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The Assembly and Interactions of Mreb in the Maintenance of Cell Shape in Caulobacter Crescentus

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THE ASSEMBLY AND INTERACTIONS OF MREB IN THE MAINTENANCE OF CELL SHAPE IN CAULOBACTER CRESCENTUS A DISSERTATION SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Natalie Anne Dye May, 2010 © 2010 by Natalie Anne Dye. All Rights Reserved. Re-distributed by Stanford University under license with the author. This work is licensed under a Creative Commons Attribution- Noncommercial 3.0 United States License. http://creativecommons.org/licenses/by-nc/3.0/us/ This dissertation is online at: http://purl.stanford.edu/bg008yn0701 ii I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Julie Theriot, Primary Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Lucille Shapiro, Co-Adviser I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. James Spudich I certify that I have read this dissertation and that, in my opinion, it is fully adequate in scope and quality as a dissertation for the degree of Doctor of Philosophy. Aaron Straight Approved for the Stanford University Committee on Graduate Studies. Patricia J. Gumport, Vice Provost Graduate Education This signature page was generated electronically upon submission of this dissertation in electronic format. An original signed hard copy of the signature page is on file in University Archives. iii ABSTRACT This work focuses on the mechanism by which MreB contributes to the maintenance of cell shape in the gram-negative alpha-proteobacterium Caulobacter crescentus. The gene mreB encodes a protein that resembles actin, a eukaryotic cytoskeletal protein. Previously, it was shown that mreB is required to maintain a rod-like shape and localizes to a helical pattern near the cytoplasmic membrane. Here, we show that MreB is associated with regions of active growth in Caulobacter, as mutant strains that mislocalize MreB to the cell poles direct new growth at or near the poles. We present evidence to suggest that MreB contributes to the determination of proper length, width, and curvature through partially distinct mechanisms. The determination of proper width involves the essential proteins MreC and Pbp2, which are encoded in the mreB operon. While MreB and MreC are both required to position the cell wall transpeptidase Pbp2 along the lateral sidewalls and away from midcell, the two do not colocalize and each can maintain its localization in the absence of the other. When MreB is mislocalized to the poles, MreC and Pbp2 do not follow. These data argue against the idea that MreB provides a scaffold-like structure to localize enzymes that directly modify the cell wall. The determination of proper curvature, involves the intermediate filament-like protein, Crescentin. We identify a putative binding site on MreB for Crescentin or other curvature-mediating factors. We also show that the extent to which the subcellular localization of MreB changes over the cell cycle is correlated with cell size, indicating that MreB is involved in the coordination between elongation and division. In addition, we show that in vitro purified MreB spontaneously forms very stable polymers in the presence or absence of nucleotide. These polymers are globular or amorphous and only filamentous when placed on a highly positively charged surface of Poly-L-lysine. These in vitro data suggest that MreB is likely to be regulated at the disassembly step in the cell and that the cellular environment may influence the structure of MreB polymers. Lastly, we present biochemical evidence to support the existence of a disassembly factor in cytoplasmic Caulobacter extract. Together our data suggest that the maintenance of the crescent-rod cell shape in Caulobacter is the result of a complicated balance between MreB’s dynamic subcellular localization, polymeric structure, and communication with cellular components. iv For my always loving and supportive parents John and Donna Dye v ACKNOWLEDGEMENTS This thesis is the culmination of almost seven years of work. During this time, I have had the pleasure of working with so many truly brilliant, creative, and knowledgeable scientists. I would not be the scientist I am today without the generous help of my mentors and colleagues. I would like to take this opportunity to thank the following persons. First and foremost, I must thank my joint thesis advisors, Julie Theriot and Lucy Shapiro. These two women are phenomenal scientists and individuals, and it has been a privilege and a pleasure to work with them. While they have very different scientific perspectives, styles, and interests, they share an inspiring enthusiasm for scientific research and a genuine care for their students. Thank you both for the guidance, mentorship, motivation and inspiration you have given me during this process. In addition to my advisors, I received considerable guidance from the other members of my committee Aaron Straight and Jim Spudich. I very much appreciate the advice and feedback that I received from each of them over the years. I am also thankful for KC Huang, who agreed to be my chair and has given me significant feedback on this work. Harley McAdams, though not an official committee member or advisor, has also provided an interesting and important point of view on this project. I’d also like to thank John Perrino at the EM facility in the Beckman center here at Stanford for guidance with the electron microscopy. I must acknowledge my collaborators in this project. Zachary Pincus was a graduate student with Julie when I first started this project, and even after he left Julie’s lab to become a postdoc thousands of miles away, he remained an active participant in this research. His contributions to the work presented in Chapters 2 and 3 were significant, and these stories would not have been as mature as they are now without his expertise in computation and statistics. Isabelle Fisher contributed to this project as a high school student. Though lacking the many years of formal training in biology, Isabelle was able to grasp complicated concepts and make me think about this subject in new ways. She was a joy to teach and work with, and I hope to see her continue in science. Enrique de la Cruz and his graduate student Kendra Frederick hosted me in their lab at Yale vi University for a short time to work with purified MreB. While this collaboration was brief and did not end up yielding publishable data, it was a valuable experience for me to learn specific techniques from them and get their perspective on this project. I have received countless ideas, feedback, reagents, protocols, and emotional support from the members of the Theriot and Shapiro labs, and as such I would like to thank everyone with whom I overlapped. In particular in the Shapiro/McAdams lab: Zemer Gitai, who was my rotation advisor, Antonio Iniesta, Grant Bowman, Erin Goley, Jerod Ptacin, Esteban Toro, Joseph Chen, Patrick McGrath, and Virginia Kalogeraki. In the Theriot lab: Kinneret Keren, Karine Gibbs, Aretha Fiebig, Guy Ziv, David Hallidan, Greg Allen, Mark Tsuchida, and Steph Weber. A very special thank you goes to Susanne Rafelski, who was my mentor in the Theriot lab when I first started and has remained a dear friend. I owe a huge huge huge debt of gratitude to Matthew Footer. He has taught me so much about biochemistry, protein purification, optics, cooking, and more. Thank you for always being so friendly and generous with your time. I think all of us agree that the lab would fall apart without you! I must also especially thank my friend and labmate Erin Barnhart, who has sat next to me almost my entire time in graduate school. She has been my sounding board, my colleague, my friend, and at times my entertainment . Erin also read almost this entire thesis and gave me thoughtful feedback, all while writing her own thesis. You are a talented, fun, intelligent, courageous, and goofy individual and it’s been a pleasure sharing this experience with you. Thank you. I would also like to thank all of the past and present members of the Biochemistry and Developmental Biology departments here at Stanford for making it a collegial and enjoyable place to work. I must specifically acknowledge the friendly, patient, and helpful support staff in these departments, in particular Joella Ackerman, Karen Butzman, Tara Trim, Todd Galitz, and Shedrick Watts. Special thanks to my classmates and friends in the department Ryan Nottingham, Ian Brennan, Kirstin Milks, Eric Espinosa, and especially Kristina Godek. Kristina has been a dear friend and colleague. Her endurance and dedication to science is an inspiration, and I wish her all the success her hard work deserves. I will miss having her vii right down the hall. She also provided significant feedback on this thesis. I’d also like to thank my friends Dina Finan, Duane Baxter, Michael Costa, and Corey Meyer, who provided me with much wine and amazing home-cooked meals. In my time at Stanford, I have been a part of a wonderful network of female scientists and engineers, and I would like to thank the following for their considerable advice and support in both professional and personal manners: Megan Young, Jennifer Zamanian, Hyejun Ra, Davie Yoon, Misty Davies, Esther Chen, Liv Walter, Sabina Stefania, and Lisa Moore. Through my involvement with the Alpine club, I became part of a fantastic climbing community, which has enriched my experience here at Stanford.
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