University of Arkansas, Fayetteville ScholarWorks@UARK Theses and Dissertations 5-2019 Characterization of Microtubule Organizing Centers in the genus Protostelium, Including Evolutionary Implications Ethan Taylor Ozment University of Arkansas, Fayetteville Follow this and additional works at: https://scholarworks.uark.edu/etd Part of the Cell Biology Commons, Developmental Biology Commons, Environmental Microbiology and Microbial Ecology Commons, and the Evolution Commons Recommended Citation Ozment, Ethan Taylor, "Characterization of Microtubule Organizing Centers in the genus Protostelium, Including Evolutionary Implications" (2019). Theses and Dissertations. 3136. https://scholarworks.uark.edu/etd/3136 This Thesis is brought to you for free and open access by ScholarWorks@UARK. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of ScholarWorks@UARK. For more information, please contact [email protected]. Characterization of Microtubule Organizing Centers in the genus Protostelium, Including Evolutionary Implications A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Cell and Molecular Biology by Ethan Ozment Utah Valley University Bachelor of Science in Biotechnology, 2015 May 2019 University of Arkansas This thesis is approved for recommendation to the Graduate Council. _________________________________ Frederick W. Spiegel, Ph.D. Thesis Director _________________________________ ________________________________ Andrew J Alverson, Ph.D. Burton H. Bluhm, Ph.D. Committee Member Committee Member _________________________________ Jeffrey A. Lewis, Ph.D. Committee Member Abstract Microtubule organizing centers (MTOCs) are cellular regions of microtubule nucleation. The best known MTOCs are those associated with the centrosome, but several non-centrosomal MTOCs are known in eukaryotes, especially in land plants. MTOCs are poorly characterized across the breadth of amoebozoan diversity, but are well-known in certain amoebozoan lineages, including the genus of protosteloid slime molds Protostelium. The structure of the MTOC is known for two non-ciliated species, P. nocturnum and P. mycophaga, as well as P. aurantium, which can reversibly become ciliated under appropriate conditions. P. nocturnum and P. mycophaga have acentriolar centrosomal MTOCs while P. aurantium has a centriole-bearing pro-kinetid that differentiates into a kinetid when the cell becomes ciliated. It was previously thought that the MTOCs of P. mycophaga and P. nocturnum were homologous to each other, and were derived from a structure reminiscent of the kinetid of P. aurantium, but recent changes in our understanding of the group’s phylogeny, as well as the realization that most isolates of P. aurantium cannot become ciliated, have called this hypothesis into question. In this thesis, a new strain of P. aurantium was isolated. This strain, which was unable to produce cilia when isolated, was characterized ultrastructurally and found to have an MTOC typical of non-ciliated Protostelium spp. After ultrastructural work was complete, ciliated cells were unexpectedly found in one culture of the new isolate. The significance of these findings, and their implications for the evolutionary history of Protostelium, are discussed. 2019 by Ethan Ozment All Rights Reserved Acknowledgements Had I been on my own, I never would have reached this point in my career. I am indebted to several people for their assistance and support in completing this thesis. My mother, Ann Harrison, who raised me to have a curious mind. She, and my father, John, refused to let me be satisfied with anything less than my best. They were always there to comfort and support me, and to correct my course when I lost sight of my goal. My advisor, Dr. Fred Spiegel, helped me to set aside my preconceived ideas and think about things in new ways. Fred struck a perfect balance of assisting me and making me figure things out for myself. Dr. Betty Martin did more than just train me in electron microscopy. She was there for the struggles and the triumphs. She always believed in me, and I thoroughly enjoyed our conversations. This work would not have been possible without the Institute for NanoSciences & Engineering - Arkansas Nano & Bio Materials Characterization Facility for the supplies and equipment needed for electron microscopy, and funding from NSF-DEB grant 1456054. Table of Contents Chapter 1: Introduction ............................................................................................................... 1 LITERATURE CITED .......................................................................................................... 12 Chapter 2: Evidence for a Novel Life History in the Protosteloid Genus Protostelium ....... 17 ABSTRACT ............................................................................................................................. 17 INTRODUCTION................................................................................................................... 17 MATERIALS AND METHODS ........................................................................................... 19 RESULTS ................................................................................................................................ 22 DISCUSSION .......................................................................................................................... 25 LITERATURE CITED .......................................................................................................... 35 Chapter 3: Conclusion ................................................................................................................ 38 LITERATURE CITED .......................................................................................................... 43 Chapter 1: Introduction Introductory biology courses need to lay the foundation for more advanced courses, and often must be accessible to students not entering a biology-related field. Possibly in an attempt to satisfy these requirements, such courses focus on examples that will be familiar to students: animals, especially humans, and to a lesser extent, flowering plants. Although this approach helps ease students into what can be a difficult subject, it leaves them with the notion that these organisms are “typical” and represent a standard for what living things are supposed to be like. They often lack an appreciation for how unusual multicellularity is, and tend to see unicellular organisms as “primitive” or transition states to “higher organisms”. There tends to be an assumption that evolution always progresses toward complexity, and that simpler organisms are primitive while more complex organisms are more evolved. This type of thinking is further reinforced by the fact that vertebrates are among the most structurally complex organisms known, which allows us as humans to think of ourselves as being highly evolved. Many people don’t consider the fact that any two extant organisms are separated from their last common ancestor by the same length of time. As such, no extant organism is any more or less evolved than any other extant organism. Multicellularity is simply a characteristic of certain lineages, metazoans and embryophytes among them; it is not some end goal that all organisms are striving for. Evolution does not always lead toward complexity over time. Parasites for instance, are well known to often be highly reduced. The model organism Saccharomyces cerevisiae is almost certainly descended from a more complex filamentous fungus with a mycelium in its life cycle (Dee et al. 2015). Amoebozoa is major clade of eukaryotes composed almost entirely of organisms that are amoeboid during at least part of their life cycle (Adl et al. 2019; Kang et al. 2017). The group 1 includes “naked” amoebae, testate (shelled) amoebae, ciliated forms, and several so-called slime molds that produce spore-bearing fruiting bodies. Protosteloid amoebae or protosteloid slime molds are amoeboid organisms in which a single cell can develop into a fruiting body with one to a few spores atop an acellular stalk. Although all known protosteloid slime molds are amoebozoans, they are widely spread in the group and do not form a monphyletic group or a paraphyletic group within a monophyletic Eumycetozoa sensu Olive, along with the myxogastrid and dictyostelid slime molds (Olive, 1975; Spiegel, 1990; Spiegel et al. 1995). This view was refuted by Shadwick et al. (2009). These organisms were formerly referred to as protostelids when they were thought to be closely related, but that term has fallen into disuse to emphasize this fact (Shadwick et al. 2009; Spiegel et al. 2017; Tice et al. 2016). Simplification and trait loss are major evolutionary trends in Amoebozoa (Adl et al. 2019; Kang et al. 2017; Spiegel et al. 2017). The group’s last common ancestor was a sexual, ciliated organism that may have possessed other traits as well, including the ability for single cells to develop into spore-bearing fruiting bodies, a behavior known as sporocarpy (Adl et al. 2012; Spiegel et al. 2017). Sexuality may have involved an alternation of haploid and diploid generations (Adl et al. 2019; Kang et al. 2017). Today, only a few lineages in Amoebozoa contain organisms in which ciliated cells have been observed or in which a sexual cycle has been fully characterized (Adl et al. 2012; in press; Kang et al. 2017; Spiegel et al. 2017), although there is molecular evidence for sex in