Investigating the cellular substructure during three stages of EGF stimulation by comparing pCF carpets.

(A) Three snapshots of FLIM data before, 3 minutes after and 6 minutes after EGF stimulation. We aim to replicate features in the pCF carpets from the in vivo data (Fig 2E–2G) using our in silico simulations. (B—C) Uniform actin islands, as shown in Fig 1C and 1H. (D) Actin islands across the axis of the cell bind Rac1 with different affinities. These affinities range from 3.12% to 12.15% from the back to the front, which is less than the simulation in Fig 3G. This arrangement of islands is similar to what we expect is present in vivo. (E) pCF analysis reveals four arc features of differing lengths, co-localized with the actin islands. Rac1 mobility is slower towards the front of the cell. (F) Modeling two populations of Rac1 by the superposition of two actin island gradients: one with twice the affinity (Fig 3G) as the other (Fig 4D). We combine the intensity carpets of the two simulations and perform pCF analysis. Although the two sets of islands are superimposed, here, we separate them for illustrative purposes. (G) The resulting pCF carpet for a cell with two populations of Rac1. We find two distinct gradients as highlighted by the curves: one for each population. The red curve corresponds to the Rac1 population with the higher actin affinity.