Thousands of diverse proteins interact giving life to cells. Understanding such interactions involves on the one hand answering what proteins can do in well-defined in vitro studies containing only one or a few macro-molecular species, and on the other hand answering what proteins actually do in in vivo studies inside the largely unknown environment of the live cell.
The technology for studying such interactions falls under the field of Cytometry, a family of methodologies for measuring the molecular and structural properties of large numbers of cells either suspended or attached to a substrate. Cytometry has the unique advantage of revealing heterogeneities from cell to cell. With respect to molecular interactions, capabilities in the field are continuously advancing in terms of sensitivity, speed, spatial resolution, fluorescent labeling and automation. Consequently, molecular interaction studies are increasingly quantitative, and increasingly at the single molecule level thus revealing molecular heterogeneities within and across cells. Hence, a special issue of this journal, the premier journal on Cytometry, about molecular interactions is particularly pertinent and timely.
The issue begins with an historical perspective of fluorescence microscopy by Malte Renz that ends with an overview of the state-of-the-art technologies for acquiring and analyzing images of molecular dynamics. In a second review, Ammasi Periasamy and co-workers explain the well-established but still advancing technique of Förster resonance energy transfer (FRET) for measuring molecular interactions on the scale of 1 to 10 nm. Since FRET is currently the major technique for interaction analysis, a significant proportion of the special issue is centered around this topic. One of the pioneers of biological FRET, Robert Clegg, passed away during the compilation of this issue, and we would like to devote this collection of papers to his loving memory.
The Jovin lab has contributed their work on investigating dynamic conformational transitions of the EGF receptor upon ligand binding in living cells using fluorescence lifetime imaging FRET microscopy, while the Houtsmuller group presents a multi-parameter FRET imaging assay that reveals different stages of ligand induced androgen receptor activation. Then Szalóki et al. advance FRET technology for high throughput analysis of slide-supported cells, with validation using confocal microscopy and flow cytometry FRET techniques. In keeping with technological advancement, Poulsen et al. report how cytochalasin B improves over paraformaldehyde as the immobilizing agent for fast moving subcellular organelles like plant Golgi stacks when making FRET measurements using fluorescent proteins.
Katalin Toth, Jörg Langowski, and their colleagues guide us from ensemble FRET into single molecule analysis with an in vitro study of the opening of the nucleosome structure. In keeping with the quest to quantify oligomolecular complexes, Gerhard Schütz and co-workers take a different approach, protein micropatterning, to quantify the binding interaction of Lck and CD4 in cell membranes. In a third study aiming to see molecular patterns at higher than classical microscopic resolution, the Widengren group utilizes the emerging technique of stimulated emission depletion (STED) microscopy to analyze the spatial organization of adhesion sites between the cells and the extracellular matrix along with the intermediate filaments of vimentin, with data indicating this approach as a potential predictor of the metastatic characteristic of tumor cells.
Finally two papers focus on molecular dynamics both utilizing the technique of fluorescence correlation spectroscopy (FCS). In one, the Engelborghs lab applies the combination of FCS, FCCS and FRAP to conclude that the PDZ protein syntenin 2 requires the nuclear lipid, PIP2 to interact with practically immobile structures like chromatin. In the second, Malte Wachsmuth and his colleagues take FCS and combine it with point FRAP to achieve a more accurate analysis of simultaneous diffusion and binding processes.
The field of molecular interaction analysis inside and outside cells is accelerating. We hope this issue provides a solid introduction to the field and stimulates new technical advances and cytometric applications that augment our understanding of molecular mechanisms driving cell behavior.