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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    • "Endocytosis of apical membrane proteins in polarized epithelial cells with brush borders, such as the intestine, can be separated into three steps: (1) movement to and accumulation of microvillus membrane proteins in the inter-microvillus region; (2) internalization in clathrin-coated vesicles (and also nonclathrin-dependent processes) that form in the inter-microvillus region; and (3) movement of vesicles through the actin-rich terminal web to early endosomal compartments (Aschenbrenner et al., 2003; Hasson, 2003). Evidence for the involvement of myosin VI in the second and third steps has been reported by others (Gottlieb et al., 1993; Biemesderfer et al., 2002; Birn et al., 1997; Christensen and Nielsen, 1991; Aschenbrenner et al., 2003; Yang et al., 2005; Ameen et al., 2007; Yang et al., 2008; Riquier et al., 2009; Collaco et al., 2010; Kravtsov et al., 2012), and our results described so far support its requirement for both basal and agonist-stimulated endocytosis of NHE3 in Caco-2/Bbe cells. However, the localization of NHE3 along the microvillus in control and myosin VI KD cells has not been determined; consequently, we used immunoelectron microscopy to address this question. "
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    ABSTRACT: The intestinal brush border (BB) Na(+)/H(+) exchanger, NHE3, is acutely regulated through changes in its endocytosis/exocytosis. Myosin VI, a minus-end directed motor, has been implicated in endocytosis at the inter-microvillar (MV) cleft and vesicle remodeling in the terminal web. We asked if myosin VI also regulates NHE3 movement down MV. Basal NHE3 activity and surface amount, determined by BCECF/fluorometry and biotinylation, respectively, were increased in myosin VI knock-down (KD) Caco-2/Bbe cells. Carbachol (CCH) and forskolin (FSK) stimulated NHE3 endocytosis in control but not in myosin VI KD cells. Importantly, immuno-EM results showed NHE3 preferentially localized in the basal half of MV in control but in the distal half of myosin VI KD cells. Dynasore duplicated some aspects of myosin VI KD: it increased basal surface NHE3 activity and prevented FSK-induced NHE3 endocytosis; but NHE3's distribution along the MV was intermediate in dynasore-treated as compared to either myosin VI KD or untreated cells. We conclude that myosin VI is required for basal and stimulated endocytosis of NHE3 in intestinal cells and suggest that myosin VI also moves NHE3 down MV.
    Journal of Cell Science 06/2014; 127(16). DOI:10.1242/jcs.149930 · 5.43 Impact Factor
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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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