Periodic Reporting for period 5 - PArtCell (Physiologically Crowded Artificial Cells for Relevant Drug Screens)
Período documentado: 2025-03-01 hasta 2025-08-31
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 come into contact and can interact. Interactions such as hydrophobic and electrostatic interactions, as well as 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, potentially leading to 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 aimed to map and understand these crowding effects with our project PArtCell (Physiologically Crowded Artificial Cells for Relevant Drug Screens). In addition, our goal was 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 aimed 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 defined and thus controlled primarily.
We concluded with the ability to generate various relevant crowded artificial cells for studying protein aggregation and sensors to map their physiologically relevant environment, as well as validation against living cells. We further demonstrated methodology to observe aggregation in detail, displaying the impact of the environment on aggregate morphologies.
Although crowding is essential, it remains a question of how the biochemical equilibrium in cells is controlled. We aimed to map and understand these crowding effects with our project PArtCell (Physiologically Crowded Artificial Cells for Relevant Drug Screens). In addition, our goal was 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 aimed 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 defined and thus controlled primarily.
We concluded with the ability to generate various relevant crowded artificial cells for studying protein aggregation and sensors to map their physiologically relevant environment, as well as validation against living cells. We further demonstrated methodology to observe aggregation in detail, displaying the impact of the environment on aggregate morphologies.
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 included relevant macromolecular crowdin in liposomes by reconstituting the content of living cells. These crowding levels are similar to living cells. We thereby provided a relevant platform to study crowding effects.
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 included relevant macromolecular crowdin in liposomes by reconstituting the content of living cells. These crowding levels are similar to living cells. We thereby provided a relevant platform to study crowding effects.
We went beyond state-of-the-art by developing a method that enables the precise and high-throughput tracking of toxic proteins using 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 most unambiguous 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 provided the first reconstitution of macromolecular crowding of an E. coli cell using its native lysate, allowing the study of crowding effects and mapping the consequences of small molecules on crowding.
We show for the first time that cell wall damage can increase crowding in bacterial cells, providing the most unambiguous 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 provided the first reconstitution of macromolecular crowding of an E. coli cell using its native lysate, allowing the study of crowding effects and mapping the consequences of small molecules on crowding.