Fig 1: Myo5b KO mice have microvilli-lined inclusions associated with a-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 a-actinin-4 (magenta), which delineates the terminal web immediately below the apical brush border and the lateral membrane of intestinal epithelial cells and ?-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 ?-actin–positive inclusions that also were ringed by a-actinin–4. (C) The small intestine of littermate control and Myo5b KO mice were stained for Rab11a (magenta) and ?-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 after loss of Myo5b. (E) Fracture SEM was performed on neonatal small intestine from Myo5b KO mice to view microvilli-lined inclusions. A completely internalized inclusion (pseudocolored magenta) and a forming inclusion (pseudocolored purple) are shown. n = 4–6 mice per group. Scale bars (immunofluorescence images): 50 µm. Scale bars (SEM from left to right): 10 µm, 5 µm, 5 µm.