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 3

Loss of MYO5B in CaCo2-BBE cells causes the redistribution of basolateral markers.

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Loss of MYO5B in CaCo2-BBE cells causes the redistribution of basolatera...
(A–H) x-y confocal images are shown above z-axis reconstructions. (A) p120 staining in controls showed a lateral distribution, (B) while MYO5B-KD cells showed decreased p120 at the lateral membranes. (C) Na/K-ATPase staining in controls showed a lateral distribution, (D) while MYO5B-KD cells showed reduction in lateral membranes. (E) E-cadherin staining in controls showed a junctional distribution, (I) with the x-z image magnified in junctional E-cadherin (white arrows). (F) MYO5B-KD showed redistribution of E-cadherin along the apical and lateral membranes and with internal pools of E-cadherin distributed throughout the cells; x-z image magnified in J. (G and H) In control and MYO5B-KD cells, β-catenin stained lateral membranes. (K) Quantitation of basolateral mean fluorescence. MYO5B-KD cells showed a reduction of p120 and an increase in E-cadherin. (L and M) Western blot with quantitation of basolateral markers in MYO5B-KD showed an isoform switch of p120, with no decrease and an increase E-cadherin total protein. (N) Quantitation of apical-to-basolateral ratio in MYO5B-KD cells showed a redistribution of Na/K-ATPase to the apical surface, while E-cadherin was localized over both the apical and basolateral surfaces. (O) Surface biotinylation of either the apical or basolateral surfaces in control and MYO5B-KD cells with total protein, flow through from streptavidin beads (FT), and biotinylated-streptavidin-bound protein from control (Bound-Ctrl) or MYO5B-KD (Bound-MVBKD) cells showed an increase in apical and a decrease in basolateral E-cadherin in the MYO5B-KD cells. Scale bar: 10 μm. *P ≤ 0.05, **P ≤ 0.01, Mann-Whitney test. Error bars denote mean ± SEM.