Single Molecule Diffusion of Phosphatidylinositol Bisphosphate (PIP2) Lipids on Asymmetric Lipid Bilayers under the Influence of Polycationic Macromolecules

Abstract

Proteins binding with PIP2 lipids in the plasma membrane initiate and regulate many cell signaling events. Previous research suggests that proteins interact with PIP2 lipids through electrostatic interactions between polycationic protein motifs and anionic lipid head groups, leading to potential sequestration of multiple PIP2 lipids. The sequestration of PIP2 lipids will cause a change in the lipid mobility and the spatial distribution of PIP2 in the plasma membrane. We set out to understand the molecular structure and dynamics of the protein-PIP2 interface at single molecule level. In this study, a polycationic polymer, quaternized polyvinylpyridine (QPVP), and Myristoylated alanine-rich C-kinase substrate (MARCKS) peptide were chosen to mimic the membrane binding proteins. The behavior of PIP2 lipids in response to QPVP and MARCKS binding was evaluated by a time-resolved fluorescence technique, pulsed interleaved excitation fluorescence cross-correlation spectroscopy (PIE-FCCS), along with single particle tracking (SPT) of individual PIP2 molecules. The trajectories of individual PIP2 lipids indicate intermittent-type diffusion in which confined small steps are separated by steps typical of free Brownian motion. The confined movement is a result of stalling during binding events with QPVP molecules and reveals the microscopic details of the PIP2 lipid mobility decrease observed previously by FCCS measurements. FCCS measurements also indicate a collective diffusion of a cationic peptide, MARCKS, and PIP2, suggesting the existence of a large peptide-lipid assembly formed through electrostatic sequestration of PIP2 lipids by MARCKS. SPT analysis shows the diffusive behavior the peptide-lipid assemblies. The results suggest that PIP2 diffusion is significantly altered upon binding to polycationic molecules and is the first direct experimental evidence of stable lipid-peptide complexes formed through electrostatic interactions between MARCKS peptide and PIP2 lipid.