Abstract
Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and PIP3 phosphatase (PTEN) are enriched mutually exclusively on the anterior and posterior membranes of eukaryotic motile cells. However, the mechanism that causes this spatial separation between the two molecules is unknown. Here we develop a method to manipulate PIP3 levels in living cells and used it to show PIP3 suppresses the membrane localization of PTEN. Single-molecule measurements of membrane-association and -dissociation kinetics and of lateral diffusion reveal that PIP3 suppresses the PTEN binding site required for stable PTEN membrane binding. Mutual inhibition between PIP3 and PTEN provides a mechanistic basis for bistability that creates a PIP3-enriched/PTEN-excluded state and a PTEN-enriched/PIP3-excluded state underlying the strict spatial separation between PIP3 and PTEN. The PTEN binding site also mediates the suppression of PTEN membrane localization in chemotactic signaling. These results illustrate that the PIP3-PTEN bistable system underlies a cell’s decision-making for directional movement irrespective of the environment.
Highlights
Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and PIP3 phosphatase (PTEN) are enriched mutually exclusively on the anterior and posterior membranes of eukaryotic motile cells
The exclusion was always accompanied with an enrichment of PIP3, as visualized with eGFP-tagged Pleckstrinhomology domain of PKB (PHPKB-eGFP), making a clear spatial separation between PTEN and PIP3
The defects were more severe with regards to chemotaxis (Supplementary Movie 1; Supplementary Movie 2; Supplementary Movie 3; Supplementary Fig. 2; Supplementary Table 1). These results suggest that confined PIP3 enrichment requires the appropriate membrane localization of PTEN along with lipid phosphatase activity
Summary
Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and PIP3 phosphatase (PTEN) are enriched mutually exclusively on the anterior and posterior membranes of eukaryotic motile cells. PTEN produces PIP2 on the cell membrane to further recruit PTEN, which contains a PIP2binding motif[23,24,25] These two positive-feedback loops require coupling with each other to avoid merging of the PIP3enriched and PTEN-enriched domains, interactions between the anterior and posterior signaling molecules have hardly been taken into account. Single-molecule imaging reveals that the negative regulation is mediated by a specific binding site for PTEN that is inactivated by PIP3 and by cAMP stimulation These results illustrate that PTEN works as a component of the bistable system to generate a digitized signal of the confined PIP3 enrichment and thereby determine a cell’s motile behavior irrespective of the environment
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