Quantifying dynamic molecular aggregates in living cells

In cells, many vital processes take place in membraneless molecular aggregates. Such aggregates ensure that the molecules involved are present in the right concentration and proximity to each other. Researchers from the Cluster of Excellence CIBSS at the University of Freiburg and the University of Cambridge in the UK have now been able to observe and analyse the formation of such condensates in living cells. In the scientific journal Nature Communications, they describe that not only physical principles but also active control mechanisms regulate their growth. Experimental protocols and analysis programmes are available and, according to the University of Freiburg, can also make research on small aggregates possible for less specialised laboratories.

If the molecules inside a cell were randomly distributed, it would not be viable. Only by dividing it into specialised areas can many biochemical processes take place in a coordinated manner. Some of these compartments are separated from each other by membranes, but a demarcation also works without membranes. Such membraneless molecular aggregates, also known as condensates, fulfil important biological functions because their size and number are particularly flexible. It is assumed that they are formed through the physical process of liquid-liquid phase separation. „These condensates are an important control mechanism in cells, as they can accelerate or slow down biochemical processes as required“, explains Prof Dr Thorsten Hugel. He is a member of the Cluster of Excellence CIBSS – Centre for Integrative Biological Signalling Studies at the University of Freiburg and led the current study together with Prof. Dr. Aleks Reinhardt from the University of Cambridge.

Studying smaller condensates difficult
How condensates help the cell to process biological signals and environmental stimuli has not yet been sufficiently researched, says Hugel. „Research usually focusses on large and static condensates because they are easier to study. But these large condensates are usually only the final stage of a long process. Much more interesting are small condensates that grow and decay dynamically," he explains. The problem: they consist of relatively few molecules and are too small even for high-resolution microscopy methods and move too quickly to be analysed in living cells.

Imaging method
In the current study, the researchers from Freiburg and Cambridge have now described a way to circumvent these technical limitations. To do this, they used conventional high-resolution fluorescence microscopy with a narrow laser (HILO microscopy,
HILO = highly inclined and laminated optical sheet) and combined it with photobleaching, a special experimental procedure, and AI-supported analysis methods.

Condensate formation processes
The researchers compared the measurements made in living cells with theoretical assumptions about the formation of condensates. „The results surprised us,“ says Reinhardt, who conducts research at the Faculty of Chemistry at the University of Cambridge. „In the condensates that we analysed in this study, the initial growth still follows physical models. We would expect the same for such processes. From a certain size, however, this growth then suddenly stops.

Growth of aggregates regulated by stress signals
In the current study, the researchers investigated aggregates of the protein NELF. These protein aggregates form when a cell is under stress, for example due to heat, or when aggregates of other proteins form, as in dementia and other neurogenerative diseases. „By forming condensates in the cell nucleus, NELF inhibits the expression of genes“, summarises co-author Dr Ritwick Sawakar the natural function of the protein. „This inhibition is important for the cell to survive stress.“ Sawakar also worked at the CIBSS and is currently a researcher at the MRC Toxicology Unit at the University of Cambridge.

The scientists have now been able to observe that numerous small NELF condensates are also present in „non-stressed“ cells. „Outside a cell, we would expect the condensates to continue to grow once they have reached a critical size. In living cells, however, they only seem to do this when the cell is under stress," Reinhardt describes the result. The researchers conclude that NELF condensates are actively kept small until stress signals release growth again.

Protein aggregates and signal processing
According to the researchers, this seemingly complicated process is probably essential for the processing of stress signals: "Larger condensates can form particularly quickly when required and dissolve smaller ones very quickly," explains Hugel. „This enables the cell to react to stress in good time.“

Protein aggregates are thought to have many different and fundamental functions in cell signalling. Investigations using the newly developed microscopy method can provide information to understand these functions. This could also be used to research the role of protein aggregates in diseases such as dementia, Alzheimer's or Huntington's disease.

Further information:
The study was funded by the European Research Council (ERC) and the German Research Foundation (DFG).

The analysis programmes can be downloaded free of charge here: https://doi.org/10.5281/zenodo.6946007

The Cluster of Excellence CIBSS – Centre for Integrative Biological Signalling Studies at the University of Freiburg aims to gain a comprehensive understanding of biological signalling processes across scales - from the interactions of individual molecules and cells to the processes in organs and entire organisms. The researchers are using the knowledge they have gained to develop strategies that can be used to specifically control signals. Further information at www.cibss.uni-freiburg.de

Article from "LABO" from 17/08/2023