Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease
Byron C. Knowles, … , James R. Goldenring, Mitchell D. Shub
Byron C. Knowles, … , James R. Goldenring, Mitchell D. Shub
Published June 2, 2014
Citation Information: J Clin Invest. 2014;124(7):2947-2962. https://doi.org/10.1172/JCI71651.
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Research Article Gastroenterology

Myosin Vb uncoupling from RAB8A and RAB11A elicits microvillus inclusion disease

Abstract

Microvillus inclusion disease (MVID) is a severe form of congenital diarrhea that arises from inactivating mutations in the gene encoding myosin Vb (MYO5B). We have examined the association of mutations in MYO5B and disruption of microvillar assembly and polarity in enterocytes. Stable MYO5B knockdown (MYO5B-KD) in CaCo2-BBE cells elicited loss of microvilli, alterations in junctional claudins, and disruption of apical and basolateral trafficking; however, no microvillus inclusions were observed in MYO5B-KD cells. Expression of WT MYO5B in MYO5B-KD cells restored microvilli; however, expression of MYO5B-P660L, a MVID-associated mutation found within Navajo populations, did not rescue the MYO5B-KD phenotype but induced formation of microvillus inclusions. Microvilli establishment required interaction between RAB8A and MYO5B, while loss of the interaction between RAB11A and MYO5B induced microvillus inclusions. Using surface biotinylation and dual immunofluorescence staining in MYO5B-KD cells expressing mutant forms of MYO5B, we observed that early microvillus inclusions were positive for the sorting marker SNX18 and derived from apical membrane internalization. In patients with MVID, MYO5B-P660L results in global changes in polarity at the villus tips that could account for deficits in apical absorption, loss of microvilli, aberrant junctions, and losses in transcellular ion transport pathways, likely leading to the MVID clinical phenotype of neonatal secretory diarrhea.

Authors

Byron C. Knowles, Joseph T. Roland, Moorthy Krishnan, Matthew J. Tyska, Lynne A. Lapierre, Paul S. Dickman, James R. Goldenring, Mitchell D. Shub

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Figure 8

MYO5B-WT and MYO5B-YE/QR rescue the MYO5B-KD phenotype, while MYO5B-P660L and MYO5B-YE/QR promote microvillus inclusions.

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MYO5B-WT and MYO5B-YE/QR rescue the MYO5B-KD phenotype, while MYO5B-P660...
(A) Ezrin and (B) phalloidin staining in microvilli was rescued in MYO5B-KD–expressing MYO5B-WT. Arrowhead, high expressers; –, low expressers. (C and D) Ezrin and phalloidin staining in microvilli was not rescued in MYO5B-KD–expressing MYO5B-P660L and induced microvillus inclusions (asterisks). (E and F) Scanning electron microscopy of (E) MYO5B-KD–expressing MYO5B-WT showed reestablished mature microvilli and (F) MYO5B-KD–expressing MYO5B-P660L showed immature microvilli in the tepee-like stage. (G) Western blot of rescue cell lines showed similar expression levels of mCherry. (H) Microvillus inclusions in MYO5B-KD reexpressing MYO5B-P660L and MYO5B-YE/QR were significantly higher compared with other cell lines. (I) Expression of MYO5B-YE/QR reestablished phalloidin staining of microvilli (yellow arrow) but also induced the formation of microvillus inclusions (white arrow). (J) Expression of MYO5B-QL/YC partially recovered microvilli. (K and M) Scanning electron microscopy and TEM of MYO5B-KD cells expressing MYO5B-YE/QR showed reestablished mature microvilli. (L and N) Scanning electron microscopy and TEM of MYO5B-KD cells expressing MYO5B-QL/YC showed partial rescue of nonuniform microvilli. (O) MYO5B-KD cells expressing MYO5B-YE/QR showed subapical microvillus inclusions (red arrows), with corresponding denuded microvilli (black arrow) and surrounding brush border (blue arrow). (P) MVID patient duodenum with pathognomonic microvillus inclusion (red arrows) and denuded microvilli (black arrow). Scale bar: 10 μm (A–D, I, and J); 2 μm (E and F); 500 nm (E and F, insets); 5 μm (K and L); 1 μm (K and L, insets), 2 μm (M and N); 500 nm (O and P). *P ≤ 0.05, ***P ≤ 0.001, Mann-Whitney test. Error bars denote mean ± SEM.