Breaking the biological code

Abstract

Our present knowledge of the cell is the result of decades of cell biological research that has unravelled the biochemical reaction pathways and their compartmentalization in subcellular organelles. One of the biggest future challenges in cell biology and human medicine is to decipher the whole functional plan of a cell or a tissue (its biological code). How does the cell establish, organize and coordinate in time and space the myriads of different cellular functionalities involved, for instance, in migration, in the highly selective topologically confined cell-to-cell interactions during morphogenesis of tissues, organs and organisms? Moreover diseases are the result of the operation of large pathological molecular networks within cells and tissues. To detect and decipher these biological codes (entirety of all protein networks, also termed the toponome) of healthy and diseased organisms we must develop technologies that address the protein network structure and function directly in the cell and tissue in vivo/situ (in contrast to proteomics techniques, that rely on protein analyses ex vivo). Given, for example, the protein network architecture and function of synapses in the central nervous system (CNS), we are facing a significant bottleneck: Appr. 1,000 synaptic proteins have been found by proteomics defining the “average synaptic proteome”. However, while this number of proteins is extremely large, it is physiologically impossible that they are all expressed in every synapse. The important next step is to map all the protein clusters that are really expressed in individual synapses in the CNS to answer the question, which protein clusters are and which are not related to disease, and define their decisive role. Conceivably, the number of similar important problems to be solved in biology and medicine is quasi unlimited, but they all have in common that researchers rely on techniques allowing them to colcocalize a very large number of molecular components in the same biological structure. Traditional fluorescence microscopy using multiple dyes for colocalization studies is limited to the spectral isolation of five to maximal ten dyes (1). This is not sufficient to identify and explore large molecular networks in the identical biological structure: for example Zipf's law (2) – a power law and measure of the hierarchical architecture of molecular systems (3) – does not apply when the number of molecular components localized simultaneously is too low, e.g. <15, but applies when the number exceeds 45 (3). A powerful way to overcome this spectral limitation of traditional multicolor fluorescence microscopy and address molecular networks is to bleach a dye after imaging and re-stain the same or other structures in the identical sample with the same dye coupled to a tag having the same or another specificity, and repeat similar cycles with other tags many times resulting in multidimensional colocation patterns. In 1990 it was shown that 17 different proteins could be selectively colocalized in the same muscle tissue section by running many cycles of incubation, imaging and bleaching (4). Since this first demonstration of the feasibility and specificity of “re-staining” for the identification of more than 50 cellular phenotypes at a tissue site, the importance of re-staining techniques has been increasingly recognized (5-12). For example, investigators have combined laser scanning multicolor analysis and localization of various CD markers by performing 3 sequential cycles of de-and re-staining (7). Similar approaches were reported for the analysis of peripheral blood mononuclear cells with the emphasis that the non-consumptive nature of re-staining techniques is superior to flow cytometry (5, 6). A re-staining technique was used to colocalize 9 cellular marker proteins resulting in the identification of a new cellular transdifferentiation mechanism in skeletal muscle regeneration (12), a finding that has been confirmed by in vivo