Novel (Bio)chemical and (Photo)physical probes for imaging living cells

The living cell mediates its internal state and the exchange of substances and information with its environment primarily via protein-protein interactions. The spatio-temporal disposition of structural, catalytic, and regulatory proteins defines the nature and functional state of the cell. Signaling mechanisms, as a prominent example, occupy a central role in this process, leading to a set of canonical questions, challenges and strategies (Table 1). In applying fluorescence microscopy in cell biology to a particular system, one is faced with a multiplicity of molecules at every level of organization (external, membrane, cytoplasm). The elucidation of such an extensive degree of vertical and horizontal networking, extending into the downstream signaling cascades, requires imaging technology in addition to the classical biochemical and molecular biological methods based largely on classical "divide (separate) and conquer" protocols (Table 2). For example, the "orphan" (ligand-less) erbB2/HER2 receptor tyrosine kinase (RTK) is overexpressed and highly activated in a large fraction of breast tumors, forming characteristic homo- and heterodimers with three other members of this RTK family1. These are targets for the only anti-tumor immunotherapies in present clinical use, exemplified by the antibody specific for HER2, Herceptin2. Unfortunately, the modes of action of such agents are poorly understood. Thus in order to elucidate the repertoire of the RTKs under normal and pathological conditions one must evaluate their localization and molecular structural and functional state(s) in defined cell populations be it cell culture lines or primary patient-derived cells. The thermodynamic and kinetic complexity is evident from the minimal scheme defining the interplay between ligand binding conformational states (2) and association states (2) for a prototypic growth factor receptor (Figure 1). Although Table 2 cannot be regarded as comprehensive it emphasizes that in addition to established biochemical and genetic approaches physico-chemical techniques offer the versatility required for assessing molecular interactions in the cell. In particular fluorescence unites the features of great sensitivity and selectivity with high contrast even under conditions of low local molecular density i.e. concentration.