COFILIN AND ACTIN DEPOLYMERIZING FACTOR PLAY DIFFERENT ROLES IN THE REGULATION OF INTESTINAL BRUSH BORDER STRUCTURE AND SODIUM-HYDROGEN EXCHANGER 3 ACTIVITY

cofilin;actin;depolymerizing;factor;adf;destrin;nhe3;nhe;sodium;hydrogen;exchanger;transporter;terminal;web;microvilli;intestine;intestinal;brush;border;trafficking;traffick;Biomedical Engineering

Gao, Lucy BDonowitz, Mark ;

Johns Hopkins University

关键词: cofilin;    actin;    depolymerizing;    factor;    adf;    destrin;    nhe3;    nhe;    sodium;    hydrogen;    exchanger;    transporter;    terminal;    web;    microvilli;    intestine;    intestinal;    brush;    border;    trafficking;    traffick;    Biomedical Engineering;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60365/GAO-THESIS-2015.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

The brush border Na+/H+ exchanger 3 (NHE3) accounts for most small intestinal Na+ absorption which occurs across the surface of microvilli. NHE3 surface expression and activity are acutely regulated by endocytosis and exocytosis. Cofilin-1 (COF) and actin depolymerizing factor (ADF) are highly conserved isoforms of the cofilin family of actin severing proteins. COF/ADF are ubiquitously expressed, though ADF is more primarily expressed in intestinal tissue. We hypothesized that COF and/or ADF are necessary for basal and regulated NHE3 activity by remodeling the terminal web actin structure to allow endocytosis/exocytosis. COF and ADF knockdown (KD) cells were engineered with lentivirus (shRNA) / puromycin in Caco-2 cells, a polarized intestinal Na+-absorptive cell line. COF-KD reduced COF by 57±5% (p≤0.01) without altering ADF, while ADF-KD cells reduced ADF by 73±5% (p≤0.01) without altering COF. By transmission electron microscopy, ADF-KD cells had straighter and a greater number of microvilli (MV) than control, and COF-KD cells had bent and a fewer number of MV. By immunofluorescence, neither COF nor ADF had a predominantly MV distribution but are expressed sub-apically, laterally, in the nucleus, and throughout the cytosol. NHE3 activity was quantified by measuring pH change by BCECF / fluorometry under basal and inhibited (adenylyl cyclase elevation by exposure to forskolin (FSK), 25 uM) conditions. COF-KD expressed less NHE3 activity than control cells (p≤.10). ADF-KD showed higher NHE3 activity than control cells (p≤.10). FSK inhibited NHE3 similarly in all three conditions. Conclusions: 1. Using KD studies we conclude that COF is necessary for microvillar rigidity while ADF normally reduces apical microvillar number. The lack of COF/ADF in MV suggest that they affect the MV through intermediate proteins. 2. COF normally increases and ADF decreases basal NHE3 activity, but neither are involved in cAMP induced inhibition of NHE3.