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Abstract

Cells are classified based on the presence (eukaryotes) or absence (prokaryotes) of the nucleus, a double-membrane bound organelle housing the genome and nucleolus. Exchange between the nucleus and cytoplasm happens across the nuclear envelope exclusively via the nuclear pore. The nuclear envelope and pores are dramatically remodelled during cell division or mitosis via several different strategies ranging from global envelope breakdown and pore disassembly (open mitosis) to local envelope breakdown with intact pores (closed mitosis). Depending on the mitotic strategy, the mitotic spindle composed of the kinetochore, microtubules, and the microtubule-organizing center are built differently. This “mitotic” cytoskeleton is a very phenotypically diverse machinery regulating a process that stretches unbroken back to the very first protocells. Current model organisms do not adequately represent this extant diversity and are inadequate for investigating its evolution. Studying this cytoskeleton in non-model organisms in the lab is non-trivial and requires extensive investment of resources. However, we are in a Golden Era for comparative genomics with the advent of low-cost sequencing and community efforts like the Darwin Tree of Life project, 1000 plant transcriptomes, and many more. Phylogenetics and comparative genomics present optimal strategies for asking questions about how different parts of this cytoskeleton evolved. In my PhD thesis, I built a semi-automated workflow to retrieve protein orthologs at the tree-of-life scale using well-known, widely-used statistical models of protein sequence variation called Hidden Markov Models and trace the evolutionary history of different parts of this mitotic cytoskeleton across the eukaryotic tree of life.

I use my workflow to investigate different biological questions across different parts of the eukaryotic tree of life. I also demonstrate that my workflow works across biological questions and across large timescales. Specifically, I show that the mitotic cytoskeleton, composed of the nuclear pore complex, other nuclear envelope proteins, the microtubules and microtubule-organizing center, and the kinetochore, is conserved in Opisthkonta, and that it evolved by increased duplications at three key ancestral nodes. I demonstrate that the proteins involved in nuclear pore formation in animals (Clcc1) and fungi (Brl1/Brr6) evolved independently. I investigate the evolution of the S. cerevisiae kinetochore alongside its centromere, and show that the kinetochore is conserved and that it can support centromere diversification. Across over 2 billion years of eukaryotic evolution, I show that the proteins of the transmembrane ring of the nuclear pore evolved separately, that the centriole cartwheel protein Sas6 has an ancient duplication and likely has novel roles beyond the centriole, and that the 8-protein Augmin complex, involved in microtubule branching, is near ubiquitously present, and identify all 8 subunits in the green algae C. reinhardtii. Finally, I demonstrate that my workflow produces reliable results within the Archaeplastida clade where I investigate the complex evolutionary history of the centriole and flagellum.