Patterning of Transcription Factor Activity in T cells During Influenza Infection

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This is the first year of the lab setup. Lab renovations were completed on the February 1st 2008. As mentioned in the goals and objectives of the project we are interested in understanding the temporal dynamics of transcription factors in response to TCR stimulation. We will be performing these experiments in primary mouse T cells from TCR transgenic mice. We will be setting up a glass supported lipid bilayer system to perform these stimulation experiments. Purified proteins are incorporated in such bilayers such that they are free to diffuse. Such substrates are ideal for quantitatively controlling the ligand densities as well as ligand types. We will be incorporating proteins such as MHC class II, ICAM-1, CD80 and CD48. Such bilayers are ideal for optical imaging and studying molecular interactions that take place between cell-cell contacts. These proteins are either expressed in mammalian cells as GPI-anchored proteins or in insect cells as histidine tagged proteins. GPI-anchored proteins are incorporated in phospholipid liposomes by method of dialysis. These liposomes can fuse with a acid cleaned glass surface to form a glass supported bilayer. Ni-NTA lipids are also incorporated in liposomes which can be used to form bilayers. Histidine tagged proteins are made to interact with Ni-NTA lipid bilayers. The proteins will be purified using affinity chromatography. To this end we have setup tissue culture to grow large amounts of mammalian cells. We purchased two carbon-dioxide incubators to this end. We have also purified several antibodies from hybridomas. We have grown several grams of cells for purifying each of these proteins. We have also setup a system to grow insect cells. To this end we have purchased to stacked shaking incubators. Soluble MHC class II molecules purified from insect cells will be loaded with specific peptides in solution and the separation of free peptide with loaded protein will be done by subjecting the protein to gel filtration using FPLC. To this end we have purchased a FPLC machine. To make liposomes out of bilayers we need to treat the lipids under high vacuum for which we have purchased a lyophilizer. To visualize the transcription factors we will be genetically tagging them to different fluorescent proteins. NFATc1 will be tagged to CFP, c-Fos and c-Jun will be tagged to YFP and p65 subunit of NF-kB will be tagged to tag-RFP, a monomeric version of red fluorescent proteins. Molecular biology work is underway to produce these constructs. These constructs will be introduced in primary T cells using electroporation using kits developed by a company called Amaxa. We have purchased the electroporator developed by this company. We have setup a state of the art fluorescence microscope capable of wide field fluorescence microscopy, total internal reflection fluorescence microscopy (TIRFM), spinning disc confocal microscopy and fluorescence correlation spectroscopy. Wide field fluorescence microscopy will allow us to perform calcium measurements to test the functionality of the T cell interaction with the bilayers. TIRFM will allow us to visualize single peptide loaded MHC class II molecules and also cytoplasmic signaling molecules that get recruited to activated receptors. Spinning disc confocal microscopy will allow us to follow the spatial and temporal dynamics of transcription factors in single cells. And lastly, fluorescence correlation spectroscopy will allow us to study the dynamics of individual subunits in multi-subunit receptor systems. This microscope is also equipped with 5 lasers (wavelengths 405, 440, 488, 514, 561, 640 nms), an automated XYZ stage and incubator systems to keep cells at 37 degrees. All components of the microscope are controlled via software allowing precise control of the experiments. Individual components of this microscope were purchased, assembled and tested. Using a combination of these different techniques we will start understanding the spatial and temporal dynamics of transcription factors.