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Telling new from old – visualization of newly made proteins in a cell
March 2015. Cells carry out specialized functions. Some cells in the pancreas, for example, are programmed to produce insulin whereas brain cells are responsible for sensing, perceiving and acting on environmental stimuli. This “specialization” of cells occurs not by different genetic material (this is more-or-less the same in all cells) but rather by the synthesis (assembly) of distinct sets of proteins. Protein synthesis is a dynamic and fundamental process exploited during the development and the plasticity of all cells to tune the kind and amount of proteins cells need to respond effectively to internal and external demands. Aberrations in protein synthesis are integral to diseases as diverse as cancer and mental retardation. In recent years, we have discovered that not only the rate but also the site of a protein’s synthesis within a cell is of importance. This is especially true for neurons with their long information receiving and sending extensions from the cell body. Synthesis of proteins near synapses – communication sites between neurons often located far away from the cell body – has been shown to be important for cellular processes associated to learning and memory. However, the identification and visualization of identified newly synthesized proteins has not been possible directly in a cell. Now Susu tom Dieck and Lisa Kochen with others from the Schuman lab have developed a strategy to visualize within cells specific recently synthesized proteins with both spatial and temporal resolution.
Erin Schuman (photo: Winfried Denk)
The strategy uses a clever combination of several techniques: the identity of a protein of interest is probed with a specific antibody. To identify the protein as recently made the researchers used either of two different labeling techniques: for the first method they applied an artificial amino acid resembling the natural amino acid methionine to the growth medium. The cells are fooled by this trick and incorporate the artificial amino acid into new proteins. The fact that the artificial analog harbors a small chemically reactive group allows the researcher to later modify the newly synthesized proteins in a chemical reaction – e.g. by attaching a tag that is recognized by another antibody. Alternatively newly made proteins were labeled by low concentrations of the antibiotic puromycin that is detectable by puromycin-specific antibodies.
To detect the coincident binding of the two antibodies - a protein specific antibody and one for the newly synthesized tag – the team used a proximity-ligation assay. Only close spatial proximity of the antibodies leads to a puzzle-like assembly of small DNA molecules in a way that allows subsequent amplification of the signal. The amplification product creates hundreds of binding sites for fluorescent probes and this can then be detected under a microscope as a bright colored spot. The strategy can be used to visualize identified newly synthesized proteins within minutes or hours of their synthesis and to track their position within cells over time. This technique can be used to study any newly synthesized protein in any cell, potentially leading to an understanding of events that lead to memory formation or even contribute to diseases, such as cancer. More...
Contact:
Erin Schuman, Max Planck Institute for Brain Research, Max-von-Laue Str. 4, 60438 Frankfurt am Main, Germany, e-mail: erin.schuman(at)brain.mpg.de, tel. ++49 69 850033-1001
Publication
S. tom Dieck, L. Kochen, C. Hanus, M. Heumüller, I. Bartnik, B. Nassim-Assir, K. Merk, T. Mosler, S. Garg, S. Bunse, D. A. Tirrell, E. M. Schuman (2015) Direct visualization of newly synthesized target proteins in situ. Nature Methods, published online on 16 March 2015. Link