Cilia and flagella extend from human cell surfaces and function in motility and signaling. Many disorders, including polycystic kidney diseases and primary ciliary diskinesia, result from loss or malfunction of cilia. Ciliary structure and function are highly conserved among animals and many unicellular eukaryotes, including the green alga Chlamydomonas reinhardtii, the premier model organism for the study of ciliary and flagellar structure and function. In both human and Chlamydomonas cells, assembly of cilia and flagella is accompanied by up regulation of genes encoding flagellar proteins. While little is understood about the mechanism of induction, Chlamydomonas has a unique combination of experimental tools for addressing this problem. Genes encoding Chlamydomonas flagellar proteins are synchronously induced following flagellar detachment either in large cultures or in multi-well plates, making biochemical analysis and large- scale mutant screens possible. Experimentally introduced DNA integrates throughout the Chlamydomonas genome making it possible to use selectable markers both to create mutations and to identify the mutated genes. The long-term goal of this research is to develop a better understanding of the mechanism of gene induction during ciliary and flagellar assembly using Chlamydomonas as a model organism. Identified transcriptional regulators will become candidates in the search for disease-causing mutations. The work in this proposal will test the hypothesis that activation of flagella-specific transcriptional regulators is required for deflagellation-induced gene expression in Chlamydomonas. A strain that upregulates a luciferase/flagellar dynein light chain (LC8) promoter construct following deflagellation will be mutagenized by transformation with a selectable marker and the transformants will be screened for defects in upregulation. The mutant genes will be identified and predicted transcriptional regulators will be further characterized. In an independent line of investigation, the LC8 promoter will be functionally characterized by generating mutations in the LC8/luciferase construct and reintroducing these mutant constructs into wild- type cells. The functionally important regions of the promoter will be used to purify DNA-binding proteins from cells assembling flagella. These proteins will be further characterized both biochemically and by knocking down their expression using RNA interference. Although Chlamydomonas has been the subject of many studies on gene induction during flagellar assembly, no proteins involved in this regulation have been identified. These experiments will combine the unique advantages of Chlamydomonas as a model organism with established techniques for studying gene regulation to overcome this barrier to progress in the field.
Cilia extend from the surfaces of cells to receive signals and facilitate movement. Since defective cilia cause human disorders affecting millions of people, the potential of this research to determine how genes are regulated to allow normal growth of cilia makes the proposed work of great importance to public health.
| Brown, Jason M; Cochran, Deborah A; Craige, Branch et al. (2015) Assembly of IFT trains at the ciliary base depends on IFT74. Curr Biol 25:1583-93 |
| Brown, Jason M; Dipetrillo, Christen G; Smith, Elizabeth F et al. (2012) A FAP46 mutant provides new insights into the function and assembly of the C1d complex of the ciliary central apparatus. J Cell Sci 125:3904-13 |
