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 5

Coimmunostaining of DPPIV and LAMP2a in CaCo2-BBE cells with redistribution of DPPIV to large vesicles in MYO5B-KD cells.

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Coimmunostaining of DPPIV and LAMP2a in CaCo2-BBE cells with redistribut...
(A–L) x-y confocal images are shown above z-axis reconstructions. Control and MYO5B-KD cells were immunostained for DPPIV (green) and LAMP2a (red). (A–F) Control cells showed a normal apical distribution of DPPIV and diffusely cytoplasmic LAMP2a. (G–L) In MYO5B-KD cells, DPPIV was seen in internal vesicles, some of which also stained for LAMP2a (white arrow). (M) Magnified x-z image from control cells in F. (N) Magnified x-z image from MYO5B-KD cells in L showing cytoplasmic distribution of DPPIV. (O) Control cells immunostained for DPPIV (green) and RAB11A (red) showed apical DPPIV and subapical RAB11A. (P–R) MYO5B-KD cells, immunostained for DPPIV (green) and RAB11A (red), showed dispersal of RAB11A from the subapical surface and localization in large DPPIV positive vesicles (white arrows). (S) DPPIV reduction in MYO5B-KD cells shown via mean fluorescence in maximum-intensity Z-stack projections. (T) Western blot comparing control and MYO5B-KD cell lines probed for DPPIV and α-tubulin demonstrating reduction of DPPIV expression. (U) Apical surface biotinylation in control and MYO5B-KD cells showing DPPIV immunoreactivity in total protein, flow through from streptavidin beads, and biotinylated streptavidin-bound protein, demonstrating an increase in the nonbiotinylated cytoplasmic pool and a decrease in DPPIV on the apical surface in the MYO5B-KD (Bound-MVBKD) versus control (Bound-Ctrl) cells. Scale bar: 10 μm. *P ≤ 0.05, Mann-Whitney test. Error bars denote mean ± SEM.