Cells use membranes as their boundary, shielding their inside from the outside world, and to create internal structure. The different membranes in a cell have large variations in chemical... Show moreCells use membranes as their boundary, shielding their inside from the outside world, and to create internal structure. The different membranes in a cell have large variations in chemical composition, elasticity, shape and function. In contrast with the standard static picture often shown in cartoons, membranes are moreover one of the most dynamic components of the cell. Based on a detailed study of the structure and shape of various membranes we have developed techniques to measure the relevant physical parameters. Using these, we can directly couple the structure and shape to the function of the membrane. Combining these studies with studies of the membrane dynamics we find that membranes can spontaneously demix in different domains, which can interact with each other by forces mediated by the membrane itself. This interaction results in a sorting of the domains by size. Introducing an active element, molecular motors, into the system, we find that new structures are formed. An example of such a structure is a long membrane tube. These tubes also exhibit rich dynamics, and oscillating growth and shrink patters, which makes them suitable length and shape regulators in living cells. Show less
Key cellular processes such as cell division, internal cellular organization, membrane compartmentalization and intracellular transport rely on motor proteins. Motor proteins, ATP-based... Show moreKey cellular processes such as cell division, internal cellular organization, membrane compartmentalization and intracellular transport rely on motor proteins. Motor proteins, ATP-based mechanoenzymes, actively transport cargo throughout the cell by walking on cytoskeletal filaments. Motors have been studied in detail on the single motor level such that information on their step size, ATP turnover rate, stall force, average run length and processivity are well known. However, in vivo, motors are often found working together, raising the question of how motors work together in transport. In their native environment, motors are bound to membrane material so that they can diffuse through a lipid bilayer, suggesting that their collective behavior may rely more on dynamic self-organization than experiments until now have allowed. In this thesis, an in vitro approach is presented to study collections of motors as they self-organize to actively transport membrane along microtubule tracks. Motors are specifically attached to a membrane reservoir and when they encounter cytoskeletal tracks, the motors walk on the track and extract membrane tubes from the reservoir. We find that dynamic fluctuations in motor densities and effects such as cooperative binding are key players in the collective action of motors in membrane transport. Show less
Nucleosome Dynamics Resolved with Single-Pair Fluorescence Resonance Energy Transfer Spectroscopy Het oprollen van DNA in een nucleosoom is de eerste stap van DNA condensatie in de eukariotische... Show moreNucleosome Dynamics Resolved with Single-Pair Fluorescence Resonance Energy Transfer Spectroscopy Het oprollen van DNA in een nucleosoom is de eerste stap van DNA condensatie in de eukariotische celkern. Nucleosomen vormen obstakels voor enzymen die het opgevouwen DNA binden, en spelen om die reden een belangrijke rol in genregulatie. Om te begrijpen hoe de toegankelijkheid van nucleosomaal DNA wordt gecontroleerd, is het noodzakelijk om de moleculaire mechanismen te ontrafelen die hieraan ten grondslag liggen. Dit proefschrift doet verslag van een experimentele studie met behulp van enkel-paar Fluorescentie Resonantie Energie Overdracht Spectroscopie (single-pair Fluorescence Resonance Energy Transfer, ofwel spFRET). De technieken die we hebben ontwikkeld zijn met name geschikt om de dynamica te bestuderen van heterogene DNA-eiwitcomplexen. Met behulp van spFRET was het mogelijk om de structuur van het DNA in __n enkel nucleosoom te volgen in de tijd. Daarmee laten we zien dat nucleosomaal DNA spontaan loskomt van de histon kern. Doordat dit frequent gebeurt, is het DN A in nucleosomen toegankelijk voor enzymen op biologische relevante tijdschalen. Show less
Biopolymers are essential for cellular organization. They bridge the cell interior forming a framework that is used as a reference frame for different cellular organelles. Interestingly this... Show moreBiopolymers are essential for cellular organization. They bridge the cell interior forming a framework that is used as a reference frame for different cellular organelles. Interestingly this framework, called the cytoskeleton, is not static but constantly reorganizes. The dynamics of the cytoskeleton allow the cell to rearrange its interior for various processes, such as cell division. This dynamic reorganization relies, at least partly, on forces that arise from assembly and disassembly of cytoskeletal biopolymers. The work presented in this thesis focuses on microtubules, a particular biopolymer. In vivo microtubules regularly grow into physical boundaries, like the cell cortex or cellular organelles, where assembly and disassembly forces are generated. We study different mechanisms of microtubule force generation and the regulation of microtubule dynamics by these generated forces. The interactions with physical boundaries are assessed in minimal in vitro experiments that allow for systematic analysis of isolated mechanisms. In addition, simple computer simulations and mathematical modeling are performed to explain the experimental findings and to investigate the consequences of the findings for other in vitro and in vivo systems. Show less
This thesis is composed of two parts part one: The study on anti-estrogen resistance and defining criteria a cell has to meet in order to become resistant to anti-estrogenic compounds. part two:... Show moreThis thesis is composed of two parts part one: The study on anti-estrogen resistance and defining criteria a cell has to meet in order to become resistant to anti-estrogenic compounds. part two: the study of antigen-loading, vesicle positioning and costimulation. Show less
