Quantitative study of mother-daughter asymmetry in Ace2 localization in yeast

LUCÍA DURRIEU; GUNNAR CEDERSUND; ALAN BUSH; ALEJANDRO COLMAN-LERNER

Montevideo, Uruguay

Congreso; International society for computational biology-latin america; 2010

International society for computational biology

Saccharomyces cerevisiae reproduces by budding, a process that is intrinsically asymmetric. Mother cells form buds that separate to become daughters. Daughters and mothers are genetically identical but constitute distinct cell types. Two asymmetric gene expression programs have been discovered so far: mating type switching and, more recently, Ace2-driven daughter cell-specific gene expression, which is the focus of this work. At the end of anaphase, localization of Ace2 shifts from cytoplasmic to nuclear. Later, between telophase and cytokinesis, Ace2 becomes asymmetric and accumulates in the daughter cell nucleus. The cause for this asymmetry and the molecular mechanism that leads to the activation of daughter cell-specific gene expression remains unclear. Localization of a protein to the nucleus is the net result of import and export reactions. However, it is very difficult to assess the effect of each of these reactions by means of genetic analysis and chemical inhibition without disrupting the whole system. Thus, to illuminate the nature of Ace2 asymmetry, we decided to measure these rates in mother-daughter pairs before, during and after asymmetry is established. The measurement of import and export rate constants allows us to determine, for example, if localization of Ace2 in the cell is regulated by changes in import, export of both. For proteins that shuttle continuously between the nucleus and the cytosol, it is impossible to deduce this type of information from conventional time-lapse experiments. We have developed a method to extract nuclear import and export rate constants from individual live cells by FRAP (fluorescence recovery after photobleaching). We constructed a simple mass action kinetics model to describe import and export reactions. Analysis of simulated data using this model revealed that the rates obtained by FRAP are too sensitive to the noise, albeit low, expected in our data (~1% of the signal), but also suggested that introducing multiple perturbations to the system should overcome this problem. Thus, we set up a protocol involving sequential FRAPs on the same cell (“train FRAP” technique) that allowed us to estimate with good confidence the desired rates. The measured import and export rates of Ace2 in mothers and daughters shows significant differences between these cell types. We also analyzed Ace2 localization mutants that showed a correspondingly altered transport dynamics. These results are presented as an introduction to a new approach for the study of Ace2 asymmetry and a as a method for measuring import and export rates.