Restoration of cytoskeletal and membrane tethering defects but not defects in membrane trafficking in the intestinal brush border of mice lacking both myosin Ia and myosin VI

Abstract

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 cross links 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.

Identification of intestinal ion transport defects in microvillus inclusion disease

“…Thus it is possible that, in the absence of Myo5b, CFTR delivery into the crypt BBM is facilitated by MT motors. Alternatively, as we recently demonstrated in a Myo6/Myo1a double-knockout mouse model, loss of endocytic (Myo6) and exocytic (Myo1a) myosin motors in the enterocyte BB leads to rapid upregulation of compensatory myosin members (21). A similar scenario could explain CFTR delivery to the BBM in MVID in the absence of Myo5b.…”

Mice lacking myosin IXb, an inflammatory bowel disease susceptibility gene, have impaired intestinal barrier function and superficial ulceration in the ileum

“…Moreover, the relatively WT‐like response to DSS‐induced colitis in the Myo9b KO suggests that the cells of the submucosal immune system are not in a primed inflammatory state. This is in contrast mice lacking several other components of the actin based IEC cytoskeleton including villin [Ferrary et al, ; Wang et al, ] myosin 1a and Myo6 [Hegan et al, ]. However, bone marrow transplant chimeras to generate WT mice with Myo9b KO gut immune response cells and KO mice with submucosal immune response cells from WT mice will be required to definitively parse the relative contributions of the IEC and cells of the submucosal immune system to the observed mucosal damage in the KO.…”

“…Sets (10 mice/genotype each) WT and Myo9b KO male mice were transferred from drinking water to distilled water containing 3% w/v DSS (MP Biomedicals, Solon, OH) for 14 days as described in [Hegan et al, ]. Mice were monitored daily using a disease index score that included the following assessments: (1) Weight loss: no change or weight increase; score of 0; weight loss of 1–3% of body weight at day 0, score of 1; weight loss of greater than 3–6%, score of 2; weight loss greater than 6–9%, score of 3; weight loss of greater than 9%, score of 4; weight loss of equal or greater than 20%, score of 5—and mortality end point.…”

Role of Polarity Proteins in the Generation and Organization of Apical Surface Protrusions

“…Brush-border MYO1A forms a helically arranged series of cross bridges that connect the actin bundles of the microvillus to the plasma membrane (Tyska et al 2005). Other associated myosins include MYO6, which links the apical surface with the underlying actin cytoskeleton (Ameen and Apodaca 2007; Hegan et al 2015a), and MYO7B, which is associated with the electron dense distal tips of microvilli (Wolfrum et al 1998). The latter also forms interactions between the cytoplasmic scaffolding protein USH1C (alias harmonin-a) and the transmembrane protocadherins PCDH24 (alias protocadherin-24) and CDHR5 (alias the mucin-like protocadherin) (Fig.…”