| Popis: |
Cilia and flagella are hair-like projections found on the surface of virtually every vertebrate cell. These microtubule-based organelles are historically known for their striking motility, a valuable tool for the manipulation of fluid environments. In addition, immotile (or 'primary') cilia play critical roles in cell signaling. More than ten human diseases have been linked to cilia function, with pleiotropic phenotypes including obesity, kidney and liver disease, skeletal abnormalities, situs defects, mental retardation, and sterility. In this dissertation, I first examine the function of Cep290, a putative master regulator of cilia biology, which is mutated in five human ciliopathies. I found that the zebrafish Cep290 protein was localized in a cell-type specific fashion to two distinct ciliary compartments: transition zones and centriolar satellites. Through morpholino knockdown, I demonstrated that Cep290 regulates the length of photoreceptor, Kupffer’s vesicle, and spinal canal cilia, while it was dispensable for normal cilia length in other tissues. Rescue of Cep290 associated cilia length defects by overexpression of cilia membrane proteins implicated Cep290 in cilia vesicle trafficking. Unexpectedly, I found that Cep290 deficiency in Kupffer’s vesicle and spinal canal resulted in cilia paralysis, accounting for left right asymmetry and hydrocephalus phenotypes, and identifying a novel function for Cep290 in dynein arm assembly. In the second chapter I identify and characterize three novel ciliopathy genes. We performed a small-scale morpholino screen to test the function of predicted cilia proteins. Three genes essential to cilia motility were identified: c21orf59, ccdc65, and c15orf26. Parallel studies in other systems revealed that C21orf59 was a component of the flagellar matrix required for the assembly of outer dynein arms, while Ccdc65 was part of the dynein regulatory complex, which regulates ciliary beat patterns. Importantly, we discovered that both C21ORF59 and CCDC65 were mutated in patients diagnosed with the human motile cilia disorder primary ciliary dyskinesia, identifying two novel human disease genes. Taken together, this work analyses multiple requirements for the assembly of motile and primary cilia and highlights the utility of the zebrafish system in investigations of cilia biology, particularly in the discovery and characterization of human disease genes. |
