176 Loss of Functional MYO5B in CaCo2-BBe Cells and Navajo MVID Patient Enterocytes Leads to Loss of Polarity and Deficits in Maintenance of Microvilli

Abstract

Myosin Vb (MYO5B) is an actin based motor that localizes specific Rab small GTPases (Rab8a, Rab10, Rab11, and Rab25) to sub-apical domains in polarized epithelial cells. Microvillus Inclusion Disease (MVID) represents a pathophysiologic window into the apical trafficking process, because it arises as a result of inactivating mutations in MYO5B that leads to the loss of microvilli in intestinal enterocytes and chronic unremitting diarrhea in the affected newborns. Understanding how mutations in MYO5B lead to aberrant enterocyte phenotype will provide novel insights into the fundamental mechanisms governing apical recycling system trafficking, microvilli formation, and MVID. We hypothesize that MYO5B plays a crucial role in apical trafficking and polarization in enterocytes, and that defective MYO5B in MVID patients mislocalizes Rab small GTPases leading to disruption in apical transport and loss of polarity in enterocytes. To test this hypothesis we have established cellular models of aberrant enterocyte apical trafficking by stably knocking down MYO5B in CaCo2-BBE cells. The cells grown on permeable filters for 15 days were analyzed using immunostaining, Scanning EM, and Transmission EM to examine markers of apical and basolateral polarity, intracellular trafficking, and the establishment of apical microvilli. MYO5B KD knockdown (KD) caused a loss of microvilli and dispersal of Rab8a and Rab11acontaining vesicles from their usual subapical distribution to diffusely cytoplasmic in the cells. MYO5B KD elicited an increase in claudin-2 and a decrease in claudin-1 expression as well as a decrease in lateral membrane staining for p120-catenin (p120). MYO5B KD also caused a loss of apical active GTP-bound cdc42 in the cells. Rescue of theMYO5B KDwith synthetic MYO5B wild type elicited recovery of normal microvilli. However, re-expression of MYO5B(P660L), the Navajo MVID mutation, failed to rescue and in addition caused the formation of microvillus inclusions. Navajo MVID MYO5B(P660L) patient samples demonstrated a loss of p120 and a pseudostratified columnar epithelium at the villus tips where ezrin-staining microvillus inclusions were most predominant. Patient biopsies also showed aberrant localization of MYO5B, Rab8a, Rab11a, Rab11-FIP2, DPPIV, and Ezrin. Rab11a and MYO5B localized around microvillus inclusions, while Rab8a staining was diffuse. Importantly, while a severe loss of brush border was observed in cells at the tips of microvilli, the cells in the mid-villus appeared to have a preserved normal brush border. Our results suggest that MYO5B regulates the polarity of intestinal epithelial cells, thereby maintaining the functional brush border. The normal brush border seen in the proximal villi in the Navajo MVID patients suggests that the pathogenesis of MVID lies in a loss of polarity in enterocytes at the tips of intestinal villi.