Spatiotemporal organization of proteins in living cells

Fluorescence microscopy is a well established tool to study dynamic processes in vitro as well as in live cells and living organisms. Our lab employs a wide-range of different fluorescence microscopy approaches to quantify processes in different biological systems.

To understand how cells respond to extracellular cues, detailed knowledge about the underlying signaling network is crucial. In the past, much work was performed to assemble the individual components of such signaling networks and their interactions. Increasing knowledge about these signaling networks has led to the realization that the spatiotemporal organization of signaling proteins within cells is non-random and highly important for reliable signal transduction. The aim of research in our group is to characterize these dynamics of signaling proteins at the single-molecule level in individual, living cells. Ongoing projects in our lab include the analysis of signal transduction pathways in bacteria as well as in eukaryotic cells.

We use the prokaryotic chemotaxis system as a model for signal transduction. Particularly, we measure the frequency of binding of single CheY molecules to its interaction partners. Studies at the single-molecule level allow us to extract the kinetics of individual binding processes. In eukaryotic cells extracellular signals are processed via receptors at the cell surface, which in turn get activated and translate the external signal into a cellular response. The temporal characteristics of intracellular signaling induced by active receptors are controlled by the kinetics of several processes: receptor internalization and intracellular trafficking, intracellular inactivation of receptors, and the degradation and the recycling of receptors and ligands. By studying the dynamics of intracellular receptor transport at the single-cell level, we were able to analyze cell-to-cell variability in these processes which is not accessible to bulk assays at the population-level. Furthermore we investigate the dynamics of signaling proteins within the cell after receptor activation by extracellular ligands. Here, we especially focus on binding kinetics of different transcription factors before and after stimulation and on the mobility of these proteins in different sub-compartments of the cell. Among other techniques, we employ multicolour total internal reflection microscopy (TIRFM), fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET) and confocal imaging in our studies.

A well-designed and -characterized labeling approach is critical for any fluorescence microscopy-based experiment and many different technologies to label specific (bio)-molecules with fluorophores have been developed over the past decades. Due to the wide range of applications and the diversity of labeling approaches, there is no general approach suited for every application but the choice of fluorophore and labeling procedure has to match the experimental system as well as the biological system under study. We are mainly using fluorescent proteins (FPs) and enzyme-based covalent labeling strategies with synthetic fluorophores (SNAP/CLIP/HALO-tag) for live-cell studies. While FPs are easy to use and inherently circumvent problems with non-stoichiometric labeling, their use is often limited by their relative low photostability. A number of different systems employing enzymatic reactions for covalent labeling with synthetic fluorophores have recently been introduced. All of these methods allow using bright and photostable synthetic fluorophores for live-cell studies which largely simplify single-molecule studies in a live-cell environment. At the same time, these methods require careful evaluation of the labeling procedure since non-stoichiometric and unspecific labeling can significantly influence the outcome of an experiment.