Imaging Newly Synthesized Proteins Or Special Lipids Or Acids In Cells

As the direct participant in and executor of various life functions, proteins play a great role in retaining the balance of cells and in sustaining different cell activities. A further exploration into the dynamic process between proteins and other small molecules such as lipids and fatty acid will facilitate a better understanding of the different roles of proteins in various life processes. Studies have also shown that many diseases like cancer, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) are closely related to certain newly synthesized proteins. Therefore, to spatiotemporally visualize the dynamic process between newly synthesized proteins and certain small molecules and to image the distribution of certain newly synthesized proteins are of great importance in understanding and curing particular diseases. Despite the fact that the normal fluorescence imaging mode is satisfactory in imaging newly synthesized proteins, it could not image newly synthesized proteins and other small molecules simultaneously, furthermore few progresses have been made in imaging specific newly synthesized endogenous proteins, and the only achievement was found to have too many fake signals. Therefore, it seems especially urgent and necessary to develop methods for visualization of newly synthesized proteins and lipids simultaneously or visualization of particular newly synthesized endogenous proteins. In this study, by combining the advantages of SILAC (or click technology) and TOF-SIMS (or fluorescence lifetime imaging mode), we established 2 analytical systems that could image newly synthesized proteins and lipids simultaneously and 1 system that could image particular newly synthesized endogenous proteins. The details are as follows:1. Simultaneous Imaging of Newly Synthesized Proteins and Lipids in Single CellWe demonstrate a new method which combines the advantages of SILAC and TOF-SIMS to label newly synthesized proteins during cell growth and division, and to image proteins and special lipids with subcellular spatial resolution. The sensitivity and signal-to-noise ratio of major characteristic peaks of arginine and lysine are compared. TOF-SIMS could quantify cellular newly synthesized proteins in different time periods by using characteristic peaks of 15N arginine and 15N lysine. Moreover, we simultaneously image sphingomyelin and cholesterol during the cell division, since TOF-SIMS is especially suitable for this kind of molecules. With the proposed strategy,2-dimension and 3-dimension images of proteins and lipids are obtained during cell division. This technique might be a valuable tool to study the molecular behaviours and their spatial distribution in single cell.2. Simultaneous Imaging of Newly Synthesized Proteins and Fatty acid in Single CellOn the basis of click chemistry, we develop a new method to image newly synthesized proteins and special fatty acid or nucleic acid simultaneously in single cell. Metabolic labeling of newly synthesized proteins with AHA, then connect azide with alkynyl bromine or iodine though the click reaction. As TOF-SIMS is highly sensitive to the features of certain fatty acids or nucleic acid, the method could be used to image newly synthesized proteins and special fatty acid or nucleic acid simultaneously. This novel analytical imaging method could be used to visualize dynamic process between newly synthesized proteins and lipids in certain activities of the imaged cells.3. Imaging Specific Newly Synthesized Proteins within Cells by Fluorescence Resonance Energy TransferWe develop a FRET-based strategy for imaging the newly synthesized endogenous POI within cells:a FRET acceptor is installed onto the newly synthesized proteins via click chemistry, and a FRET donor onto POI via immunocytochemistry. We found that photobleaching based FRET efficiency imaging mode and fluorescence lifetime imaging mode showed the distribution of newly synthesized proteins more accurately compared to direct observation of FRET signals mode. We demonstrated the capability of this FRET-based imaging method by visualizing several newly synthesized proteins including TDP-43, tubulin and CaMK II a in different cell lines. Furthermore, our imaging revealed the redistribution and turnover of newly synthesized proteins endogenous TDP-43 in the HEK293T cells at different intervals. This novel analytical imaging method could be used to visualize other special endogenous proteins of interest in situ.