Inclusion formation following loss of Myo5b results from endocytosis of the brush border. (A and B) Myo5b KO mice were crossed with LifeAct mice expressing fluorescent F-actin to allow for visualization of the brush border and inclusion formation. (A) Live explants of control LifeAct mice showed moderate subapical expression of F-actin (green). (B) LifeAct;Myo5b KO mice had large F-actin–rich inclusions, many of which appeared to be attached to the apical membrane. Scale bars = 10 µm. (C and D) To determine whether inclusions form through endocytosis of the brush border, 70-kD FITC-dextran was added to the apical side of proximal small-intestine explants (duodenum and jejunum) from neonatal control (C) and Myo5b KO mice (D). Scale bars = 25 µm for first panel of C (low-magnification image). In control mice, no subapical F-actin–positive inclusions were observed; however, a small number of cells had FITC-dextran accumulation (C). In Myo5b KO enterocytes, numerous F-actin inclusions were observed containing concentrated FITC-dextran, providing evidence that inclusions form via endocytosis (D). n = 3–5 mice per group (groups: LifeAct, LifeAct;Myo5b KO, control, and Myo5b KO). Scale bars for C and D = 2 µm.