Working in the Hill Lab at UCLA

Hi Everyone!

My name is Jason Melehani. I just joined MARC this summer so I haven't had the opportunity to meet many of you but I look forward to being able to. I am spending this summer at UCLA working in Dr. Kent Hill's lab in the MIMG department. I have actually work with Dr. Hill sense Fall 06 and love the research and the people in the lab. I am being trained by a post-doc named Pius Kabututu. Our lab is studying the parasitic eukaryotic Trypanosoma brucei. Specifically we are looking at the flagellum and motility of the parasite. We have many exciting projects going on right now in our lab including cryoelectron microscopy and tomography of flagella, social motility and chemotatic experiments, and isolating complexes to identify their components. I am working on two projects which focus on the regulation of motility through the central pair apparatus, radial spokes, dynein regulatory complex and dynein motors.
For the first, I am isolating the flagellar skeleton from the rest of the cell body by using a combination of detergent and salts. Most of the work comes in verifying the purity of this extraction by western blotting and electron microscopy. Once satisfactorily enriched for flagella, I will send samples of wild-type and two mutants (a central pair apparatus mutant and a dynein regulatory complex mutant) to collaborators in Maryland for iTRAQ mass spec analysis. iTRAQ is a system of tags used to quantitatively compare different samples and has been very valuable in seeing altered regulation in cancers and in identifying post-translational modification differences.
The second project is a little more exciting because it is my own design. I had previous done work on identifying the components of the dynein regulatory complex by immunoprecipitation of the only known component, trypanin. This was successful in showing that a protein, CMF70, interacts with trypanin. However, the current extraction protocol for solublizing the dynein regulatory complex is probably too harsh and likely rips apart most of the complex. There are thought to be at least 7 proteins in this complex but without a more efficient system of isolating these proteins we are unable to identify them. So my new project will hopefully overcome that problem. I will be using a recently developed system (2005) of in vivo crosslinking of proteins through endogenous incorporation of UV reactive Photo-Leucine and Photo-Methionine. While this system has worked in mammalian cells, it has never been tried in trypanosomes. Hopefully, the trypanosomes will use the UV reactive amino acids in building all their proteins. Then, when I expose them to UV light the amino acids will cross link adjacent proteins at the protein interaction interface. Because the proteins will be covalently linked, the extraction buffer will not break them apart and I will be able to isolate the complex and identify the components by mass spectrometry.

Sorry for that long winded summary of my research. Besides lab work, life in LA is great. The weather has been wonderful every day and the water is cool. Today there was an earthquake but luckily no damage occurred on campus.

I hope everyone is having a wonderful summer and I look forward to meeting you all soon!
Jason Melehani