Loss of Myosin Vb Results in Decreased Apical P‐Glycoprotein in vivo and in vitro

Background & Aims P‐glycoprotein (p‐gp), also known as multidrug resistance protein 1, is a xenobiotic efflux pump. In the small intestine, p‐gp is localized to the apical membrane of enterocytes. Changes in p‐gp expression is linked to inflammatory bowel diseases (IBD) and impairment of drug response in cancer. Regulating the trafficking of p‐gp to the plasma membrane represents a plausible approach to improve therapy for cancer treatment. At present, the trafficking mechanism responsible for delivery of p‐gp to the apical membrane of epithelial cells is unclear. Myosin Vb (Myo5b) is a molecular motor that is crucial for the proper transport of vesicles and proteins to the apical membrane of epithelial cells. Loss of functional Myo5b results in a congenital diarrheal disorder termed Microvillus Inclusion Disease (MVID). Individuals with MVID present with severe, unremitting diarrhea requiring parenteral nutrition and eventual small bowel transplantation. Studies using animal models of MVID and MVID patient tissue have revealed that loss of Myo5b results in a dramatic decrease in key transporters on the apical membrane of enterocytes. Therefore, we investigated whether proper localization of p‐glycoprotein was impeded by loss of Myo5b. Methods Intestinal tissue was analyzed from neonatal control and germline Myo5b knockout (Myo5b KO) mice. Additionally, a tamoxifen inducible, intestinal specific mouse model of loss of Myo5b was also used (VillinCre ERT2 ;Myo5b flox/flox ). Adult Myo5b flox/flox mice treated with tamoxifen were used as controls for VillinCre ERT2 ;Myo5b flox/flox mice. Intestinal organoids were generated from VillinCre ERT2 ;Myo5b flox/flox mice and treated with either ethanol as a control or 4 hydroxy tamoxifen to induce loss of Myo5b. Results Immunostaining of gamma actin staining was performed to delineate the actin rich microvilli and identify the apical membrane. In control mice, p‐gp was restricted to the apical membrane and co‐localized with gamma actin as expected. In germline Myo5b KO mice, p‐gp had increased immunofluorescence intensity and was observed below the apical membrane of small intestinal enterocytes. Lamp1 immunostaining also revealed that subapical p‐gp was surrounded by lamp1 positive lysosomes in Myo5b KO mice. The association of lysosomes with p‐gp suggests that improper localization of p‐gp resulting from loss of Myo5b may promote lysosomal degradation of p‐gp. Adult Myo5b flox/flox control mice treated with tamoxifen showed normal distribution of p‐gp on the apical membrane of small intestinal enterocytes demonstrating co‐localization with gamma actin. In contrast, tamoxifen induced VillinCre ERT2 ;Myo5b flox/flox mice showed no p‐gp at the apical brush border and no overlap with gamma actin immunostaining. Tamoxifen induced VillinCre ERT2 ;Myo5b flox/flox mice displayed a large accumulation of p‐gp below the apical membrane. Complementing our in vivo data, in vitro induction of Myo5b loss using 4 hydroxy tamoxifen resulted in a similar subapical localization of p‐gp in induced VillinCre ERT2 ;Myo5b flox/flox intestinal organoids compared to ethanol treated organoids. Conclusions Loss of Myo5b results in improper localization of p‐gp. Collectively, these data suggest that Myo5b is necessary for the proper trafficking of p‐gp to the apical membrane of intestinal cells.