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RESEARCH

Overarching Goal

The interior of the cell is unlike a dilute buffer. However, traditionally, most biochemistry experiments have been performed in a dilute buffer. To understand how the interior of the cell influences biochemical processes, such as the organization of the biopolymers, we need to know the important parameters in the cell and quantify their contribution to the intracellular organization.

We aim to improve our understanding of the intracellular molecular self-organization by:

1) measuring relevant parameters with novel probes, such as crowding and (pathogenic) protein self-assembly/aggregation

2) perturbing and measuring such parameters in living cells,

3) Reconstruct aspects of the living cell organization in artificial systems.

Macromolecular Crowding

We have an extensive track record on studying macromolecular crowding with our unique sensors, which we developed. The sensors provide a readout for the excluded volume in bacteria under stress, aging yeast cells, and mammalian cells. Our efforts are to improve these sensors and map macromolecular crowding in different species and conditions.

Some interesting applications were to show how crowding changes in Escherichia coli and mammalian cells upon osmotic stress and how the crowding recovers. Furthermore we have seen large increases in macromolecular crowding upon cell wall damage. In these cases, there is likely a major change in the biochemical organization that can have large implications for the cell.

Next to this, we could apply our sensor during replicative aging of yeast cells to see that the crowding remained overall stable, especially in the longer-lived cells.

Finally, we gained extensive insights into how these sensors work through multiple biophysical and photophysical studies.

Artificial cells

To better understand the consequences of macromolecular crowding and other interactions arising from a high concentration of macromolecules, we reconstitute aspects of the living cells in artificial cells.

We have been able to generate highly concentrated liposomes by means of microfluidics. We use droplet-based microfluidics based on PDMS. The lipid-stabilized double emulsions allow concentrating the macromolecules to the desired concentration with osmotic upshift, and subsequent removal of the oil phase by e.g. centrifugation.

In addition, we constructed artificial cells containing cell lysates using the classical phase-transfer method, which provided highly concentrated vesicles by careful osmotic upshift, to reach the same crowding values as Escherichia coli and human HEK293T cells.

Protein Self-organization

We applied genetically-encoded fluorescent protein FRET pairs optimized for intermolecular FRET to monitor the self-assembly of their fusion partner in cells. The method provides a measure for structure in foci/aggregates which can be followed continuously. Moreover, self-assembly can be determined without visible foci. This probing method gives a fast readout, for example by FACS, and we applied it to aggregating proteins (mutants of Huntingtin protein associated with Huntington's disease) and condensate forming proteins (such as mutants of the FUS protein associated with some forms of ALS).