Inclusion formation in neonatal enterocytes lacking Myosin Vb is associated with apical accumulation of tight junction proteins

Abstract

BackgroundMicrovillus inclusion disease (MVID) is caused by inactivating mutations in the molecular motor Myosin Vb (Myo5B). MVID is characterized by the presence of intracellular inclusions lined by microvilli in enterocytes. In germline Myosin Vb knockout mice (Myo5b KO) inclusions form from invagination of the apical brush border via apical bulk endocytosis. Crumbs 3 regulates apical membrane formation, intestinal epithelial polarity and tight junction assembly. As a result, we speculate that Crumbs 3 and junctional proteins are involved in inclusion formation in our Myo5b KO mice. While some aspects of inclusion formation and altered enterocyte polarity have been previously characterized in Myo5b KO mice, the localization and role of tight junction proteins during inclusion formation is currently unknown. Thus, we sought to elucidate the role of junctional proteins during the formation and excision of inclusions from the apical membrane in Myo5b KO mice.Methods & ResultsIntestinal tissue collected from neonatal control and Myo5b KO littermates were analyzed by immunofluorescence to determine the localization of apical proteins and tight junction proteins. In the neonatal intestine the tight junction protein, Claudin 2, provides a pathway for the paracellular transport of sodium, potassium and water. Immunostaining for Claudin 2, which is expressed in neonatal villus enterocytes, showed normal localization at the apical junctional complex of neighboring intestinal epithelial cells in control mice. In Myo5b KO mice, Claudin 2 was present intracellularly as puncta and was associated with inclusions, identified by gamma‐actin staining, that were forming at the apical membrane. Occludin immunofluorescence revealed a similar pattern in Myo5b KO mice in which occludin appeared to be concentrated immediately above the site of inclusion formation. In control mice, zona occludin‐1 (ZO‐1) was observed at the apical junctional complex between intestinal cells as expected. In contrast, ZO‐1 appeared concentrated over inclusions that were still associated with the brush border in Myo5b KO mice. Finally, immunostaining for Crumbs 3 showed Crumbs 3 present on the apical brush border in enterocytes in control mice, while Myo5b KO mice exhibited Crumb 3‐positive inclusions below the apical membrane.ConclusionsCollectively, these data demonstrate that inclusion formation in neonatal Myo5b KO enterocytes is associated with collection of junctional proteins. The data suggest two possible mechanisms: either inclusions are forming at the apical junction of intestinal epithelial cells or alternatively inclusion formation recruits junctional components.(A) Claudin 2 (red) and Occludin (green) are restricted to the apical junctional complex and gamma‐actin (white) labels the apical membrane in control enterocytes. In Myo5b KO enterocytes gamma‐actin rich inclusions forming from the brush border had an accumulation of Claudin 2 and Occludin. (B) Myo5b KO enterocytes showed ZO‐1 enrichment (red) immediately above forming inclusions. (C & D) In Myo5b KO mice Crumbs 3 (red) was closely associated with forming and internalized inclusions.Figure 1