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Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl(-)) and sodium (Na(+)) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na(+)), CFTR (Cl(-)), and SLC26A3 (DRA) (Cl(-)/HCO3 (-)) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl(-) and Na(+) stool loss in MVID diarrhea.

Myosin Ia (Myo1a), the most prominent plus-end directed motor and myosin VI (Myo6) the sole minus-end directed motor, together exert opposing tension between the microvillar (MV) actin core and the apical brush border (BB) membrane of the intestinal epithelial cell (IEC). Mice lacking Myo1a or Myo6 each exhibit a variety of defects in the tethering of the BB membrane to the actin cytoskeleton. Double mutant (DM) mice lacking both myosins revealed that all the defects observed in either the Myo1a KO or Snell's waltzer (sv/sv) Myo6 mutant mouse are absent. In isolated DM BBs, Myo1a crosslinks between MV membrane and MV actin core are absent but the gap (which is lost in Myo1a KO) between the MV core and membrane is maintained. Several myosins including Myo1c, d, and e and Myo5a are ectopically recruited to the BB. Consistent with the restoration of membrane tethering defects by one or more of these myosins, upward ATP-driven shedding of the BB membrane, which is blocked in the Myo1a KO, is restored in the DM BB. However, Myo1a or Myo6 dependent defects in expression of membrane proteins that traffic between the BB membrane and endosome (NaPi2b, NHE3, CFTR) are not restored. Compared to controls, Myo1a KO, sv/sv mice exhibit moderate and DM high levels of hypersensitivity to dextran sulfate sodium-induced colitis. Consistent with Myo1a and Myo6 playing critical roles in maintaining IEC integrity and response to injury, DM IECs exhibit increased numbers of apoptotic nuclei, above that reported for Myo1a KO., (© 2015 Wiley Periodicals, Inc.)

Microvillus inclusion disease (MVID) is an autosomal recessive condition resulting in intractable secretory diarrhea in newborns due to loss-of-function mutations in myosin Vb (Myo5b). Previous work suggested that the apical recycling endosomal (ARE) compartment is the primary location for phosphoinositide-dependent protein kinase 1 (PDK1) signaling. Because the ARE is disrupted in MVID, we tested the hypothesis that polarized signaling is affected by Myo5b dysfunction. Subcellular distribution of PDK1 was analyzed in human enterocytes from MVID/control patients by immunocytochemistry. Using Myo5b knockdown (kd) in Caco-2BBe cells, we studied phosphorylated kinases downstream of PDK1, electrophysiological parameters, and net water flux. PDK1 was aberrantly localized in human MVID enterocytes and Myo5b-deficient Caco-2BBe cells. Two PDK1 target kinases were differentially affected: phosphorylated atypical protein kinase C (aPKC) increased fivefold and phosohoprotein kinase B slightly decreased compared with control. PDK1 redistributed to a soluble (cytosolic) fraction and copurified with basolateral endosomes in Myo5b kd. Myo5b kd cells showed a decrease in net water absorption that could be reverted with PDK1 inhibitors. We conclude that, in addition to altered apical expression of ion transporters, depolarization of PDK1 in MVID enterocytes may lead to aberrant activation of downstream kinases such as aPKC. The findings in this work suggest that PDK1-dependent signaling may provide a therapeutic target for treating MVID., (Copyright © 2014 the American Physiological Society.)

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel critical to intestinal anion secretion. In addition to phosphorylation, vesicle traffic regulates CFTR in some epithelial cells. Studies of cultured intestinal cells are conflicting regarding the role of cAMP-dependent vesicle traffic in regulating chloride transport. Whether CFTR is present in vesicular compartments within chloride secretory cells in the intestine is unknown and the role of cAMP-dependent vesicle insertion in regulating CFTR and intestinal fluid secretion remains unclear. The purpose of this study was to: (1) examine and quantify the subcellular distribution for CFTR in rat intestine, (2) further define the ultrastructure of the previously identified CFTR High Expresser (CHE) cell, and (3) examine the cellular distribution of CFTR following cAMP stimulation in vivo. Using the sensitive techniques of cryoimmunogold electron microscopy we identified CFTR in subapical vesicles and on the apical plasma membrane in crypt, Brunner glands, and CHE cells. cAMP stimulation in rat proximal small intestine produced a fluid secretory response and was associated with an apical redistribution of CFTR, supporting a physiologic role for cAMP-dependent CFTR vesicle insertion in regulating CFTR in the intestine.

The striking similarities between microvillus inclusions (MIs) in enterocytes in microvillus inclusion disease (MID) and vacuolar apical compartment in tissue culture epithelial cells, led us to analyze endoscopic biopsies of duodenal mucosa of a patient after the samples were used for diagnostic procedures. Samples from another patient with an unrelated disease were used as controls. The MID enterocytes showed a decrease in the thickness of the apical F-actin layer, and normal microtubules. The immunofluorescence analysis of the distribution of five apical membrane markers (sucrase isomaltase, alkaline phosphatase, NHE-3 Na+/H+ exchanger, cGMP-dependent protein kinase, and cystic fibrosis trans-membrane conductance regulator), showed low levels of these proteins in their standard localization at the apical membrane as compared with normal duodenal epithelium processed in parallel. Instead, four of these markers were found in a diffuse distribution in the apical cytoplasm, below the terminal web (as indicated by co-localization with F-actin and cytokeratin 19), and in MIs as well. The basolateral protein Na(+)-K+ATPase, in contrast, was normally localized. These results support the hypothesis that MID may represent the first genetic defect affecting apical membrane traffic, possibly in a late step of apical exocytosis.