Dynamic Formation of Microvillus Inclusions During Enterocyte Differentiation in Munc18-2-Deficient Intestinal Organoids

Abstract : BACKGROUND and AIMS Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID. METHODS Intestinal organoids from Munc18-2IStxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions. RESULTS Loss of Munc18-2IStxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin-positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane. CONCLUSIONS We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border.
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Mohammed H. Mosa, Ophélie Nicolle, Sophia Maschalidi, Fernando E. Sepulveda, Aurelien Bidaud-Meynard, et al.. Dynamic Formation of Microvillus Inclusions During Enterocyte Differentiation in Munc18-2-Deficient Intestinal Organoids. Cellular and Molecular Gastroenterology and Hepatology, Philadelphia, PA : American Gastroenterological Association, [2015]-, 2018, 6 (4), pp.477-494. ⟨10.1016/j.jcmgh.2018.08.001⟩. ⟨hal-01972441⟩

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