Excitable dynamics of Ras triggers spontaneous symmetry breaking of PIP3 signaling in motile cells.

Spontaneous cell movement is underpinned by an asymmetric distribution of signaling molecules including small G proteins and phosphoinositides on the cell membrane. However, the molecular network necessary for spontaneous symmetry breaking has not been fully elucidated. Here, we report that, in Dictyostelium discoideum, the spatiotemporal dynamics of GTP bound Ras (Ras-GTP) breaks the symmetry due its intrinsic excitability even in the absence of extracellular spatial cues and downstream signaling activities. A stochastic excitation of local and transient Ras activation induced phosphatidylinositol (3,4,5)-trisphosphate (PIP3) accumulation via direct interaction with Phosphoinositide 3-kinase (PI3K), causing tightly coupled traveling waves that propagated along the membrane. Comprehensive phase analysis of the waves of Ras-GTP and PIP3 metabolism-related molecules revealed the network structure of the excitable system including positive-feedback regulation of Ras-GTP by the downstream PIP3. A mathematical model reconstituted a series of the observed symmetry-breaking phenomena, illustrating the essential involvement of Ras excitability in the cellular decision-making process.

Single-molecule imaging of PI(4,5)P2 and PTEN in vitro reveals a positive feedback mechanism for PTEN membrane binding.

PTEN, a 3-phosphatase of phosphoinositide, regulates asymmetric PI(3,4,5)P3 signaling for the anterior-posterior polarization and migration of motile cells. PTEN acts through posterior localization on the plasma membrane, but the mechanism for this accumulation is poorly understood. Here we developed an in vitro single-molecule imaging assay with various lipid compositions and use it to demonstrate that the enzymatic product, PI(4,5)P2, stabilizes PTEN's membrane-binding. The dissociation kinetics and lateral mobility of PTEN depended on the PI(4,5)P2 density on artificial lipid bilayers. The basic residues of PTEN were responsible for electrostatic interactions with anionic PI(4,5)P2 and thus the PI(4,5)P2-dependent stabilization. Single-molecule imaging in living Dictyostelium cells revealed that these interactions were indispensable for the stabilization in vivo, which enabled efficient cell migration by accumulating PTEN posteriorly to restrict PI(3,4,5)P3 distribution to the anterior. These results suggest that PI(4,5)P2-mediated positive feedback and PTEN-induced PI(4,5)P2 clustering may be important for anterior-posterior polarization.

A Computational Framework for Bioimaging Simulation.

Using bioimaging technology, biologists have attempted to identify and document analytical interpretations that underlie biological phenomena in biological cells. Theoretical biology aims at distilling those interpretations into knowledge in the mathematical form of biochemical reaction networks and understanding how higher level functions emerge from the combined action of biomolecules. However, there still remain formidable challenges in bridging the gap between bioimaging and mathematical modeling. Generally, measurements using fluorescence microscopy systems are influenced by systematic effects that arise from stochastic nature of biological cells, the imaging apparatus, and optical physics. Such systematic effects are always present in all bioimaging systems and hinder quantitative comparison between the cell model and bioimages. Computational tools for such a comparison are still unavailable. Thus, in this work, we present a computational framework for handling the parameters of the cell models and the optical physics governing bioimaging systems. Simulation using this framework can generate digital images of cell simulation results after accounting for the systematic effects. We then demonstrate that such a framework enables comparison at the level of photon-counting units.