Abstract
Eukaryotic amoeboid cells generate an intracellular asymmetric signal through excitable dynamics of chemotactic signaling pathways, by which GTP-bound Ras (Ras-GTP) and PI(3,4,5)P3 phosphatidylinositol lipid (PIP3) accumulate in the anterior part of a migrating cell even in the absence of external asymmetric cues. In order to clarify key determinants in the signaling pathways for the spontaneous excitable dynamics, we have studied spatiotemporal dynamics of the major signaling components with genetic and pharmacological inhibition. Here we report that spontaneous excitability in the activation of Ras triggers symmetry breaking without the activation of downstream pathways. Quantitative live imaging of pairs of Ras-GTP and one of PIP3, PI(4,5)P2, PI3K or PTEN revealed that all these components followed the dynamics of Ras-GTP. Furthermore, the interaction between Ras-GTP and PI3K was essential for the asymmetric accumulation of downstream PIP3 on the membrane, which in turn enhanced Ras excitability via a positive feedback mechanism. Mathematical modeling of the excitable dynamics reconstituted the observations, illustrating that the Ras GTPase regulatory network is central for internal symmetry breaking. The results also suggest cellular decision-making mechanisms in response to external guidance cues.
Author contributions:
S.F. conducted the experiments; all authors analyzed the data and wrote the manuscript.
- Abbreviations
- GAP
- Guanosine Triphosphate hydrolase activating protein
- GEF
- Guanine nucleotide exchange factor
- GFP
- Green fluorescent protein
- PHD
- Pleckstrin homology domain
- PI3K
- Phosphoinositide 3-kinase
- PIP2
- Phosphatidylinositol (4,5)-bisphosphate
- PIP3
- Phosphatidylinositol (3,4,5)-trisphosphate
- PKB&Akt
- Protein kinase B
- PLCδ1
- Phospholipase C delta 1
- PTEN
- Phosphatase and tensin homolog
- RBD
- Ras binding domain
- RFP
- Red fluorescent protein