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.
View: Text | PDF
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

×

Figure 2

Immunostaining of duodenum samples from Navajo patients with MVID reveals that mutation of MYO5B causes loss of apical microvilli, microvillus inclusion formation, and accumulation of F-actin into the terminal web.

Options: View larger image (or click on image) Download as PowerPoint
Immunostaining of duodenum samples from Navajo patients with MVID reveal...
(A–C) Normal duodenum immunostaining for MYO5B (green) and ezrin (magenta) showed subapical MYO5B with increased staining at the tips of villi and ezrin localization to the brush border. (D–F) MVID patient duodenum immunostaining for MYO5B and ezrin showed dispersal of MYO5B from the subapical region of enterocytes and ezrin localization to microvillus inclusions in cells at the villus tips. Note that cells near the crypts (white arrowhead) showed a normal apical ezrin distribution when compared with cells at the villus tips (red arrowhead). (G) SIM imaging of β-actin (red) and MYO5B (green) in normal duodenum showed actin staining in the apical brush border (yellow arrow), discrete staining of the terminal web (white arrow), and MYO5B localization above the terminal web and below the apical surface (blue arrow). (H) SIM imaging of the samples from patients with MVID revealed an accumulation of β-actin (red) in the terminal web and dispersal of MYO5B (green) from its normal subapical position above the terminal web. (I) TEM in normal duodenum showed normal apical microvilli. (J) TEM of the MVID patient duodenum revealed a decrease in microvilli and enlarged subapical vesicular structures. Scale bar: 50 μm (A–F); 10 μm (A–F, insets); 5 μm (G and H); 2 μm (I and J).