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Reactions in living cells possible to measure without disturbance

Researchers at Uppsala University and Stockholm University have developed a new method for measuring bonding reactions in living cells without subjecting them to an outer disturbance. The results help researchers to understand how the biochemistry in a living cell differs from what can be measured in a test tube.

This contributes to better models of cells’ regulatory systems which in turn can be used to understand how diseases arise.

The new method is presented in an article in scientific journal Nature Methods. The method uses a combination of high frequency and high resolution light microscopy and statistical calculations.

Traditional biochemical measurements of cellular kinetics are carried out by pushing millions of molcules out of equilibrium using some form of outer disturbance and then observing how quickly they regain their equilibrium. That type of experiment can rarely be performed on living cells since the molecules exist in different states and all information about how quickly the molecules switch between states is lost if only averages are observed.

“By looking at one molecule at a time and seeing how it moves around the cell we can get information about how often the molecule switches bonding states”, says researcher Fredrik Persson at Uppsala University.

Molecules and other small objects in a water solution move randomly in a movement pattern called diffusion and are driven by constant collisions with molecules in the surrounding liquid. How quickly the molecules move through diffusion depends on their size. Small molecules diffuse faster than large ones, and it is this relation that the researchers have made use of to detect when the molecules switch bonding partners.

“The change is hard to see with the naked eye, but with the help of statistical computer calculations it is possible to work out how many diffusion states a certain molecule displays. You can also see how often the molecule switches between different states and thereby measure chemical reaction rate constants in living cells without exposing them to outer disturbances”, says Martin Lindén, researcher at Stockholm University.

So what can the new method be used for?

“Knowledge about which bonding states the proteins have inside the cell and how quickly they bind to different complexes makes it possible to understand how biochemistry in a living cell differs from what is measurable in a test tube”, says Johan Elf, professor of physical biology at Uppsala University.

The new method can also be utilised to create better models of the cells’ regulatory systems. These models can then be used for example to understand how diseases arise and in the best case, how they can be cured.

Johan Elf et al., Extracting intracellular diffusive states and transition rates from single-molecule tracking data, Nature Methods, DOI: 10.1038/nmeth.2367

Uppsala University

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