This dissertation aims to advance understanding of amoeboid single cell migration under the influence of changing physicochemical environmental properties. The particular focus is on the relatively... Show moreThis dissertation aims to advance understanding of amoeboid single cell migration under the influence of changing physicochemical environmental properties. The particular focus is on the relatively unknown influence of differing physical properties in the extracellular environment, such as the migratory response to changes in confinement and availability of space (topotaxis) or to locally varying material properties like stiffness (durotaxis) and deformability. The experiments discussed in this dissertation are performed in especially designed in vitro cell migration assays, that provide the necessary control over the chemical and physical stimuli presented to the cells. Although experiments are performed outside natural cell environments, where possible, certain stimuli are combined to asses the relevance of one cell guidance cue to another. The cell lines used to asses the influence of various physicochemical properties vary from simple unicellular organisms like amoeba, often used to model movement of more complex metazoan cells, to human cancer cell lines that employ the same amoeboid movement to invade tissue. Show less
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
The behavior of single G-protein coupled receptor molecules were studied with single-molecule microscopy in the plasmamembrane during Dictyostelium discoideum chemotaxis. The mobility of the... Show moreThe behavior of single G-protein coupled receptor molecules were studied with single-molecule microscopy in the plasmamembrane during Dictyostelium discoideum chemotaxis. The mobility of the receptor was different in the anterior and posterior regions of living cells migrating towards the source of chemoattractant. This difference of mobility can account for an amplification of the extracellular signal, necessary for chemotaxis. It was also found that the receptor molecules are internalized. Show less
