Fluorescence microscopy for Cdc42 quantification in Saccharomyces Cerevisiae

Abstract

In budding yeast, a certain concentration of the GTPase Cdc42 is optimal for cell division. This optimal concentration depends on the phenotype and genetic background of the cells. Due to bet hedging, the Cdc42 concentration is different between cells in a population and it is expected that the concentration, measured for many cells, is gamma distributed. This study describes a method for determining the concentration of Cdc42 proteins depending on the phenotype of individual cells. Fluorescence microscopy images were analyzed using custom designed software for tracking single cells and detecting budding and polarization events, based on existing segmentation software. This allows for estimating the cell volume and determining the concentration distribution based on the fluorescence intensity. The estimated cell volume is larger than was expected which is proven to be caused by segmentation errors. The intensity is shown to scale linearly with the number of fluorescent sources and using this result, the distribution for the Cdc42 concentration and copy number can be determined. For calculating the absolute concentration and copy number values, a constant still needs to be determined. To assess the reliability of the obtained results, validation measurements should be performed, for example by using different galactose concentrations to control the Cdc42 production by means of a galactose promoter or by using different genetic backgrounds. Based on the results, this method shows a possible way of determining the budding and polarization events and measuring the protein concentration for individual cells.