Chemotaxis, the process in which cells detect a concentration gradient of a specific substance, interpret that information, and subsequently initiate movement towards the source is an essential... Show moreChemotaxis, the process in which cells detect a concentration gradient of a specific substance, interpret that information, and subsequently initiate movement towards the source is an essential part of many biological phenomena. It___s central to the processes in wound healing, in immune defense and in the formation of a viable embryo. In this thesis I used the well characterized social amoeba Dictyostelium discoideum to investigate, in depth, the dynamics that govern the first steps in the detection of a chemical gradient. D. discoideum detects cyclic adenosine mono-phosphate (cAMP) by a special receptor protein, cAMP receptor 1 (cAR1). Inside the cell this receptor activates a G protein which subsequently initiates a complex signaling cascade. Using fluorescence single-molecule microscopy I investigated the movements of both cAR1 and its associated G protein. During chemotaxis both proteins show striking differences in mobility between the leading and trailing edge of the cell. Those differences are presumably key to our understanding of gradient sensing by cells that have been ignored in models so far. Show less
Membrane heterogeneity on the micro- and nanometer scale plays an important role for a large number of biological processes. In parallel to the conception of refined membrane models, new... Show moreMembrane heterogeneity on the micro- and nanometer scale plays an important role for a large number of biological processes. In parallel to the conception of refined membrane models, new experimental techniques to determine membrane microstructure were developed in recent years. Single molecule fluorescence has emerged as one of the leading technologies since it delivers the required spatial resolution and can be employed in living cells. In a complementary approach artificial model systems are used to study specific biophysical aspects of membranes in isolation and in a controllable way. In this thesis we show how phase separated artificial membranes can be used to gain fundamental insight into lipid composition based heterogeneity (Chap. 2) and membrane mediated interactions (Chap. 3). We demonstrate that those interactions can lead to lipid domain sorting (Chap. 4). Experiments with artificial membranes are complemented with live cell studies. We develop a robust analysis method for single molecule position data (Chap. 5) and use it to study the role of heterogeneity in cell signaling (Chap. 6). Finally, we show how protein cluster formation can be measured by counting single molecules in live cells (Chap. 7). Show less
Proteins and enzymes play a key role in all biological systems. Understanding the mechanism of biological functions and reactions in which proteins and enzymes are involved requires a detailed... Show moreProteins and enzymes play a key role in all biological systems. Understanding the mechanism of biological functions and reactions in which proteins and enzymes are involved requires a detailed characterization of protein structure and dynamics. Structure refers to geometrical structure, as a result of the local arrangement of amino-acid side chains, and electronic structure, in particular at the active site of proteins and enzymes. Dynamics refers to structural changes that proteins undergo to perform their function. The work reported in this thesis concerns both methodological developments and the application of electron paramagnetic resonance (EPR) to study protein structure and dynamics. To this end, both continuous wave (cw) and pulsed microwave excitation have been applied. In the research described in this thesis transition-metal ions, such as Cu(II) and Fe(III), and nitroxide spin labels have been used as paramagnetic probes. Show less
