MOLECULAR CHARACTERIZATION OF CELL DIVISION MACHINERY IN CAULOBACTER CRESCENTUS A DISSERTATION SUMITTED TO THE DEPARTMENT OF CHEMISTRY AND THE COMMITTEE ON GRADUATE STUDIES OF STANFORD UNIVERSITY IN PARTIAL FULFILLMENT OF THE REQUREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY Yi-Chun Yeh December 2010 © 2011 by Yi-Chun Yeh. 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/js518vh3162 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. Harley McAdams, 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. William Moerner 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 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 Cell division is a major developmental event in the life cycle of a bacterial cell. Caulobacter crescentus division is asymmetric, producing daughter cells that differ in morphology and polar features: a sessile stalked cell and a motile swarmer cell that subsequently differentiates into a stalked cell. In this work we investigate the assembly of the Caulobacter cell division machinery (the divisome) using genetics, biochemistry, and microscopy. The bacterial divisome mediates the constriction of the cell membranes and the inward growth of the cell wall in coordination with cell elongation and chromosome segregation. In Caulobacter , the cell division process requires a set of approximately twenty-three proteins localizing from the cytoplasm to the outer membrane. To understand divisome assembly as a function of the cell cycle, we generated fluorescent fusions to analyze the temporal regulation of 19 divisome and division-site localized proteins. In Chapter 2, we identified a series of stages and transitions in divisome assembly and the associated events yielding a comprehensive temporal picture of the process. First, the FtsZ binding proteins appear at midcell about 10 minutes after the initial assembly of Z ring near midcell. Second, proteins involved in cell growth and morphology specification localize at midcell. Next, an apparent transition to constriction occurs wherein TolQ and FtsA are recruited to the division site. Subsequently, the arrival of five core divisome proteins to midcell is followed by the appearance of the cell polarity marker protein, TipN. The assembly interdependency for divisome formation in Caulobacter appears to involve cooperative rather than sequential recruitment, suggesting that it is a multiprotein subcomplex model. iv Cell division in Caulobacter involves constriction and fission of the inner membrane (IM) followed about 20 min later by fission of the outer membrane (OM) and daughter cell separation. In Chapter 3, we describe our investigation of the Tol-Pal complex where we demonstrated that it plays a vital role for membrane integrity maintenance and that it is essential for viability. Cryo-electron microscope images of the Caulobacter cell envelope exhibited outer membrane disruption, and cells failed to complete cell division in TolA, TolB, or Pal mutant strains. In wild type cells, components of the Tol-Pal complex localize to the division plane in early predivisional cells and remain predominantly at the new pole of swarmer and stalked progenies upon completion of division. The Tol-Pal complex is required to maintain the position of the transmembrane TipN polar marker, and indirectly the PleC histidine kinase, at the cell pole, but it is not required for the polar maintenance of other transmembrane and membrane associated polar proteins tested. Co- immunoprecipitation experiments showed that both TolA and Pal interact directly or indirectly with TipN. We propose that disruption of the trans-envelope Tol-Pal complex releases TipN from its subcellular localization. The Caulobacter Tol-Pal complex is thus a key component of cell envelope structure and function, mediating outer membrane constriction at the final step of cell division, as well as the positioning of a protein localization factor. FtsZ, a relative of tubulin, is the most highly conserved divisome protein. It is a GTPase that polymerizes into a contractile ring near midcell, defining the future site of cell division. Various proteins have been shown to stimulate FtsZ ring assembly while negative regulators dissociate it. In Chapter 4, we describe our examination of v the FtsZ binding protein, ZapA. We showed that ZapA is required for proper FtsZ ring formation. Using a fluorescent fusion to the zapA gene, we found that ZapA is colocalized with FtsZ to the division plane and new pole. In addition, a ZapA- mCherry fusion is dependent on the localization of FtsZ to the division plane. ZapA is required to maintain a normal cell length, and it acts at midcell to promote Z ring assembly. A ZapA deletion strain is filamentous, showing that ZapA is required for normal divisome assembly. These biochemical and functional studies suggest that Caulobacter ZapA is a positive regulator of Z ring assembly. In summary, we have addressed three major stages in developments of the divisome in Caulobacter : Z ring assembly, divisome maturation and outer membrane invagination. These experiments have provided a new understanding of how the Caulobacter cell temporally executes the cell division program to propagate reliably and how Caulobacter cell division is performed. vi Acknowledgements I would like to express my immense gratitude to my advisor, Harley McAdams, for all his support and suggestions over the years. Harley's guidance has been indispensable for my conversion to the biology field without any prior bench experience. I am also grateful to Lucy Shapiro for all her advice and mentoring toward research. Lucy’s enthusiasm and encouragement made the lab an enjoyable place to work. I appreciate the advice given by the other members of my thesis committee, Drs. W. E. Moener, Robert Simoni and Andrew Spakowitz. I am grateful to current and former members of the McAdams and Shapiro labs. Throughout grad school, I have had the pleasure of working with many brilliant, creative, and knowledgeable scientists. I would not be the scientist I am today without the generous help of my mentors and colleagues. In particular, I would like to take this opportunity to thank Erin Goley who collaborated with me on many projects and provided most of the experimental support. The work described in this thesis would not have been possible without her. I am also thankful to Sun-Hae Hung, Martin Thanbichler, Jay Lesley, Eduardo Abeliuk, Monica Schwartz, Paola Mara, Grant Bowman, Nathan Hillson, Antonio Iniesta, Virginia Kalogeraki, Masaki Kato and Yong Jae Chong for their friendship including many delightful discussions and collaborations. I would also like to thank Luis Comolli at LBNL for his help the for cryo-electron microscopy. I also want to thank my boyfriend Yi-Ju, who has enriched my life. Finally, I want to acknowledge my sister, brother-in-law, my parents and my grandparents for their continual support. vii Table of Contents Chapter 1. General introduction ..................................................................................... 1 1.1 The Caulobacter cell cycle ................................................................................... 2 1.2 FtsZ ...................................................................................................................... 5 1.3 Regulation of Z-ring formation ............................................................................ 6 1.3 Overview of the late cell division proteins ......................................................... 12 1.4 Assembly of the divisome .................................................................................. 16 1.5 Overview of other modes of bacterial cell division ............................................ 18 1.6 Constriction and invagination of the cell envelope ............................................ 20 Chapter 2. Assembly of a bacterial cell division machine ........................................... 22 Introduction .............................................................................................................. 23 Materials and methods .............................................................................................. 28 Results ...................................................................................................................... 31 Discussion ................................................................................................................. 57 Chapter 3. The Caulobacter Tol-Pal complex is essential for outer membrane integrity and is required for the completion of cell division and polar protein localization ....... 71 Introduction .............................................................................................................