The ability to accurately measure the effects of molecular crowding within cells is an area that has eluded researchers for many years. However, scientists at the University of Groningen in the Netherlands have developed a clever new molecular sensor that provides quantitative information about the concentration of macromolecules in mammalian and bacterial cells.
This new sensor, developed by Bert Poolman, Ph.D., professor of biochemistry at the University of Groningen and Arnold Boersma, Ph.D., first author of the current study and research fellow in Dr. Poolman’s laboratory, is characterized in an article entitled “A sensor for quantification of macromolecular crowding in living cells”, which was published today in Nature Methods.
Cells contain an array of macromolecules such as proteins, lipids, nucleic acids, and carbohydrates that impact biochemical events like diffusion and enzyme kinetics. For example, it has been well established that DNA replication is considerably more efficient under conditions that closely mimic the cellular environment than under attenuated laboratory conditions. For this reason, researchers often add crowding agents to biological reactions in order to achieve optimal results.
Despite the importance of molecular crowding, it is often neglected in biochemical studies due to a lack of dependable measurement techniques. In this current study, scientists are able to measure molecular congestion inside live cells at high resolution and in real time. This is a considerable step forward, since previously it was only possible to estimate measurements from average concentrations and average cell volumes.
The investigators designed a protein spring that takes advantage of Förster resonance energy transfer (FRET) technology. Specifically, the protein spring, which contains fluorescent markers on both ends, is excited by a laser that causes one end to fluoresce blue. The blue light than excites the second marker on the opposite end, which will then emit a yellow fluorescence. The transfer of energy from one marker to the other is directly proportional to the distance between the markers.
Since macromolecules contained within a cell will exert mechanical stress on each other, due to the molecular crowding effect, Dr. Poolman’s team can quantitatively measure the force pushing on their protein spring under various cellular conditions. The researchers have ruled out that sensor protein is affected by other forces, such as ionic strength or chemical affinity, in a serious of control experiments.
“We will use this sensor to map the structure of the cytoplasm during the cell cycle,” stated Dr. Poolman. “Our interest is in volume regulation, which obviously affects crowding. But it's easy to envision many other applications for the sensor.”
The protein sensor was generated by applying some elegant molecular biology techniques. Dr. Boersma and Dr. Poolman designed the protein structure to contain a series of α-helical peptide linkers that act like a hinge spring, allowing the whole protein to change conformation under crowded conditions. Furthermore, they encoded the sensor on two separate artificial genes, one for bacterial and one for mammalian cell systems.
“We want to know how cells work, and how we can engineer designer cells,” explained Dr. Poolman. He believes that the crowding sensor will significantly contribute to a comprehensive understanding of cellular architecture and function.