Simultaneous Imaging of Single-Molecule and Bulk Localization of Pten

How does a cell decide it's front or back? According to studies of chemotaxis in Dictyostelium discoideum, an important factor is the mutually exclusive localization of PIP3 and PIP2, which are interconvertible phosphostates of the phosphatidylinositol membrane lipid. PTEN, an enzyme that catalyzes dephosphorylation of PIP3 to PIP2, plays an important role in regulating dynamic self-organizing distributions of PIP2 and PIP3. In a previous study, we demonstrated self-organizing patterns by analyzing bulk PIP3 and PTEN fluorescence distributions at the membrane. In addition, we measured the single-molecule level membrane association kinetics of PTEN. In order to understand the self-organization mechanism at the level of individual molecules, it is necessary to couple these results.To achieve this, we set up a new measurement method, dual fluorescence detection TIRFM (Total Internal Reflection Fluorescence Microscopy) that allows us to measure single-molecule kinetics and multi-molecule localization simultaneously. This method enable us to examine correlations between single-molecule PTEN kinetics and the local bulk density, or fluctuations in density, to elucidate the role of PTEN in self-organization. We find that PTEN kinetics, in particular the membrane binding on-rate and off-rate vary as a function of bulk PTEN localization and fluctuation patterns. This indicates that some mechanism promotes or restrains PTEN binding to the membrane. To identify the molecular causes of local changes in PTEN kinetics, we are now performing this analysis with other molecules involved in this self-organizing molecular network.