Loss of myosin Vb promotes apical bulk endocytosis in neonatal enterocytes

Abstract

In patients with inactivating mutations in myosin Vb (Myo5B), enterocytes show large inclusions lined by microvilli. The origin of inclusions in small-intestinal enterocytes in microvillus inclusion disease is currently unclear. We postulated that inclusions in Myo5b KO mouse enterocytes form through invagination of the apical brush border membrane. 70-kD FITC-dextran added apically to Myo5b KO intestinal explants accumulated in intracellular inclusions. Live imaging of Myo5b KO–derived enteroids confirmed the formation of inclusions from the apical membrane. Treatment of intestinal explants and enteroids with Dyngo resulted in accumulation of inclusions at the apical membrane. Inclusions in Myo5b KO enterocytes contained VAMP4 and Pacsin 2 (Syndapin 2). Myo5b;Pacsin 2 double-KO mice showed a significant decrease in inclusion formation. Our results suggest that apical bulk endocytosis in Myo5b KO enterocytes resembles activity-dependent bulk endocytosis, the primary mechanism for synaptic vesicle uptake during intense neuronal stimulation. Thus, apical bulk endocytosis mediates the formation of inclusions in neonatal Myo5b KO enterocytes.

Recruitment of Polarity Complexes and Tight Junction Proteins to the Site of Apical Bulk Endocytosis

“…We previously showed that apical bulk endocytosis mirrors neuronal activity–dependent bulk endocytosis requiring Pacsin2 and dynamin2 for inclusion formation. 16 The work presented here indicates that endocytic trafficking and polarity complexes are closely associated with the formation of inclusions via bulk endocytosis. Based on our findings, we postulate that MYO5B regulates multiple aspects of epithelial homeostasis, including cell polarity, tight junction protein distribution, and localization of ion transporters.…”

“…Intestinal organoids require differentiation to develop robust inclusion formation. 16 , 65 Inclusion formation in DMSO and ζ-Stat treatment resulted in a similar number of inclusions identified by F-actin staining ( Figure 9 A ). Therefore, treatment of Myo5b KO intestinal organoids with ζ-Stat did not prevent inclusion formation, suggesting that inclusion formation is not entirely dependent on aPKC or that there are redundancies in the functions of polarity proteins.…”

“…Microvillus inclusions are formed over 3–4 hours through a novel mechanism of apical bulk endocytosis, which requires both Pacsin2 and dynamin2. 16 This mechanism in enterocytes has overlapping characteristics with activity-dependent bulk endocytosis described during neuronal hyperstimulation. 77 , 78 , 79 , 80 , 81 The accumulation of tight junction proteins (claudin-2, ZO-1, ZO-2, and occludin) at the site of inclusion formation suggests that tight junction proteins may be recruited to sites of apical bulk endocytosis to promote excision and internalization within Myo5b KO enterocytes.…”

“… 31 We recently showed that inclusions form through a novel process of apical bulk endocytosis. 16 However, the exact mechanisms regulating inclusion formation are incompletely understood and currently understudied. To address these gaps in knowledge, we used germline Myo5b KO mice and systematically compared them with neonatal control (wild-type or heterozygous) littermates that lacked inclusions.…”

“…These findings support our previous studies suggesting that microvillus inclusions in Myo5b KO mice form through invagination of the apical membrane of enterocytes in a process termed apical bulk endocytosis . 16

Figure 1 Myo5b KO mice have microvilli-lined inclusions associated with α-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.

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