Genetic Analysis of Centrioles and Cilia

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Project Summary Cilia and centrioles are evolutionarily conserved organelles that require over 2000 proteins for their assembly and function. We use the unicellular alga, Chlamydomonas, to study these organelles. Chlamydomonas allows us to use both haploid and diploid mutant strains obtained from unbiased forward genetic screens together with outstanding genomics, biochemistry and microscopy to gain knowledge about these organelles. The conservation of these organelles allows us to translate our findings to other organisms where it is harder to do biochemistry and or genetic screens. The goal of my lab is to understand how these organelles are built and function. As we and others have shown, ciliopathies can exhibit a wide range of phenotypes in people that include retinal degeneration, polydactyly, cystic kidneys, diabetes, obesity, respiratory clearance defects, shortened bones, congenital heart disease and infertility. Defects in centrioles result in microcephaly and dwarfism. Duplication of centrioles once per cell cycle is key; many cancers have more than two centrioles and form transient multipolar spindle structures with more than two poles that can generate aneuploidy. I propose to address three questions using genetics, genomics, biochemistry, and microscopy. We want to know how inner dynein arms are correctly placed. How do they find the right address in the cilia? How are inner dynein arms assembled in the cytoplasm? Using genes that we identified in patients with primary ciliary dyskinesia, we are examining proteins that are needed to first assemble and then dock the large megadalton inner dynein arms at the right address so that a functional waveform is produced. We will also perform a large-scale mating scheme to ask if digenic inheritance can identify genes involved in dynein arm assembly and function. We will use proteomics and cryo-EM tomography to characterize these mutations. We want to explore new and novel pathways for regulating the precise duplication of centrioles; what redundancies are present to guarantee only two centrioles per cell? We think that cells have layers of control of centriole duplication. We will determine if splice site isoforms of centriolar proteins sequester other centriolar proteins to regulate duplication. We will use proximity mapping to ask if the mother centriole provides another level of regulation via a unique licensing site on specific triplet microtubules. We want to examine the role of the ciliary necklace, which is a unique membrane compartment at the transition zone; does it may play a role in membrane protein trafficking and/or in production of extracellular vesicles? Using mutants that have a greatly reduced ciliary necklace, we will ask if extracellular vesicle production is altered and what proteins make up the cargo of these vesicles.