Quantitative Measurement of Protein Relocalization in Live Cells

Abstract

Microscope cytometry provides a powerful means to study signaling in live cells. Here we present a quantitative method to measure protein relocalization over time, which reports the absolute fraction of a tagged protein in each compartment. Using this method, we studied an essential step in the early propagation of the pheromone signal in Saccharomyces cerevisiae: recruitment to the membrane of the scaffold Ste5 by activated Gβγ dimers. We found that the dose response of Ste5 recruitment is graded (EC50 = 0.44 ± 0.08 nM, Hill coefficient = 0.8 ± 0.1). Then, we determined the effective dissociation constant (K(de)) between Ste5 and membrane sites during the first few minutes when the negative feedback from the MAPK Fus3 is first activated. K(de) changed during the first minutes from a high affinity of < 0.65 nM to a steady-state value of 17 ± 9 nM. During the same period, the total number of binding sites decreased slightly, from 1940 ± 150 to 1400 ± 200. This work shows how careful quantification of a protein relocalization dynamic can give insight into the regulation mechanisms of a biological system.

Feedforward Regulation Ensures Stability and Rapid Reversibility of a Cellular State

“…During exposure to mating pheromone, the Ste5-YFP scaffold reversibly localizes to the membrane at the shmoo tip to mediate pheromone signaling (Garrenton et al, 2009). Since membrane localization of Ste5 is required for pheromone signaling, Ste5-localization allows us to asses MAPK activity in single cells (Bush and Colman-Lerner, 2013; Strickfaden et al, 2007; Yu et al, 2008). To examine the kinetics of signaling reversibility, we grew cells in 240nM pheromone for 2 hours and then acutely removed all pheromone.…”

“…Bistability implies a multi-valued input-output relationship that in turn, necessarily implies a loss of information about the input signal. In sharp contrast, feed-forward regulation does not compromise the linear input-output relationship measuring the extracellular environment through the entire duration of a pheromone arrest (Bush and Colman-Lerner, 2013; Paliwal et al, 2007; Takahashi and Pryciak, 2008; Yu et al, 2008). Thus, to the best of our knowledge, feed-forward regulation is the only network structure that achieves the twin aims of stability and reversibility with minimal tradeoffs (Fig.…”

Push-Pull and Feedback Mechanisms Can Align Signaling System Outputs with Inputs

“…This may explain an otherwise puzzling finding: PRS gene expression aligns well with receptor occupancy (Figure 1B) despite the fact that the dose-response for Ste5 scaffold recruitment is more sensitive than that for receptor occupancy (Bush and Colman-Lerner, 2013) and blocking the Fus3 MAPK negative feedback, which sensitizes the dose-response curve for Fus3 MAPK phosphorylation (Yu et al, 2008), does not sensitize recruitment further (Bush and Colman-Lerner, 2013). A pull reaction originating upstream of Ste5 recruitment would explain these results.…”

“…Each quantifiable molecular event in this chain defines the system output at that particular measurement point (Brent, 2009). We and others have quantified these outputs, including for G-protein dissociation (Yi et al, 2003), reporter gene induction (Colman-Lerner et al, 2005; Yu et al, 2008), and scaffold recruitment (Bush and Colman-Lerner, 2013). …”

Utilization of extracellular information before ligand-receptor binding reaches equilibrium expands and shifts the input dynamic range

“…For image cytometry, we affixed exponentially growing cells to the bottom of wells in a glass-bottomed 96-well plate, as described (12), and stimulated them with 1 μM αF. We performed image acquisition essentially as described (78) and quantified the results as described in SI Appendix, section 6.2.…”