Defective CFTR Apical Endocytosis and Enterocyte Brush Border in Myosin VI-Deficient Mice

Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Traffic (Impact Factor: 4.35). 09/2007; 8(8):998-1006. DOI: 10.1111/j.1600-0854.2007.00587.x
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In polarized epithelial cells such as those that line the inner ear, kidney and gut, myosin VI has been localized to the intermicrovillar domains where it is proposed to regulate clathrin-dependent endocytosis; however, a direct role for myosin VI in apical endocytosis has not been shown. We examined the apical membrane distribution and endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in myosin VI-deficient Snell's Waltzer Myo6((sv/sv)) mice. Confocal microscopy and cell-surface biotinylation confirmed that surface levels of CFTR in the intestine of Myo6((sv/sv)) mice were markedly higher, and CFTR internalization from the apical plasma membrane was reduced compared with heterozygous controls. Consistent with a defect in CFTR endocytosis and accumulation at the cell surface, exaggerated CFTR-mediated fluid secretion was observed in Myo6((sv/sv)) mice following treatment of isolated jejunum with the cyclic GMP-activated heat stable enterotoxin. These data establish that myosin VI modulates apical endocytosis and may be an important physiological modulator of CFTR function and CFTR-associated secretory diarrhea in the gut.

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Available from: Nadia Ameen, Dec 18, 2013
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    • "WB analysis was conducted as described previously [20]. Briefly, jejunal mucosa was obtained by gently scraping the longitudinally opened loops with a glass slide. "
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    ABSTRACT: Increased intestinal chloride secretion through chloride channels, such as the cystic fibrosis transmembrane conductance regulator (CFTR), is one of the major molecular mechanisms underlying enterotoxigenic diarrhea. It has been demonstrated in the past that the intracellular energy sensing kinase, the AMP-activated protein kinase (AMPK), can inhibit CFTR opening. We hypothesized that pharmacological activation of AMPK can abrogate the increased chloride flux through CFTR occurring during cholera toxin (CTX) mediated diarrhea. Chloride efflux was measured in isolated rat colonic crypts using real-time fluorescence imaging. AICAR and metformin were used to activate AMPK in the presence of the secretagogues CTX or forskolin (FSK). In order to substantiate our findings on the whole tissue level, short-circuit current (SCC) was monitored in human and murine colonic mucosa using Ussing chambers. Furthermore, fluid accumulation was measured in excised intestinal loops. CTX and forskolin (FSK) significantly increased chloride efflux in isolated colonic crypts. The increase in chloride efflux could be offset by using the AMPK activators AICAR and metformin. In human and mouse mucosal sheets, CTX and FSK increased SCC. AICAR and metformin inhibited the secretagogue induced rise in SCC, thereby confirming the findings made in isolated crypts. Moreover, AICAR decreased CTX stimulated fluid accumulation in excised intestinal segments. The present study suggests that pharmacological activation of AMPK effectively reduces CTX mediated increases in intestinal chloride secretion, which is a key factor for intestinal water accumulation. AMPK activators may therefore represent a supplemental treatment strategy for acute diarrheal illness.
    PLoS ONE 07/2013; 8(7):e69050. DOI:10.1371/journal.pone.0069050 · 3.23 Impact Factor
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    • "Myosins are known to be critical for the maintenance of the highly ordered structure of the brush border, nutrient uptake, and prevention of enteropathogenic bacteria at microvilli on the surface of enterocytes (McConnell et al., 2009; Müller et al., 2008; Ameen and Apodaca, 2007; Heintzelman et al., 1994). A yeast 2-hybrid screening revealed that Myosin IV (Myo6) is a binding partner of PLCd3. "
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    ABSTRACT: Phospholipase C (PLC) is a key enzyme in phosphoinositide turnover, and in the regulation of various cellular events. Among the 13 PLC isozymes, PLCδ1 and PLCδ3 share a high sequence homology, and similar tissue distribution. Recent studies with genetically manipulated mice have clarified the importance of these PLC isozymes in a number of tissues. PLCδ1 is required for maintenance of homeostasis in skin and metabolic tissues, while PLCδ3 regulates microvilli formation in enterocytes and the radial migration of neurons in the cerebral cortex of the developing brain. Furthermore, simultaneous loss of PLCδ1 and PLCδ3 in mice causes placental vascular defects, leading to embryonic lethality. Taken together, PLCδ1 and PLCδ3 have unique and redundant roles in various tissues.
    07/2013; 53(3). DOI:10.1016/j.jbior.2013.07.003
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    • "For example, defects identified in the myosin VI knockout mouse show that myosin VI regulates endocytosis of the cystic fibrosis transmembrane conductance regulator from the apical domain of enterocytes. Also, in renal proximal tubule cells, myosin VI mediates the movement of two prominent sodium phosphate cotransporters , NaPi2a and NaPi2c, down the microvilli, and their subsequent removal from the brush-border membrane in response to parathyroid hormone stimulation (Ameen and Apodaca, 2007; Blaine et al., 2009; Lanzano et al., 2011). In polarised epithelial cells, myosin VI LI specifically associates – and can be coimmunoprecipitated – with the tumour suppressor Dab2, an endocytic adaptor protein that functions in cell signalling and the degradation of cell surface receptors (Inoue et al., 2002; Morris et al., 2002). "
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    ABSTRACT: The coordinated trafficking and tethering of membrane cargo within cells relies on the function of distinct cytoskeletal motors that are targeted to specific subcellular compartments through interactions with protein adaptors and phospholipids. The unique actin motor myosin VI functions at distinct steps during clathrin-mediated endocytosis and the early endocytic pathway - both of which are involved in cargo trafficking and sorting - through interactions with Dab2, GIPC, Tom1 and LMTK2. This multifunctional ability of myosin VI can be attributed to its cargo-binding tail region that contains two protein-protein interaction interfaces, a ubiquitin-binding motif and a phospholipid binding domain. In addition, myosin VI has been shown to be a regulator of the autophagy pathway, because of its ability to link the endocytic and autophagic pathways through interactions with the ESCRT-0 protein Tom1 and the autophagy adaptor proteins T6BP, NDP52 and optineurin. This function has been attributed to facilitating autophagosome maturation and subsequent fusion with the lysosome. Therefore, in this Commentary, we discuss the relationship between myosin VI and the different myosin VI adaptor proteins, particularly with regards to the spatial and temporal regulation that is required for the sorting of cargo at the early endosome, and their impact on autophagy.
    Journal of Cell Science 06/2013; 126(12). DOI:10.1242/jcs.095554 · 5.43 Impact Factor
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