UC Berkeley

Naegleria gruberi is a free-living eukaryote that has been described as a unicellular "Jekyll-and-Hyde. Most of its time it can be found as a small (15 µm) amoebae, common to freshwater environments throughout the world. However, when exposed to stressful conditions the amoebae quickly and synchronously differentiate into flagellates. This dramatic change involves the formation of an entire cytoplasmic microtubule cytoskeleton, including de novo assembly of the centriole-like basal bodies, `9+2' flagella, and a cortical microtubule array. This `quick-change act' offers an unprecedented opportunity to study the assembly of an entire microtubule cytoskeleton, particularly the beautifully complex structures of centrioles. However, utilization of Naegleria as a model organism has been frustrated by lack of sequence information and molecular tools. This dissertation describes my efforts during my graduate studies to coordinate the Naegleria genome project, analyze the resulting sequence data, and develop tools with which to study Naegleria's amoeba-to-flagellate transition, with a focus on basal body assembly.

Although the analysis of the Naegleria genome revealed many interesting aspects of both Naegleria biology and the evolution of eukaryotes, the results presented here are limited to those that pertain to Naegleria's actin and microtubule cytoskeletons. In particular, detailed manual inspection of individual Naegleria genes uncovered an extensive repetoire of previously characterized actin and microtubule cytoskeletal components. This indicates that despite Naegleria's extremely distant relationship to animals, the transient nature of its cytoplasmic microtubule cytoskelelon, and that the amoeboid actin cytokeleton functions independently of microtubules, Naegleria has the capacity to to form a canonical cytoskeleton.

Additionally, we took advantage of Naegleria's distant relationships to other sequenced organisms to identify ancient genes that we predict to be involved with amoeboid or flagellar motility. To do this, we compared all the genes from a diversity of sequenced eukaryotes, and selected protein families conserved only in eukaryotes with flagellar motility (Flagellar-Motility associated genes; FMs) or amoeboid motility (Amoeboid-Motility associated genes; AMs). Along with the expected gene families, Naegleria's 182 FM's include 36 novel flagellar-associated genes. The 63 AMs include genes known to be involved in amoeboid motility, membrane differentiation, and 19 novel genes. As far as we know, this is the first catalog of genes predicted to be associated specifically with amoeboid motility.

During the amoeba-to-flagellate transition, Naegleria synchronously assembles centrioles from scratch, providing nearly limitless amounts of material for both proteomics and microarrays, and an unparalleled oppourtunity to study how these structures assemble. Although we know that centrioles and basal bodies are composed principally of a cylinder of nine microtubule triplets, their protein composition and method of assembly remain largely mysterious. Animal centrioles usually duplicate via "templated" assembly, with the new centriole developing perpendicularly from the side of a preexisting centriole. Centrioles can also be formed "de novo", in cytosol devoid of preexisting centrioles in some plant and animal cells, as well as Naegleria. How Naegleria makes exactly two basal bodies de novo remains an open question. During my graduate studies, I have developed antibodies and used them to describe the order of expression and incorporation of three Naegleria centriole proteins (SAS-6, γ-tubulin, and centrin). I also used these to provide the first evidence that Naegleria has templated, as well as de novo, basal body assembly, and suggest that having both capacities allows Naegleria, and other organisms (e.g. mouse embryos), to make the correct number of centrioles.

Finally, I have tracked the expression of Naegleria's genes during differentiation to identify novel centriolar and flagellar proteins. Although about a third of Naegleria genes are induced and another third are repressed during differentiation, I focused on the evolutionarily conserved FM genes, and use the timing of induction to subdivide them into a subset of 55 genes enriched in known basal body proteins (induced early) and a subset of 82 genes enriched in axonemal proteins (induced late). The centrosome-enriched set includes nearly every conserved basal body component that has been previously characterized, many components required for microtubule nucleation (a process that occurs largely at centrosomes) and ten novel genes that are conserved across eukaryotes. As a proof of principle, the human ortholog of one of the novel genes was tagged, and indeed localizes to the centrosomes of human cells.