Figure 1.
Standard Model of the Extracellular and Intracellular Distribution of Key Components of Dictyostelium Chemotactic Signaling
(A) In an unstimulated cell PTEN is homogeneously distributed at the membrane. The cell membrane contains very little PIP3.
(B) Stimulation of the cell leads to the membrane recruitment and activation of PI3K, as indicated by the arrows (1) leading from inactive, mainly cytosolic PI3K (yellow) to membrane-proximal, active PI3K (orange). Activated PI3K transforms PIP2 into PIP3. PTEN is deactivated following cAMP stimulation and leaves the membrane. This process is indicated by arrows (2) connecting active PTEN (dark green) and the mainly cytosolic inactive PTEN (light green). Regulatory processes lead to reactivation of PTEN (3). Differences in the speed and degree of cAMP receptor ligation between front and back of the cell lead to preferential accumulation of PTEN at the back of the cell. As a result, the front experiences a higher concentration of PI3K and a lower concentration of PTEN than the back and accumulates PIP3. Note: To emphasize the changes in PIP3 content, the amount of PIP3 relative to that of PIP2 has been overstated. Even after cAMP stimulation, the actual amount of PIP2 will be much higher than that of PIP3.
Figure 2.
PI3K and PTEN Regulatory Modules
(A) Elements controlling the activity of PI3K and upstream components. In addition to the basic, “excitatory,” signaling elements like the cAMP receptor, Gβγ, and Ras, we introduced further elements controlling the activity of PI3K and upstream components. “PI3Ktp” (module 1) stands for a tyrosine phosphatase that deactivates PI3K. This phosphatase becomes enzymatically activated and is recruited to the membrane after interaction with Gβγ. “RasGAP” (module 2) translocates to the membrane and deactivates Ras after activation by Gβγ. RAK blocks Gβγ, Gα, and the receptor, thereby reducing all signals (module 3).
(B) Elements controlling the activity and localization of PTEN. In our model, PTEN is phosphorylated by a Src-like kinase, here simply called “Src” (module 1). Src is activated by Gα and deactivated by Csk, which in turn is recruited by phosphoPaxillin (“pPaxillin”) (module 2). SHP2, which is membrane recruited by pGab1 bound to PIP3, dephosphorylates pPaxillin (module 3), thereby leading to increased activation of PTEN.
Figure 3.
Defining Quantitative Interactions between Molecular Binding Sites Using Simmune (Screenshot)
The screenshot of Simmune's modeling interface shows the graphical representations of PI3K and PIP2 and the binding sites through which they interact, as well as the sites of membrane attachment. The turquoise circle around the upper binding site of PI3K identifies it as an enzymatically active site. The dotted line represents the possibility of a binding interaction between these two molecules. Selecting the interaction by clicking on the handle on the dotted line allows entry of the relevant binding parameters.
Figure 4.
Comparison of the Simulated Activities of PI3K, Membrane-Bound PTEN, and the Resulting Behavior of PIP3 (Composite Screenshot)
Stimulation of a cell in a 2:1 cAMP gradient (mean concentration 500 nmol) leads to a rapid 3-fold increase in the membrane proximal activity of PI3K (green) and to a loss of membrane-bound PTEN (blue; tracked as GFP-PTEN in real cells). This results in a rapid accumulation of PIP3 (red; reported by the location of PH-GFP in real cells). Subsequently, the PI3K activity is strongly quenched by the recruitment of regulatory components to the membrane and falls below its prestimulus level in less than 20 s. PTEN returns more slowly to the membrane. During the phase of downregulation of PI3K activity and reattachment of PTEN to the membrane, the concentration of PIP3 decays. In the front of the cell (which experiences a high cAMP concentration), membrane-associated PTEN only returns to a fraction of its prestimulus level and then enters a second phase of decline. After approximately 50 s, the low level of membrane-bound PTEN that is reached in the front of the cell allows PIP3 to increase again, even though the amount of active PI3K in this region is modest. In the back of the cell (low cAMP concentration), membrane-bound PTEN increases beyond its prestimulus level, resulting in a decrease of PIP3 below its resting state concentration. The circular inset shows a two-dimensional representation of the dynamics of membrane-bound PTEN and PIP3 in different regions of the three-dimensional simulation of a cell.
Figure 5.
Correspondence in Time and Space between the Predicted and Measured Changes in PIP3 at the Front and Back of Cells Exposed cAMP Gradients
Experimental data from exposure of Dictyostelium to a 2:1 gradient with a mean cAMP concentration of 100 nmol were used to adjust model parameters. The other two responses are predictions of the model. (A), (B), and (C) are simulated responses. (D), (E), and (F) are experimental measurements, using PH-GFP to monitor PIP3 levels in single cells exposed to gradients with a mean cAMP concentration of 1 μmol, 100 nmol, and 10 nmol. See Figure S6 for details on the full dataset of experimental replicates.
Figure 6.
Correspondence in Time and Space between the Predicted and Measured Changes in Membrane-Bound PTEN at the Front and Back of the Cells Exposed to cAMP Gradients
These dose-dependent dynamics of PTEN were produced by the simulation after the dynamics of PIP3 for one cAMP concentration (100 nM) had been used to adjust model parameters. (A), (B), and (C) are simulated responses. (D), (E), and (F) are experimental measurements based on GFP-PTEN analysis in single cells exposed to gradients with a mean cAMP concentration of 1 μmol, 100 nmol, and 10 nmol. See Figure S7 for details on the full dataset of experimental replicates.