Investigating Cell Polarity Through a Screen for Small GTPase Interacting Proteins
Speaker: Derek Rains
During early embryonic development, zygotes of the brown alga Fucus distichus develop a growing cell tip, which is oriented away from the display of a nearby unilateral light source. Development of polarity in the cells provides both a direction for zygotic growth as well as an orientation for the first plane of cell division. Localized in the growing tip of the polarizing zygotes is the Fucus protein FdRacA, which is a small GTPase. FdRacA is likely to be a signaling molecule that aids in the establishment of cellular polarity, participating in the signal transduction pathway that allows the cell to orient polar growth with respect to the light signal. Therefore of particular interest to the understanding of the pathway is the identification of Fucus genes encoding proteins that specifically interact with FdRacA small GTPase. Candidate interacting proteins could either act upstream of FdRacA by assisting the conversion of the protein to its active GTP bound form, or may act downstream of the active FdRacA to trigger cellular processes that lead to polar growth. To investigate these possibilities, I used a yeast two hybrid screen to isolate over 120 candidate interactors. The yeast two hybrid screen works on the premise that only FdRacA interacting proteins from a Fucus cDNA library will be selected for by growth of transformed yeast colonies on a selective medium. Analysis of the DNA sequence of these candidate interactors has identified several proteins for further study including a likely trans-membrane protein and a myosin like protein.
One of the most interesting candidates isolated in my screen was a gene fragment that showed sequence similarity to Notch, a trans-membrane receptor protein that acts in signal transduction and development in animal cells. The specific sequence present in this protein, designated FNL1 (Fucus Notch-like 1), and animal Notch proteins lies in a series of three NL repeats. These repeats are composed of a consensus sequence conserved for all Notch like proteins, and they provide a specific calcium-binding motif that is crucial in the formation of protein structure, and in Notch function. This consensus sequence can be clearly seen in FNL1, suggesting that FNL1 could also bind calcium. This provides an exciting potential link to another signaling component in Fucus cells, in that calcium also localizes in the growing cell tip and is essential in the early establishment of growth and cell tip formation. Interaction between a calcium-binding FNL1 and FdRacA could provide an important mechanism assisting in the establishment of polarity, as well as an exciting find in that no Notch-like receptor protein has been found in plants to date. I am currently working to further investigate this possibility, by isolating a larger FNL1 clone for sequence analysis and eventual biochemical characterization.