoprotein are still being explored. The molecular motor Myosin Vb (Myo5b) traffics diverse cargo to the apical membrane of intestinal epithelial cells. We hypothesized that Myo5b was responsible for delivery of P-glycoprotein to the apical membrane of enterocytes. METHODS: We used multiple murine models that lack functional Myo5b or the myosin binding partner Rab11a to analyze P-glycoprotein localization. Pig and human tissue were analyzed to determine P-glycoprotein localization in the setting of MYO5B mutations. Intestinal organoids were used to examine P-glycoprotein trafficking and to assay P-glycoprotein function when MYO5 is inhibited. RESULTS: In mice lacking Myo5b or the binding partner Rab11a, P-glycoprotein was improperly trafficked and had decreased presence in the brush border of enterocytes. Immunostaining of a pig model lacking functional Myo5b and human biopsies from a patient with an inactivating mutation in Myo5b also showed altered localization of intestinal P-glycoprotein. Human intestinal organoids expressing the motorless MYO5B tail domain had co-localization with P-glycoprotein, confirming that P-glycoprotein was trafficked by MYO5B in human enterocytes. Inhibition of MYO5 in human intestinal cell lines and organoids resulted in decreased P-glycoprotein capacity. Additionally, inhibition of MYO5 in human colon cancer cells diminished P-glycoprotein activity and increased cell death in response to a chemotherapeutic drug. CONCLUSIONS: Collectively, these data demonstrate that Myo5b is necessary for the apical delivery of P-glycoprotein.

Identification of Differentiated Intestinal Epithelial Cells Using Immunostaining and Fluorescence Microscopy.

Immunofluorescence imaging enables visualization of a wide range of molecules in diverse cells and tissues. Determining the localization and endogenous protein levels in cells using immunostaining can be highly informative for researchers studying cell structure and function. The small intestinal epithelium is composed of numerous cell types including absorptive enterocytes, mucus-producing goblet cells, lysozyme positive Paneth cells, proliferative stem cells, chemosensing tuft cells, and hormone-producing enteroendocrine cells. Each cell type in the small intestine has unique functions and structures that are critical for maintaining intestinal homeostasis and identifiable by immunofluorescence labeling. In this chapter we provide a detailed protocol and representative images of immunostaining of paraffin-embedded mouse small intestinal tissue. The method highlights antibodies and micrographs that identify differentiated cell types. These details are important because quality immunofluorescence imaging can provide novel insights and a greater understanding of healthy and disease states.

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

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 VillinCreERT2;MYO5Bflox/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.