Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

University of Münster

Solid-supported lipid bilayers (SLBs) mimicking a biological membrane are commonly used to investigate lipid-lipid or lipid-protein interactions. Simple binary or ternary lipid systems are well established, whereas more complex model membranes containing biologically important signaling lipids such as phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and cholesterol have not been extensively described yet. Here we report the generation of such bilayers and their relevant biophysical properties and in particular the accessibility of PI(4,5)P2 for protein binding. Ternary mixtures of POPC with 20% cholesterol and either 3 or 5 mol % dioleoyl-phosphatidylinositol-4,5-bisphosphate were probed by employing the quartz crystal microbalance and atomic force microscopy. We show that these mixtures form homogeneous solid-supported bilayers that exhibit no intrinsic phase separation and are characterized by long-term stability (>8 h). Bilayers were formed in a pH-dependent manner and were characterized by the accessibility of PI(4,5)P2 on the SLB surface as shown by the interaction with the PI(4,5)P2 binding domain of the cortical membrane-cytoskeleton linker protein ezrin. A time-dependent reduction of PI(4,5)P2 levels in the upper leaflet of SLBs was observed, which could be effectively inhibited by the incorporation of a negatively charged lipid such as phosphatidylserine. Furthermore, quartz crystal microbalance measurements revealed that cholesterol affects bilayer adsorption to the solid support.

Formation and characterization of supported lipid bilayers containing phosphatidylinositol-4,5-bisphosphate and cholesterol as functional surfaces.

Ezrin, a protein of the ezrin, radixin, moesin (ERM) family, provides a regulated linkage between the plasma membrane and the cytoskeleton. The hallmark of this linkage is the activation of ezrin by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and a threonine phosphorylation at position 567. To analyze the influence of these activating factors on the organization of ezrin on lipid membranes and the proposed concomitant oligomer-monomer transition, we made use of supported lipid bilayers in conjunction with atomic force microscopy and fluorescence microscopy. Bilayers doped with either PIP2 as the natural receptor lipid of ezrin or a Ni-nitrilotriacetic acid-equipped lipid to bind the proteins via their His6-tags to the lipid membrane were used to bind two different ezrin variants: ezrin wild-type and ezrin T567D mimicking the phosphorylated state. Using a combination of reflectometric interference spectroscopy, atomic force microscopy, and Förster resonance energy transfer experiments, we show that only the ezrin T567D mutant, upon binding to PIP2-containing bilayers, undergoes a remarkable conformational change, which we attribute to an opening of the conformation resulting in monomeric protein on the lipid bilayer. 

Mode of Ezrin-Membrane Interaction as a Function of PIP2 Binding and Pseudophosphorylation.

Tailor-made ionic liquids based on imidazolium salts have recently attracted a large amount of attention because of their extraordinary properties and versatile functionality. An intriguing ability to interact with and stabilize membranes has already been reported for 1,3-dialkylimidazolium compounds. We now reveal further insights into the field by investigating 1,3-dimethyl-4,5-dialkylimidazolium (C-IMe·HI, n = 7, 11, 15) and 1,3-dibenzyl-4,5-dialkylimidazolium (C-IBn·HBr, n = 7, 11, 15) salts. Diverse alkyl chain lengths and headgroups differing in their steric demand were employed for the membrane interface interaction with bilayer membranes imitating the cellular plasma membrane. Membrane hydration properties and domain fluidization were analyzed by fluorescent bilayer probes in direct comparison to established model membranes in a buffered aqueous environment, which resembles the salt content and pH of the cytosol of living cells. Membrane binding and insertion was analyzed via a quartz crystal microbalance and confocal laser scanning microscopy. We show that short-chain 4,5-dialkylimidazolium salts with a bulky headgroup were able to disintegrate membranes. Long-chain imidazolium salts form bilayer membrane vesicles spontaneously and autonomously without the addition of other lipids. These 4,5-dialkylimidazolium salts are highly eligible for further biochemical engineering and drug delivery.

Imidazolium Salts Mimicking the Structure of Natural Lipids Exploit Remarkable Properties Forming Lamellar Phases and Giant Vesicles.

Cholesterol is an essential component of most biological membranes and serves important functions in controlling membrane integrity, organization, and signaling. However, probes to follow the dynamic distribution of cholesterol in live cells are scarce and so far show only limited applicability. Herein, we addressed this problem by synthesizing and characterizing a class of versatile and clickable cholesterol-based imidazolium salts. We show that these cholesterol analogs faithfully mimic the biophysical properties of natural cholesterol in phospholipid mono- and bilayers, and that they integrate into the plasma membrane of cultured and primary human cells. The membrane-incorporated cholesterol analogs can be specifically labeled by click chemistry and visualized in live-cell imaging experiments that show a distribution and behavior comparable with that of endogenous membrane cholesterol. These results indicate that the cholesterol analogs can be used to reveal the dynamic distribution of cholesterol in live cells.

Addressable Cholesterol Analogs for Live Imaging of Cellular Membranes.

The protein-mediated formation of membrane contacts is a crucial event in many cellular processes ranging from the establishment of organelle contacts to the docking of vesicles to a target membrane. Annexins are Ca regulated membrane-binding proteins implicated in providing such membrane contacts; however, the molecular basis of membrane bridging by annexins is not fully understood. We addressed this central question using annexin A2 (AnxA2) that functions in secretory vesicle exocytosis possibly by providing membrane bridges. By quantitatively analyzing membrane contact formation using a novel assay based on quartz crystal microbalance recordings, we show that monomeric AnxA2 can bridge membrane surfaces Ca dependently. However, this activity depends on an oxidative crosslink involving a cysteine residue in the N-terminal domain and thus formation of disulfide-linked dimers. Alkylated AnxA2 in which this cysteine residue has been modified and AnxA2 mutants lacking the N-terminal domain are not capable of bridging membrane surfaces. In contrast, a heterotetrameric complex comprising two membrane binding AnxA2 subunits linked by a S100A10 dimer can provide membrane contacts irrespective of oxidation status. Thus, monomeric AnxA2 only contains one lipid binding site and AnxA2-mediated linking of membrane surfaces under non-oxidative intracellular conditions most likely requires AnxA2-S100 complex formation.

David Grill

Institute of Medical Biochemistry,
Center for Molecular Biology of Inflammation,
Cluster of Excellence 'Cells in Motion',
Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation

University of Münster

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

David Grill

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute of Medical Biochemistry,Center for Molecular Biology of Inflammation,Cluster of Excellence 'Cells in Motion',Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation

University of Münster

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

Institute of Medical Biochemistry,
Center for Molecular Biology of Inflammation,
Cluster of Excellence 'Cells in Motion',
Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation