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Investigating the Role of Chloride in Endocytic Organelle Acidification

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Investigating the role of chloride in endocytic organelle acidification By Mary R. Weston A dissertation submitted to Johns Hopkins University in conformity with the requirements for the degree of Doctor of Philosophy Baltimore, Maryland December 2017 © Mary R. Weston All rights reserved ABSTRACT Endocytic organelles maintain their characteristic, essential internal pH using the V-type ATPase H+-pump. Accumulation of H+ generates a voltage across the membrane; an additional ion, known as a counterion, must move to dissipate charge buildup. Chloride (Cl-) has been hypothesized to be an important counterion in the endosomal pathway, but its role is still debated. This thesis seeks to explore the role of counterions and the proteins that facilitate their movements in the acidification of clathrin-coated vesicles (CCVs), a subset of early endosomes, and lysosomes. We confirmed previous work showing that isolated bovine brain CCVs acidify in the presence of external Cl-, independent of the monovalent cations present. While unsuccessful at identifying the protein responsible for the observed anion transport, we used a new approach to confirm that most brain CCVs are synaptic vesicles. Secondly, because acidification in isolated lysosomes is Cl--dependent and the lysosomal protein ClC-7, a Cl-/H+ antiporter, moves Cl- into the organelles, this protein has been suggested to mediate such counterion movement. However, live cell ClC-7 knockout (KO) mouse lysosomes have the same pH as WT. We generated mice with a liver-specific deletion of ClC-7 to test these results in isolated lysosomes and live cells in parallel. While isolated ClC-7 KO lysosomes showed a drastic decrease in Cl--facilitated acidification, lysosomal pH was similar in WT and KO hepatocytes, even during metabolic and base challenges. Lastly, we present two patients with an identical pathogenic de novo variant in CLCN7, who display hypopigmentation, a delay in motor skills, abnormal liver/spleen/kidneys, and failure to thrive, but no osteopetrosis. Patient fibroblasts contain large vacuoles scattered throughout the cytoplasm that do not consistently stain for lysosomal markers, and ii possess a vast increase in the lysosomal-like compartments that stain for Lamp1, a lysosomal protein. Lysosomal pH in cultured patient fibroblasts are more acidic than the neonatal controls and heterologous expression of the mutant ClC-7 in Xenopus oocytes revealed substantial increases in transport activity. These results suggest a novel gain-of- function for this Cl-/H+ antiporter, one of the first reported instances of lysosomal hyperacidification, and shows that ClC-7 plays a role in lysosomal pH. Overall, this work contributes to the field of organelle acidification and brings us closer to understanding how organelles use counterions to assist in and regulate pH. Advisor/primary thesis reader: Joseph Mindell, M.D., Ph.D. Secondary thesis readers: Kenton Swartz, Ph.D. Beverly Wendland, Ph.D. iii Acknowledgements Graduate school has been a great challenge and an adventure. I have learned so much and am grateful to all those who helped and supported me over the years. First and foremost, I would like to thank my mentor, Dr. Joseph Mindell, for providing me with support and guidance throughout my graduate work. I am so thankful that I had the opportunity to work with someone who constantly challenged me to develop a thoughtful and rigorous approach to science. Thank you for your patience, encouragement, and for knowing that I would come out on the other side. The Mindell lab has been a fun place to scientifically “grow up”. Thank you to the current and past members for the atmosphere, the advice, and the food (specifically, birthday cakes, bagels, and babkas). I am especially grateful to Dr. Sara Lioi for teaching me about cells and to Dr. Christopher Mulligan for acting as my surrogate "older graduate student" when I first started. Additional thanks go to the members of the 3B and 3D pods. It has been amazing to be surrounded by so many kind people who foster a creative and collaborative scientific environment. Thank you to all my collaborators. It has been lovely working with and learning from you all, especially Dr. Anowarul Amin and Dr. Ralu Nicoli. Thank you to my wonderful thesis committee. Dr. Kenton Swartz and Dr. Beverly Wendland have provided constant support and guidance throughout my graduate work and I am grateful for their helpful scientific discussions and mentorship. Additionally, I received a lot of support from the kind people in the GPP, the professors and administrators at the JHU program, and OITE. I would specifically like to thank Dr. Orna Cohen-Fix and Julia Jarvis for listening to my science woes and encouraging me to carry on. iv I had some lovely and encouraging teachers during my high school and undergrad years who encouraged me on my path. Of those, I would like to specifically thank Mr. Kennedy for sparking my interest in science and Dr. Frank Roberto for keeping that spark alive. I have been fortunate to have had so many amazing friends over the years and many of you have made a lasting impact. Throughout graduate school, I am so happy that Senta, Kristie, Annie, Angel, and Sara shared in this adventure. Thank you to Gobi Thillai for being my person. I am eternally grateful for your advice, support, and patience through this graduate process. Thank you for making me laugh, for your healthy dose of reality through this process, and the constant reminder to enjoy life and not take things too seriously. I am so blessed to have my family. Thank you to my parents, Jeff and Karen. Thank you for providing me a solid foundation in both learning and in life. You always provided unwavering support and taught me to approach the world with kindness. I am grateful for my grandparents who are so encouraging and are some of my life role models. I strive to be as wise and humble as you. Then there are my crazy/fun/weird siblings, Rachel, David, and Eve. Thank you for being my biggest, most consistent cheerleaders, especially during those late-night phone calls. They say you can't choose your family but, given the option, I could not have done better. v TABLE OF CONTENTS ABSTRACT……………………………………………………………………..…..…ii ACKNOWLEDGEMENTS………………………………………………………..…iv TABLE OF CONTENTS…………………………………………………………..…vi LISTS OF FIGURES AND TABLES……………………..……..……………….….viii Chapter 1: Introduction pH in the endocytic pathway.………………………………………...…………...……...1 The V-ATPase generates a pH gradient in endocytic organelles…………………..…….3 The electrogenic V-ATPase requires counterions to allow organelle acidification….…..6 Endosomal acidification……………………….…………………………………..……..8 Early study of endosomal acidification……………………………………….….9 The diverse family of ClC transporters and channels………..…………..……....11 ClC-5 facilitates Cl--dependent acidification in early endosomes……………….14 Additional proteins with known and suggested roles in endosomal acidification…..……………………………………………………………..…...16 CCVs display Cl--dependent acidification via an unidentified protein….….…...20 Lysosomal acidification Lysosomes are dynamic signaling centers that rely on pH for function…….......22 Acidification and counterion study in isolated lysosomes.……………………...25 Candidate proteins that facilitate lysosomal counterion flux…………………....28 CFTR…………………………………………………………………………….28 ClC-7 ClC-7 facilitates acidification in isolated lysosomes……………………29 Loss of ClC-7 causes osteopetrosis and neurodegeneration…………….30 ClC-7 is not required for lysosomal acidification in live lysosomes? .....31 The importance of ClC-7 as a transporter vs. channel…………………..31 Cation channels that may assist in lysosomal acidification……………………. 34 Other roles for ClC-7: tissue/condition specific? ……………………………….37 Other roles for ClC-7: protein/protein interactions……………………………...38 What is the role of ClC-7? ……………………………………………………... 40 Is ClC-7 important for lysosomal pH maintenance? ……………………………41 Characterizing a de novo ClC-7 mutation in patients…………………………....43 Thesis aims………………………………………………….…………………………...44 vi Chapter 2: Characterizing chloride-dependent acidification in brain clathrin-coated vesicles Abstract…………………………………………………………………………………..46 Introduction………………………………………………………………….……..….....48 Materials and methods………………………………………………………….………..52 Results……………………………………………………………………….….………..60 Discussion……………………………………………………………………...………...78 Chapter 3: Probing the role of ClC-7 in hepatocyte lysosomal pH maintenance Abstract………………………………………………………………………..…………85 Introduction……………………………………………………………………………....87 Materials and methods…………………………………………………………….……..90 Results…………………………………………………………………………………...96 Discussion………………………………………………………………………………112 Chapter 4: A de novo CLCN7 mutation decreases intralysosomal pH and leads to a novel disorder of cutaneous albinism, lysosomal storage and neurodegeneration Abstract………………….……………………………………………………………...119 Introduction……………………………………………………………………………..120 Materials and methods………………………………………..……………………….. 122 Results…………………………………………………………………………………. 128 Discussion………………………………………………………………………………143 Chapter 5: Concluding remarks ……………………………….……………………149 LITERATURE CITED…………………………………………………………….....156 CURRICULUM VITAE………………..…………………………………………….168 vii LIST OF FIGURES Chapter 1 Figure 1-1: The endosome/lysosome system……………………………………………..2 Figure 1-2: Crystal structure of a eukaryotic ClC transporter…………………………...12 Figure 1-3: The ClC family of channels and transporters……………………………….13 Chapter 2 Figure 2-1: The isolated CCV sample is highly enriched
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