Hidden behind the population mean: Single cell analysis of membrane recruitment dynamics of the mating MAPK pathway scaffold protein

REPETTO M. V.; BUSH A.; WINTERS M.; PRYCIAK P.; COLMAN LERNER A.

Congreso; 50th Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2014

In S. cerevisiae, pheromone activates a GPRC coupled to a MAPK cascade pathway that, among other effects, arrests cell cycle progression in G1. However, in cells committed to a new round of cell division, Cdk activity blocks pheromone response. Plasma membrane (PM) recruitment of the mating MAPK scaffold, Ste5, is a key step in pheromone signaling. It is this membrane interaction that is inhibited by Cdk activity by phosphorylating residues flanking the Ste5 PM binding domain. Recently we developed a quantitative method to measure protein relocalization over time using microscope cytometry. Here, using this method, we studied the early dynamics of Ste5 recruitment as a function of the cell cycle position in single live cells. In contrast to our expectations, we found that initial recruitment is similar in G1 and S phase cells, but then it rapidly declines in S phase cells. Remarkably, this decline in S phase is strictly dependent on the activity of the mating MAPK Fus3. Nevertheless, Fus3 activity alone cannot displace Ste5 from the membrane, since Ste5 recruitment persists over time in G1 cells or when it lacks the Cdk sites flanking its PM domain. These findings reveal that Fus3, on top of its classic mating promoting functions, cooperates with the Cdk in a complex negative feedback, bringing about distinct patterns of temporal dynamics at different cell cycle stages.