A Study of a Mutation in Kinesin II and its Effects on Flagellar Function

William Alexander Edwards¹, Dr. Susan Dutcher², Biology Department, Washington University, St. Louis, MO^1, Department of Genetics, Washington University School of Medicine

Chlamydomonas reinhartii, or Chlamy, is a unicellular green alga that is found anywhere from in the soil, to the salt and fresh waters, to the most extreme conditions such as mountaintops. Because of its commonality, its quick growth and reproduction, and easily recognizable phenotypes, it is a great model organism on which to perform experiments. In the Genetics department, some scientists are currently doing research with Chlamy, and in particular, Susan Dutcher’s lab is researching the function of flagella and cilia in Chlamy, and applying the information that they learn to humans and other eukaryotic organisms. Mutations affecting the function of cilia and flagella in humans have severe results, including any combination of sterility, the reversal of visceral organs along the left-right axis, retinitis pigmentosa, polysystic kidney disease, and/or heart abnormalities. In retinitis pigmentosa, the functions of the rod cells in the retina are disrupted due to the deformed flagella, causing night blindness and later a loss of peripheral vision. In polysystic kidney disease, there is an overproliferation of the epithelial cells lining the tubules of the kidneys, causing deformation and loss of function. Sufferers of this disease, one in every thousand people, end up having to get kidney transplants due to these flagellar mutations. Mutations of flagella in Chlamy include, but are not limited to, baldness (no flagella), uniflagellar, variable flagella (three to eight flagella), and shortening or elongating of the two normal flagella. All of these will affect the motility of cells. In my research, I studied a specific mutation in one of the motor proteins of kinesin II. There are three proteins that make up kinesin II, which are Fla 3, Fla 8, and Fla 10. I studied a particular mutation in the gene FLA10, called fla10-14.

I sequenced most of fla10-14, and found a glutamate (E) to a lysine (K) mutation. The amino terminus of this kinesin contains the motor domain. E₂₄K is in an alpha helix. This glutamate is conserved in all kinesin II homologs from Giardia to humans, suggesting that it plays an important role in flagellar development. The mutation fla10-14 causes Chlamy to become immobile at 32⁰C after twenty-four hours because the cells have no flagella. I have also isolated revertants of this allele. These cells were mutagenized with UV light and several rounds of enrichment for swimming cells resulted in new strains that were flagellated. If time permits, I will sequence these revertants, and try to determine the type of reversion event that occurred. I may isolate intragenic or extragenic events. Among the intragenic revertants, the possibilities are: 1) true revertants, in which the lysine changes back to a glutamate 2) pseudo revertants, in which the lysine could be turned into some other acceptable amino acid and still retain function or a 3) pseudo revertant in which the lysine remains, but a change in another amino acid compensates, and allows Chlamy to swim. Hopefully future studies in Chalmydomonas will lead to a better understanding of mutations disrupting ciliary function, and possibly help create treatments for such disorders in eukaryotic organisms, especially humans.