ESTABLISHMENT OF A FUNGAL MODEL SYSTEM FOR THE STUDY OF CILIATION Linnea Tracy June 2015 ESTABLISHMENT OF A FUNGAL MODEL SYSTEM FOR THE STUDY OF CILIATION An Honors Thesis Submitted to the Department of Biology in partial fulfillment of the Honors Program STANFORD UNIVERSITY by Linnea Tracy June 2015 2 Acknowledgements A simple thank-you seems inadequate for all those who have offered their time, expertise, support, and supplies towards my project and education that is culminating in this thesis. Nevertheless, thank you to Tim Stearns, whose kindness, brilliance, and natural knack for teaching was an inspiration to me as a student, was motivation to me as a researcher, and was a great honor to get to know and work closely with over the last two years. Thank you for your time and dedication devoted to my, my peers’, and the world’s education. A special thank you to Erin Turk, who took me under her wing, learning about chytrids in order to teach and assist me, all the while completing her own graduate dissertation. You are one of the most motivated, prepared, and lovely people I have met at Stanford. Thank you for your guidance, mentorship, and always responding to my text messages. Thank you to the members of the Stearns lab, who guided me from learning the basics of the laboratory through experimental design and science writing. It has been, and continues to be, a great pleasure to have a lab family that is so intelligent and kind. I would be remiss to not also thank my parents and friends, who have loyally allowed me to discuss cilia and worry over my experiments with them, sometimes at the expense of our social life. Thank you for accepting that my life at Stanford has revolved around the chytrid life cycle for the last year, and unwaveringly encouraging my love for discovery and learning. 4 Table of contents List of Tables………………………………………………………………….…………..6 List of Figures……………………………………………………………………………..7 Abstract……………………………………………………………………………………8 Introduction………………………………………………………………………………10 Materials and Methods………………………………………………………………...…13 Results……………………………………………………………………………………20 Discussion………………………………………………………………………………..26 Bibliography…………………………………………………………………………..…30 Appendix of Figures…………………………………….……………………………….32 Appendix of Tables………………………………………………………………………38 5 List of Tables Table 1- Conservation of Nuclear Pore Proteins Relative to Human Proteins Table 2- Conservation of Tubulin Superfamily Members Relative to Human Proteins Table 3- Conservation of Centrosome and Axoneme-related Proteins Relative to Human Proteins 6 List of Figures Fig. 1- Schematic of cilium architecture and associated diseases Fig. 2- Diagram of the cellular architecture of a chytrid zoospore Fig. 3- Section of the eukaryotic tree of life Fig. 4- Light microscopy stills of cilium retraction in Rhizoclosmatium Fig. 5- Immunofluorescence images of zoospores with unretracted cilia Fig. 6- Circularized axonemes resultant from fixation and retraction Fig. 7-The proportion of circularized axonemes increases over time Fig. 9- Immunofluorescence image of a zoospore with axoneme half-retracted Fig. 8- Axoneme degradation after retraction 7 Abstract Cilia are highly-conserved structures found in all major branches of eukaryotic tree that function in cellular motility, directional movement of extracellular fluid, and sensing of chemical and mechanical stimuli. Many developmental transitions are marked by cilium-dependent signaling events, and there is much interest in determining the mechanisms by which cells extend and retract cilia, given the relevance to human diseases caused by ciliary dysfunction (ciliopathies). Here, we present a chytrid fungus, Rhizoclosmatium globosum, as a possible model system for the study of the motile cilium, and characterize the process of cilium retraction in this organism. Chytrid fungi, unlike higher fungi, have zoospores with a single posterior cilium and a pair of centrioles at its base. We found cilium retraction in these organisms to be highly reproducible, occurring at the developmental shift between the motile, ciliated zoospore life-stage and reproductive, non-ciliated sporangium stage. Cilium retraction was accompanied by a simultaneous cytoplasmic rotation, suggesting that the cilium is “reeled” into the cell body. Sodium azide does not inhibit this process, indicating retraction may not require energy produced by the electron transport chain. The retracted cilium was visualized within the zoospore by immunofluorescence; it is initially coiled around the diameter of the cell, but is disassembled or degraded within 30 min. We showed chytrid cilium- related proteins to be more related to their human orthologs than those of C. reinhardtii and T. thermophila, the most commonly used single-cell, ciliated model systems. Due to the easily observable, reproducible nature of cilium retraction in chytrid fungi, and the relatively high homology between chytrid and human cilium-related proteins, we 8 conclude that Rhizoclosmatium is an excellent candidate for a model system for the study of cilium retraction. 9 Introduction The cilium, is a highly-conserved cellular structure present in every major branch of the eukaryotic evolutionary tree. Cilia are essential to many important processes ranging from developmental signal transduction1 to cellular motility (Fig. 1). Cilia are microtubule-based structures, consisting of specialized microtubule doublets that extend from specialized centrioles, known as basal bodies. Primary cilia, found on most vertebrate cells, are immotile and participate in signaling pathways, receiving both chemical and mechanical stimuli. Motile cilia are only found on a few specialized cell types in vertebrates and can be present as a single cilium (as in the embryonic node) or tens to hundreds of cilia per cell (as in the motile cilia of the airway epithelium) that beat to enable fluid flow over the cell surface. Human diseases caused by ciliary dysfunction (termed ciliopathies) can similarly be grouped into those affecting non-motile and/or motile cilia. Motile ciliopathies are caused by defects in one or more of the mechanisms that are specific to ciliary motility (Figure 1), with most grouped under a single disease heading, primary ciliary dyskinesia (PCD). Non-motile ciliopathies, depending on the affected protein, can alter the function of both primary and motile cilia through defects in the basal body, or in trafficking of ciliary proteins. Examples of such diseases include Oral-Facial-Digital Syndrome (OFD) and Bardet-Biedl Syndrome (BBS). Given the relevance of both types of cilia to human health, many models for studying cilia have been employed. Mammalian cells in culture often form a primary cilium, although the frequency with hich they do this depends on the cell line, with non-transformed cells making a cilium more often. Motile cilia, however, are restricted to specialized cell types 10 and thus must be studied in whole organisms, such as tissues from mouse or Xenopus embryos, or organotypic models thereof. To study motile cilia in single-cell organisms, the green alga Chlamydomonas reinhardtii and ciliated protozoan Tetrahymena thermophila, amongst others, have been used. Given the evolutionary divergence of such single-cell ciliates relative to humans the identification of important proteins, and thus the relevance to human ciliary function is not always clear. Many aspects of cilium function, including ciliogenesis, signaling, and ciliary maintenance have been well studied in a variety of systems. By contrast, there is far less information regarding cilium resorption or retraction. The presence of a cilium on a mammalian cell is correlated with cell cycle state2, in that the cilium disappears prior to mitotic entry, and reappears in G1, although other temporal changes in cilium resorption have also been observed3. More recent work has evaluated the role of specific enzymes and protein expression patterns associated with cilium resorption4, particularly in Chlamydomonas reinhardtii5. Nevertheless, the actual mechanism of cilium loss during the cell cycle, or development, remains unknown, and largely unexplored. Although not yet employed in the study of cilia, a class of fungi known as Chytridiomycetes (chytrids) are unique among higher fungi in having maintained a ciliated, motile life stage. Chytrids were the focus of some interest in the mid-20th century to microscopists, and have recently experienced a resurgence of interest due to the devastating amphibian pandemic known as chytridiomycosis. Chytridomycosis is caused by one species of chytrid, Batrachochytrium dendrobatidis, that infects the skin of its host and kills amphibians by as yet unclear mechanisms. 11 Although B. dendrobatidis is the most well-characterized of the chytrids, there are many species of chytrids, and we chose to use another, Rhizoclosmatium globosum for most of our work. R. globosum grows well in the laboratory on simple solid and liquid media, at temperatures between 18 and 25°C. Cilium resorption in R. globosum occurs during a developmental shift from a motile, singly-ciliated zoospore (Figure 2) to an immotile, non-ciliated sporangium. Zoospores lack a cell wall, but upon the transition to the sporangium stage, a chitin-based cell wall forms, and fine, hair-like rhizoids are extended for adhesion and nutrient uptake. Given the close evolutionary relationship of fungi to humans relative to other single-cell ciliates (Figure 3), as well as the ability to correlate cilium retraction with a developmental