Ratiometric GPCR signaling enables directional sensing in yeast
Fig 5
Pheromone receptor density variation around the cell membrane.
(A) Single-plane inverted image of vegetatively growing cells expressing Ste2–sfGFP. Membrane signal (blue arrow) is Ste2–sfGFP, but vacuole signal (orange arrow) is probably sfGFP cleaved from Ste2-sfGFP after internalization. (B) Ste2 distribution through the cell cycle in representative cells. (C) G1 cells display Ste2 distributions ranging from almost uniform (top) to very asymmetric (bottom). (D) Quantification of Ste2–sfGFP membrane distribution in G1 cells. Individual linescans (examples in color) were normalized to have the same total fluorescence and centered on the peak of a smoothed spline fit. Black line, average (n = 71). (E–G) Particle-based simulations of receptor–G-protein interactions at the cell membrane. Receptors and G proteins were simulated as diffusing particles on a spherical surface. G proteins were activated when they encountered an active receptor, and active G proteins were spontaneously inactivated with first-order kinetics. (E) Receptors were distributed unevenly: receptor density is indicated by the thickness of the black line (inset) and reflects a 3-fold gradient, similar to the Ste2 distribution. (F) A 1.5-fold pheromone gradient was simulated along the x axis by varying the % of active receptors from 40% to 60% across the cell diameter. (G) Simulations were conducted with receptor activity and density gradients oriented as in the illustrations. The locations of all of the active G proteins were used to calculate a G-protein vector, whose angle to the direction of the pheromone gradient is plotted (y axis) against time (x axis) (left). 0° indicates perfect orientation: active G-protein vector in the same direction as the applied receptor activity gradient. The approximate range of G-protein vectors (blue wedge) is shown in the cartoon on the right, along with the pheromone gradient (green shading) and receptor density (as in E). Code and key data for Fig 5G are available at https://github.com/mikepab/ratiometric-gpcr-particle-sims. Scale bar, 3 μm. Strains: DLY20713 (A–D). A.U., arbitrary unit; GFP, green fluorescent protein; GPCR, G-protein–coupled receptor; sf, superfolder; Ste, sterile.