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Abstract
In biology, size control (or scaling) is a fundamental property at all levels of organization, from polymeric protein complexes to tissues, organs and organisms. This thesis addresses the mechanisms of scaling at the intracellular level using the ciliate Tetrahymena thermophila as a model. Tetrahymena carries a polarized cortical pattern of organelles including hundreds of cilia, on the cell surface. This cortical pattern can be used to study the intracellular scaling with high resolution. We developed a genetic pipeline for identification of causal variants in Tetrahymena mutants by comparative whole genome sequencing, with unusually high accuracy. In Chapter 3, I focused on the scaling at the organelle level, using cilium length as a model. We developed a genetic interactor screen based on a hypermorphic allele of the conserved kinase, LF4/MOK, that acts as negative regulator of cilium length. We identified another conserved cilium length kinase, LF2/CCRK/DYF-18, as an upstream regulator of LF4/MOK abundance. We adapted total internal reflection microscopy for imaging inside cilia, to show that LF4 is a ciliary protein that is distributed uniformly along cilia, mostly immobile and likely microtubule-bound. We also found that LF4 affects the intraflagellar transport (IFT), a motility mechanism that distributes components inside cilia. Most cells double their size prior to cell division to produce two daughters of equal size. In Chapter 4, I studied a Tetrahymena mutant, cdaI-1, that during cell division produces daughters of unequal size. I found that cdaI-1 results from a mutation in CdaI, an ortholog of the Hippo/Mst kinases, components of the highly conserved Hippo signaling, which in diverse eukaryotes regulate cell growth and cell division, and in animals affect embryo polarity and organ size. We conclude that in Tetrahymena, the Hippo pathway maintains the equatorial placement of the division plane. In summary, this dissertation delivers new powerful genetic and imaging tools to Tetrahymena, a large single-cell eukaryote that is highly amenable for studies of intracellular scaling. We dissected the principles of size control inside the single cell across multiple levels of organization and identified specific proteins and interactions that affect intracellular size control.