On a cellular level ageing is characterized by the accumulation of toxic ageing factors over time, which eventually leads to cell death. To cope with this accumulation the cell has several cellular mechanisms which retain and/or clear ageing factors, such as damaged proteins, from the cell. How cells achieve this is largely unknown. We want deepen the understanding of ageing by creating a large-scale kinetic models of ageing and ageing related processes. For this, we want to study how damaged proteins occur over time and how they are processed in the cell.
Currently, the conventional experimental techniques allow studying ageing on a population level only. Therefore, the dynamics of the ageing process of a single cell throughout its lifetime is largely unexplored and many age-related processes are obscure. Novel technologies which could allow lifespan analysis of single cell and its progeny are available. At present, these techniques are unused in the field of ageing research. Therefore we seek with this project to revolutionise the field of ageing research by using state-of-the-art technologies which will give an unprecedented view on the process of ageing.
A methodology to study aggregated proteins is the use of protein constructs which are prone to aggregation. A typical example is CPY*, a mutated and misfolded secretory enzyme, which aggregates under different proteotoxic stress conditions and can be targeted to several cell compartments . Initially, we will then also use this protein construct to study the diffusion of damaged proteins inside the cell.
Cells expressing this construct will be placed in a microfluidic system under the microscope which will allow us to control the cell environment. This control will be used to incubate the cells in specific conditions, such as certain concentrations of ROS agents H2O2, which leads to increased formation of damaged proteins. Imaging will be done every 10 min in order to follow protein diffusion of CPY* in real time. The imaging will be done over 3 days. This will give a considerable amount of data that will need to be analyzed.
Finally we will also use mathematical modeling and simulations on the single cell and the , that help us to describe and analyze different molecular processes connected to the research questions.
We therefore apply for computer resources to do segmentation and analysis of the acquired microscopic images and for mathematical simulations.