Current Master and Bachelor research projects (update fall 2016)
Analysing folding-driven secretion of hemoglobin protease using AFM
Haemoglobin protease (Hbp) is an autotransporter protein produced by Escherichia coli that causes intra-abdominal abscesses. It degrades hemoglobin and delivers heme to both E. coli and Bacteroides fragilis. The secretion of Hbp across the outer membrane is an ATP and proton gradient-independent process that is believed to be driven by sequential folding of the protein at the outer membrane surface. The main goal of the project would be to unravel the β-helical structure and explored the unfolding landscape of Hbp by analysing and data from atomic force microscopy (AFM). This is an established and powerful technique to study intermediate protein structures (see Figure below for an outline of the method). In the project, you will analyse the force extension profiles generated by the AFM, and aim to establish the unfolding barriers of Hbp. Additionally, you compare the experimental data, to curves generated by computational model, both full atomistic and more coarse grained models are available. By the end of the project, we will be able to model and unravel, which interactions between residues give rise to unfolding barriers which play a critical role in secretion mechanism of Hbp protein. During this project, you can opt to generate the AFM data, further develop the computational analysis, improve the current models, or a combination of these depending on the duration of your internship.
Vesicle Mechanics and Endocytosis- What’s the connection?
Extracellular vesicles (EVs) are found in all body fluids and play an important role in cell-cell communication by being able to fuse with target cells while carrying and transferring various signaling molecules. They are also involved in several types of diseases. Several studies suggest that the mechanical properties of particles play a major role in their cellular uptake. However, a systematic investigation of this effect is still lacking. This research project addresses the hypothesis that mechanical properties of EVs affect the efficiency and kinetics of their uptake by cells. This will be addressed on two fronts: by assessing mechanical properties of natural vesicles, as well as preparing synthetic vesicles with tunable mechanical properties. During this multidisciplinary project, you will learn how to prepare lipid vesicles and how to isolate natural EVs, how to measure their mechanical properties using AFM force spectroscopy and how to analyze your data using Matlab program. A better understanding of the link between EVs mechanical properties and uptake rate and mechanism will provide insight into fundamental biological processes mediated by vesicles, e.g. the release of neurotransmitters in synapses, viral infections etc. In addition, your findings will facilitate the design of efficient synthetic exosomes for future therapeutic solutions.
Proteins diffusing on chromatin in vitro
The main objective is to study how proteins search for their target sites in chromatin, as a function of the three-dimensional conformation of chromatin. The experiments should look similar to those performed in vd Broek et al. PNAS (2008): a single reconstituted chromatin fiber tethered between two beads, coupled to a fluorescence correlation spectroscopy detector, with fluorescently labelled proteins searching for their target site on the chromatin. By measuring the FCS of the labelled proteins, one should be able to explore how the chromatin environment affects the search process, by varying the tension applied to the chromatin fiber. For small tension, the configuration of the chromatin is coiled, while for higher force it is extended. What is the difference between the search process in a coiled configuration and in an extended configuration? In fact, here it would be possible to analyse the 3D chromatin conformation alone, without any contribution given by macromolecular crowding, which also has been shown to be important.
Probing tether formation in cells by AFS
The membrane of a red blood cell (RBC) is tightly attached to an underlying skeleton, a pseudo-hexagonal meshwork of spectrin, actin and other proteins. This connection is crucial for RBC function, their durability and ability to survive in circulation. Disassociation of the red blood cell membrane skeleton from the lipid bilayer is associated with pathological conditions such as sickle cell disease. When a small and localized force is applied on a cell membrane, the membrane and the underlying cytoskeleton can deform, and upon further pulling the membrane can delaminate from the cytoskeleton. If the membrane delaminates from the cytoskeleton, it may be further extruded and form a membrane tether. The forces required for tether extraction and tether dynamics are a useful tool to study membrane properties. During this project, you will study membrane-cytoskeleton interactions in RBC and other cell types by pulling membrane tethers from cells, utilizing the novel Acoustic Force Spectroscopy (AFS) technology. This multidisciplinary project will involve cell culture, surface functionalization, designing and performing experiments in the AFS setup and data analysis.
Nanomotors on DNA
DNA-processing molecular motors are refined nanomachines that move along DNA to process the genetic code. This project exploits quantitative single-molecule methods to study the physics of one of the most important of these molecular motors, the DNA copying motor DNA polymerase. DNA polymerase copies DNA rapidly, and with extraordinary accuracy. Exactly how it accomplishes this is not well understood. Key to unraveling its activity lies in its dynamics: the motor is dynamically assembled from multiple parts, it can operate in a forward DNA-copying mode as well as in a backward error-correction mode, and it can switch dynamically between these modes.
We have developed unique single-molecule instrumentation that can directly observe the motion and activity of single DNA-processing motors in a controlled, reductionist environment outside the cell. This instrumentation consists of force-measuring optical tweezers to hold and mechanically manipulate a single DNA molecule, combined with (super-resolution) fluorescence microscopy to directly visualise an individual motor and its components while it is copying DNA. This approach allows unraveling the physics of these biological nanomachines and obtain a deep understanding of the molecular foundations of DNA replication and repair. With motor proteins, DNA, and instrumentation ready and available, the student can focus on the biophysical experiments that involve DNA mechanics and fluorescence visualization.
In our lab we study the fundamental properties of the main information carrier in biology, the DNA. With highly focused laser beams we can trap microscopic objects and apply forces on them. These so called optical tweezers enable us to measure the mechanical properties of DNA and with fluorescence microscopy we can see the interactions of proteins with the DNA. In cells however, DNA is not a “bare” molecule, but is tightly compacted by a large amount of proteins into a structure called the chromosome. To transfer information from mother to daughter cells, one copy of each chromosome has to go into the two daughter cells during cell division. This is when the famous X-shaped chromosomes appear, in which the two copies of the DNA are still attached to each other at the crossing point called centromere. There are estimates that during cell division forces as high as 500 pN act on the centromere. To compare, the double helical shape of bare DNA already gets lost at forces of about 65 pN. It is a key question in biology how chromosomes can withstand such high forces.
In this project, we aim to measure the mechanical properties of chromosomes. We have a collaboration with a highly specialized cell biology group that supplies isolated chromosomes from a human cell line. Our aim is to attach these chromosomes to microscopic particles in order to trap them with the optical tweezers set up and measure forces along the centromere of the chromosome.We are looking for someone that is highly motivated to play a key role in developing this method and performing experiments on chromosomes which may include: the first direct force measurements on centromere labeled chromosomes, addition of proteins that are known for their function during cell division, 3D STED super resolution imaging of mitotic chromosomes, among others. You will learn techniques like optical tweezers, fluorescence microscopy and microfluidics. You will get hands on experience in working with biological materials such as DNA and proteins. Moreover, you will work in an international team where you will learn to collaborate with people from different backgrounds.