Periodic Reporting for period 3 - PArtCell (Physiologically Crowded Artificial Cells for Relevant Drug Screens)
Reporting period: 2022-07-01 to 2023-12-31
The cells are so densely packed with proteins and other molecules that the situation is comparable to a subway ride in a big city after work. This so-called "crowding" sounds like a lot of stress- but it is essential for the biochemical processes in the cell - and, thus, for its health: in this way, proteins and molecules get in contact and can interact. Such interactions as hydrophobic and electrostatic interactions or hydrogen bonds can lead to various chemical reactions that are essential for the cell to survive. However, we hypothesize that interactions caused by crowding could also be harmful and cause diseases such as Alzheimer's or Huntington's disease. Both are well-known progressive neurological diseases that cannot be cured yet.
Although crowding is essential, it remains a question of how the biochemical equilibrium in cells is controlled. We aim to map and understand these crowding effects with our project PArtCell (Physiologically Crowded Artificial Cells for Relevant Drug Screens). In addition, our goal is to develop physiologically relevant platforms that can be applied to screen new drugs for treating diseases such as Huntington's in these dense environments. We are developing approaches in natural and artificial cell systems that we will compare to each other to make the most relevant artificial cells possible. Compared to living cells, analyses of their synthetic counterparts have several advantages – for example, natural cells are influenced by many unknown parameters. In artificial cells, on the other hand, the initial conditions can be primarily defined and thus controlled.
Although crowding is essential, it remains a question of how the biochemical equilibrium in cells is controlled. We aim to map and understand these crowding effects with our project PArtCell (Physiologically Crowded Artificial Cells for Relevant Drug Screens). In addition, our goal is to develop physiologically relevant platforms that can be applied to screen new drugs for treating diseases such as Huntington's in these dense environments. We are developing approaches in natural and artificial cell systems that we will compare to each other to make the most relevant artificial cells possible. Compared to living cells, analyses of their synthetic counterparts have several advantages – for example, natural cells are influenced by many unknown parameters. In artificial cells, on the other hand, the initial conditions can be primarily defined and thus controlled.
In the first part of the project, we developed novel probes to better understand crowding in the cells and to follow toxic protein aggregates. We mapped crowding in cells under different stress conditions to better understand the possible window of crowding in living cells. We developed artificial cells from microfluidics to incorporate relevant crowding.
We achieved the following milestones:
- We developed a method to better follow the toxic proteins in great detail and in high throughput, which is needed for artificial cell experiments. This method can be applied to multiple (non)toxic proteins and shows the structure of how they stick together.
- We aimed to better map crowding effects in living cells and found that the organization of the molecules should play a major role in the crowding
- We developed a new platform to make artificial cells by microfluidics. This is the first method to do so by microfluidics, where crowded minimal-oil-containing vesicles are obtained.
We achieved the following milestones:
- We developed a method to better follow the toxic proteins in great detail and in high throughput, which is needed for artificial cell experiments. This method can be applied to multiple (non)toxic proteins and shows the structure of how they stick together.
- We aimed to better map crowding effects in living cells and found that the organization of the molecules should play a major role in the crowding
- We developed a new platform to make artificial cells by microfluidics. This is the first method to do so by microfluidics, where crowded minimal-oil-containing vesicles are obtained.
We went beyond state-of-the-art by developing a method to follow toxic proteins with high precision and high throughput with a single genetic construct. This technology is straightforward to implement and can be used inside and outside cells.
We show for the first time that cell wall damage can increase crowding in bacterial cells, providing the clearest indication thus far that cytoplasmic organization influences crowding.
In our artificial cell production, we developed the first microfluidic method to make these with high internal crowding and minimal additional oil present.
We expect to further these artificial cells as drug screening platforms, in which we have proteins in their toxic form induced by their crowded environment.
We show for the first time that cell wall damage can increase crowding in bacterial cells, providing the clearest indication thus far that cytoplasmic organization influences crowding.
In our artificial cell production, we developed the first microfluidic method to make these with high internal crowding and minimal additional oil present.
We expect to further these artificial cells as drug screening platforms, in which we have proteins in their toxic form induced by their crowded environment.