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Abstract
Cilia are membrane bound, microtubule-based organelles that project from the surface of cells. These organelles are located on nearly all cells in mammals, including humans, and are important for the localization of developmental signaling molecules required for signal transduction and activation of target genes, such as components of the Hedgehog pathway. Diseases caused by cilia related defects in people are described as ciliopathies. Cell cycle-related kinase, CCRK, is a Cyclin Dependent Kinase (CDK) that has been shown to be involved in regulating cilia length in mouse. CCRK phosphorylates Intestinal-Cell Kinase (ICK), which has also been linked to cilia phenotypes. CCRK and ICK have different phenotypes when knocked out in mouse, and the CCRK/ICK double mutant is more severe than either single mutant, thus indicating CCRK regulation of cilia is more complicated than a linear relationship through phosphorylating ICK. Ccrknull mice do not survive gestation due to Hedgehog Pathway defects. Ccrknull mouse embryonic fibroblasts (MEFs) produce cilia of abnormal lengths. To determine the role of CCRK in regulating ciliogenesis, I interrogated CCRKs effect on the process of intraflagellar transport and used proteomics to identify downstream effectors of CCRK. I found that CCRK regulates retrograde intraflagellar transport and mutations in CCRK cause accumulation of components at the ciliary tip. I also identified two downstream effectors of CCRK, MyosinIIa and Map4, that are involved in regulating cilia length through interactions with the actin cytoskeleton. In addition to studying the role of CCRK in ciliogenesis, I also developed a CRISPR/Cas9-based tool for generating revertable null mutations in cell culture. With the development of this tool, I was involved in the development and implementation of a small-scale course-based undergraduate research experience (CURE), where students used this tool to gain first-hand experience in a research laboratory, while gaining a deeper understanding of biological concepts.