Key role of local regulation in chemosensing revealed by a new molecular interaction-based modeling method

Abstract

The signaling network underlying eukaryotic chemosensing is a complex combination of receptor-mediated transmembrane signals, lipid modifications, protein translocations, and differential activation/deactivation of membrane-bound and cytosolic components. As such, it provides particularly interesting challenges for a combined computational and experimental analysis. We developed a novel detailed molecular signaling model that, when used to simulate the response to the attractant cyclic adenosine monophosphate (cAMP), made nontrivial predictions about Dictyostelium chemosensing. These predictions, including the unexpected existence of spatially asymmetrical, multiphasic, cyclic adenosine monophosphate-induced PTEN translocation and phosphatidylinositol-(3,4,5)P3 generation, were experimentally verified by quantitative single-cell microscopy leading us to propose significant modifications to the current standard model for chemoattractant-induced biochemical polarization in this organism. Key to this successful modeling effort was the use of "Simmune," a new software package that supports the facile development and testing of detailed computational representations of cellular behavior. An intuitive interface allows user definition of complex signaling networks based on the definition of specific molecular binding site interactions and the subcellular localization of molecules. It automatically translates such inputs into spatially resolved simulations and dynamic graphical representations of the resulting signaling network that can be explored in a manner that closely parallels wet lab experimental procedures. These features of Simmune were critical to the model development and analysis presented here and are likely to be useful in the computational investigation of many aspects of cell biology.

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.