Single Molecule Dynamics of Lipid Bilayer Associated PTEN

Abstract

ObjectiveStudy the dynamics of PTEN associated with laterally heterogeneous lipid bilayers using single molecule microscopy.BackgroundPhosphatase and tensin homologue deleted on chromosome 10 (PTEN), is one of the most frequent genes deleted/mutated in a wide variety of tumors. PTEN acts as an antagonist to PI 3‐kinase signaling, thereby affecting cellular processes such as cell proliferation and survival. PTEN activity is regulated by dynamic shuttling between the cytoplasm and the plasma membrane.MethodsWe use single‐molecule TIRF microscopy and stopped‐flow fluorescence measurements to observe PTEN molecules as they dynamically associate/dissociate and laterally diffuse along the lipid bilayer membrane. PTEN lipid binding is investigated using supported lipid bilayers of binary and ternary lipid mixtures of phosphatidylcholine (PC) with physiological relevant levels of phosphatidylserine (PS), phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P2) and/or phosphatidylinositol (PI).ResultsUsing single molecule trajectories of PTEN molecules we find significant differences in PTEN dynamic behavior when bound to different membrane environments. Interestingly, we determine the existence of three membrane bound states with different diffusion coefficients. To gain insight into the molecular mechanisms of PTEN membrane association, we compared the lipid binding of wt‐PTEN, PTEN‐(Δ1‐15aa) and the recently discovered, PTEN‐L with a 173 aa N‐terminal extension. We find profound differences in the dynamic behavior of these PTEN derivatives at the membrane. We also found that PI may replace PS as the second lipid component to which PTEN binds.