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 1

Confocal fluorescence imaging, scanning electron microscopy, and TEM of CaCo2-BBE cells demonstrating loss of microvilli with MYO5B-KD.

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Confocal fluorescence imaging, scanning electron microscopy, and TEM of ...
(A–F) x-y images are shown in the top panels with x-z images below each. (A and B) Immunostaining for ezrin (red) and phalloidin F-actin (green) in control cells showed prominent staining of microvilli. Triple overlay with DAPI nuclear staining (blue) is shown in C. (D and E) Immunostaining for ezrin (red) and phalloidin F-actin (green) in MYO5B-KD cells showed reduced apical ezrin and an accumulation of F-actin in the subapical region. Triple overlay with DAPI nuclear staining (blue) is shown in F. (G and H) Scanning electron microscopy and TEM of control cells showing normal densely packed microvilli. (I and J) Scanning electron microscopy and TEM of MYO5B-KD cells showing immature sparse microvilli. (K) Control cells x-z images from C magnified showing microvilli stained for both ezrin and F-actin. (L) CaCo2-BBE MYO5B-KD cells x-z images from F magnified showing loss of apical microvilli stained for both ezrin and F-actin and their accumulation in the terminal web. (M) Quantitation of ezrin mean fluorescence in maximum-intensity Z-stack projections showing a reduction in CaCo2-BBE MYO5B-KD cells. (N) Ezrin Western blot with quantitation showing a decrease in total ezrin in the MYO5B-KD cells. Scale bar: 10 μm (A–F, K, and L); 3 μm (G and I); 1 μm (G and I, insets); 500 nm (H and J). *P ≤ 0.05, **P ≤ 0.01, Mann-Whitney test. Error bars denote mean ± SEM.