Using fission yeast as a model organism, Wu and Pollard have taken fluorescence microscopy to a new level by devising a reliable method for quantifying the concentrations of fluorescent fusion proteins.
Knowledge of the global and local concentrations of specific proteins can be invaluable for understanding complicated cellular processes, as it can provide stoichiometric information on the molecules involved. Light microscopy has been a popular approach for measuring the concentrations of fluorescently tagged proteins. However, concentration measurements are difficult to obtain and often rely on assumptions that are difficult to verify.
Wu and Pollard integrated the coding sequence for yellow fluorescent protein (YFP) into the fission yeast genome to create 27 strains containing YFP fusions of different cytokinesis proteins. After thorough analyses of the new strains, and by varying the position of YFP and the size of the linker, functional fusion proteins were obtained. And immunoblotting verified that they were expressed at native levels. Confocal microscopy was then used to measure the global and local fluorescence in each strain. Using seven different proteins, the authors showed that the mean fluorescence per cell was directly proportional to the average number of fusion proteins per cell, as measured by quantitative immunoblotting. Therefore, the fluorescence per protein could be determined.
The authors used various experimental approaches to verify the reliability of their approach. They established a linear relationship between whole-cell fluorescence and protein concentration using microscopy and flow cytometry. And a YFP fusion of the actin crosslinking protein Fim1 (Fim1–YFP) produced consistent total fluorescence whether it was concentrated in actin patches or dispersed by inhibiting actin polymerization, which confirmed that fluorescence quenching was not an issue. Similarly, triple YFP tags produced three times the fluorescence of single YFP tags on the same proteins.
Having determined the fluorescence per molecule, Wu and Pollard were able to calculate the global and local concentrations of the remaining 20 proteins that they had tagged. Their measurements have provided the first quantitative inventory of the proteins that are involved in fission yeast cytokinesis. However, perhaps more significantly, they describe a precision measuring tool for quantitative biology that could be applied to any organism that uses homologous recombination. Indeed, given knowledge of the ratio of tagged to untagged proteins, even episomally expressed fusion proteins could be quantified in this way.