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Dynamic Formation of Microvillus Inclusions During Enterocyte Differentiation in

Dynamic Formation of Microvillus Inclusions During Enterocyte Differentiation in

Mohammed H Mosa, Ophélie Nicolle, Sophia Maschalidi, Fernando E Sepulveda, Aurelien Bidaud-Meynard, Constantin Menche, Birgitta E Michels, Grégoire Michaux, Geneviève de Saint Basile, Henner F Farin

Cell Mol Gastroenterol Hepatol. 2018 Aug 14;6(4):477-493.e1.

PMID: 30364784

Abstract:

Background & 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 Munc18-2/Stxbp2-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 Munc18-2/Stxbp2 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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP686770616	IWP-2		686770-61-6	Price

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