Biological polymers, including proteins and the genome, undergo folding processes crucial for their proper functioning. Even slight changes in the folding structure of these biopolymers can have... Show moreBiological polymers, including proteins and the genome, undergo folding processes crucial for their proper functioning. Even slight changes in the folding structure of these biopolymers can have significant implications, leading to the development of various pathological conditions, such as neurodegenerative diseases and cancer. In this thesis, we leverage the theoretical framework of Circuit Topology and expand its application to real-world scenarios. By employing this approach, we quantify the folding patterns of biological polymers, offering valuable insights for detecting harmful misfolds. Furthermore, this research holds the potential to provide fundamental design principles for molecular engineering in the realm of pharmaceutical applications. 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
In bacteria, what __drives__ the process of cell division is unknown. Possibly, forces generated by an internal protein ring (termed __the Z-ring__) are responsible for the division process, but... Show moreIn bacteria, what __drives__ the process of cell division is unknown. Possibly, forces generated by an internal protein ring (termed __the Z-ring__) are responsible for the division process, but direct evidence is lacking. Here we describe the development of a method to measure forces in a single dividing bacterium. Using optical tweezers, forces can be measured on optically trapped micron-sized beads. To attach such a bead to a living bacterium, one of its outer membrane proteins is engineered to present a binding epitope on the cell surface. We show that it is possible to attach a bead to this __molecular handle__ via a DNA-molecule, and characterize the strength of this molecular construct. Finally, we show that genetic fusion of the surface exposed protein domain to an internal domain with mid-cell affinity can alter the localization pattern of the exposed domain. Our findings suggest that it is possible to create a fusion protein that exposes a binding epitope and localizes specifically to the division site. Show less
Microtubules are highly dynamic protein polymers that and are essential for intracellular organization and fundamental processes like transport and cell division. In cells, a wide family of... Show moreMicrotubules are highly dynamic protein polymers that and are essential for intracellular organization and fundamental processes like transport and cell division. In cells, a wide family of microtubule-associated proteins (MAPs) tightly regulates microtubule dynamics. The work presented in this thesis gives a high-resolution perspective on the microtubule assembly process and on the regulation mechanisms employed by representative MAPs. We studied dynamic microtubules outside cells, in a reconstituted minimal system. To follow microtubule growth with near molecular resolution, we developed a high-resolution technique that integrates optical tweezers, micro-fabricated rigid barriers and high-resolution video tracking of microbeads. Using this technique we found, for example, that microtubule assembly does not always occur by addition of single protein subunits, but multiple subunits could be incorporated at once at the growing end. XMAP215, a protein known to dramatically enhance microtubule growth, altered these molecular details. Another intriguing protein studied here is Mal3, a protein that is able to track growing microtubule ends. We found that Mal3 interacts differentially at the growing tip and on the rest of the microtubule, influencing all the parameters describing microtubule dynamics. In conclusion, our results give new insights into the microtubule assembly process in the absence and in the presence of regulators. Show less
This thesis describes experiments, in which we used an optical-tweezers setup to study a number of biological systems. We studied the interaction between the E. coli molecular chaperone SecB and a... Show moreThis thesis describes experiments, in which we used an optical-tweezers setup to study a number of biological systems. We studied the interaction between the E. coli molecular chaperone SecB and a protein that was being unfolded and refolded using our optical tweezers setup. Our measurements clearly showed that in the presence of SecB, an unfolded protein could not refold. Molecular dynamics simulations were used to successfully explain features that were observed in our unfolding experiments. Our approach enables studies on other chaperones, as well. Next, we aimed to study translocation of single proteins through membranes by the E. coli Sec translocase. We modified an often-used model protein for our experiment. Different used experimental strategies are presented. Future experiments should enable measurements on the translocation of a single protein. The last study was on the packaging of double-stranded DNA by a single bacteriophage phi29. We aimed to study the effect of multivalent cations on the negatively-charged, tightly-packed DNA inside the bacteriophage capsid and in that way on the speed of the packaging process. A special DNA molecule was constructed and used in a number of successful packaging experiments. Future experiments should show the effect of cations on the packaging rate. With Summary in Dutch Show less
This thesis describes experiments, in which we used an optical-tweezers setup to study a number of biological systems. We studied the interaction between the E. coli molecular chaperone SecB and a... Show moreThis thesis describes experiments, in which we used an optical-tweezers setup to study a number of biological systems. We studied the interaction between the E. coli molecular chaperone SecB and a protein that was being unfolded and refolded using our optical tweezers setup. Our measurements clearly showed that in the presence of SecB, an unfolded protein could not refold. Molecular dynamics simulations were used to successfully explain features that were observed in our unfolding experiments. Our approach enables studies on other chaperones, as well. Next, we aimed to study translocation of single proteins through membranes by the E. coli Sec translocase. We modified an often-used model protein for our experiment. Different used experimental strategies are presented. Future experiments should enable measurements on the translocation of a single protein. The last study was on the packaging of double-stranded DNA by a single bacteriophage phi29. We aimed to study the effect of multivalent cations on the negatively-charged, tightly-packed DNA inside the bacteriophage capsid and in that way on the speed of the packaging process. A special DNA molecule was constructed and used in a number of successful packaging experiments. Future experiments should show the effect of cations on the packaging rate. Show less
Membrane tubes are ubiquitous within cells. They have a diameter of approximately 50 nanometers, and are formed when a sufficiently large localized force is exerted on a membrane. Important... Show moreMembrane tubes are ubiquitous within cells. They have a diameter of approximately 50 nanometers, and are formed when a sufficiently large localized force is exerted on a membrane. Important generators of this force are the motor proteins that can move along cytoskeletal filaments. We studied membrane tube formation by motor proteins from giant vesicles in an in vitro reconstituted system, and showed that motor proteins can dynamically associate to form clusters that work together. In addition, the physical parameters that determine the force required to form tubes were examined, and it was found that the force barrier for tube formation increases with the area the pulling force is exerted on. Finally, some first results are presented on the competition between motor proteins of opposite directionality. Our findings suggest regulatory mechanisms that may be used for the intracellular spatial organization of membranes. Show less
