COFILIN AND ACTIN DEPOLYMERIZING FACTOR PLAY DIFFERENT ROLES IN THE REGULATION OF INTESTINAL BRUSH BORDER STRUCTURE AND SODIUM-HYDROGEN EXCHANGER 3 ACTIVITY by Lucy B. Gao A thesis submitted to Johns Hopkins University in conformity with the requirements for the degree of Master of Science in Engineering in Biomedical Engineering Baltimore, Maryland May, 2015 © 2015 Lucy B. Gao All Rights Reserved Abstract 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 M) 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. Thesis Advisor: Dr. Mark Donowitz Thesis Readers: Dr. Hai-Quan Mao and Dr. Ming Tse ii Acknowledgements My labmates have given me so much over these years. I do not think I can ever thank everyone enough, to all those who have done me all sorts of favors from changing media to sharing reagents to general advising and insight on life both inside and outside of the lab. During my time here, so many have played a part in inspiring me and helping me grow: Rafiquel Sarker, Nicholas Baetz, Boyoung Cha, Molee Chakraborty, Tiane Chen, Denise Chesner, Ekaterine Chighladze, Jennifer Foulke-Abel, John Gibas, Julie In, Olga Kovbasnjuk, Xuhang Li, Ruxian Lin, Rakhilya Murtazina, Varsha Singh, Ming Tse, Jianbo Yang, Jianyi Yin, and Nicholas Zachos. Special thanks to my thesis readers, Dr. Hai-Quan Mao and Dr. Ming Tse. Outside of the Donowitz lab, thank you to Dr. Bobby Cherayil and his lab at the Massachusetts General Hospital, who gave me my first research experience which truly was my first stepping stone into the research world. From there, and with Dr. Cherayil and Dr. Donowitz’s help and recommendations, I received awards from the American Physiological Society that allowed me to stay at Hopkins over the summer to perform full-time research, in addition to travel to two conferences and present posters there—incredible experiences that are all sponsored by the APS. Those have been invaluable and unforgettable moments in my research career at Hopkins. Thank you also to Dr. Sandra Gabelli and the Amzel lab who have been so kind and have also taught me so much through research experiences outside of my cofilin project. Thank you also to Dr. Mao, allowing me to be a pseudo lab member while working on our collaborative project with Dr. Jeff Wang. This experience was also incredibly insightful and important to me, motivating me to work hard and multi-task on an exciting new project. Thank you so much to my family and friends for supporting me through my entire journey. Special shout out to Andrew Wang, a very important part of my time at Johns Hopkins. And above all, thank you so much to Dr. Mark Donowitz, who has changed my life entirely with this opportunity. iii Table of Contents COFILIN AND ACTIN DEPOLYMERIZING FACTOR PLAY DIFFERENT ROLES IN THE REGULATION OF INTESTINAL BRUSH BORDER STRUCTURE AND SODIUM- HYDROGEN EXCHANGER 3 ACTIVITY .................................................................................... i Abstract ............................................................................................................................................ ii Acknowledgements ......................................................................................................................... iii Table of Contents ............................................................................................................................ iv List of Tables ................................................................................................................................... v List of Figures ................................................................................................................................. vi Chapter 1: Introduction .................................................................................................................... 1 1.1 Sodium Hydrogen Exchanger 3 (NHE3) ............................................................................... 2 1.2 Enterocyte Structure............................................................................................................... 2 1.3 NHE3 Regulation and Trafficking ......................................................................................... 5 1.4 Cofilin and Actin Depolymerizing Factor ............................................................................. 7 1.5 COF/ADF in Trafficking and Cytoskeletal Regulation ....................................................... 10 Chapter 2: Methods and Model Development ............................................................................... 14 2.1 Materials .............................................................................................................................. 15 2.2 Cell Culture .......................................................................................................................... 15 2.3 Adenoviral Infection for NHE3 Expression ......................................................................... 16 2.4 Lentivirus/short hairpin RNA knockdown (KD) of COF and ADF .................................... 17 2.5 Immunoblotting.................................................................................................................... 18 2.6 Immunostaining/Immunofluorescence and Confocal Microscopy ...................................... 19 2.7 Transmission Electron Microscopy ..................................................................................... 19 2.8 Measurement of Na+/H+ Exchange Activity ........................................................................ 20 Chapter 3: Results .......................................................................................................................... 22 3.1 Knockdown Characterization by Western Blot and Immunofluorescence .......................... 23 3.2 Transmission Electron Microscopy ..................................................................................... 30 3.3 NHE3 Functional Studies .................................................................................................... 32 Chapter 4: Discussion/Conclusions ............................................................................................... 35 Chapter 5: Future Directions .......................................................................................................... 41 References ...................................................................................................................................... 44 Curriculum Vitae ........................................................................................................................... 47 iv List of Tables Table 1: Each construct will be named by the label in parentheses for this manuscript ................ 17 Table 2: Combination knockdowns using equal fractions of Table 1 constructs........................... 18 Table 3: Average percent knockdown from GFP-KD in normalized COF or ADF amount ......... 23 Table 4: Knockdown constructs resulting effects on COF/ADF expression ................................. 26 v List of Figures Figure 1: NHE3 must be expressed at the plasma membrane surface of intestinal cell microvilli in order to perform sodium absorption activity. ................................................................................... 2 Figure 2: Depiction of segment of small intestine, lined with villi throughout interior. ................. 4 Figure 3: Immunoprecipitation, NHE3 and COF are found to interact. ........................................ 11 Figure 4: Summary of COF and ADF expression/knockdown in the cell lines & mouse tissue. .. 24 Figure 5: Lenti-puromycin KD in Caco-2 Cells, COF-KD2 and ADF-KD2 constructs...............