Yu-Hsiu Wang

Divalent Cation-Dependent Formation of Electrostatic PIP2 Clusters in Lipid Monolayers

Polyphosphoinositides are among the most highly charged molecules in the cell membrane, and the most common polyphosphoinositide, phosphatidylinositol-4,5-bisphosphate (PIP(2)), is involved in many mechanical and biochemical processes in the cell membrane. Divalent cations such as calcium can cause clustering of the polyanionic PIP(2), but the origin and strength of the effective attractions leading to clustering has been unclear. In addition, the question of whether the ion-mediated attractions could be strong enough to alter the mechanical properties of the membrane, to our knowledge, has not been addressed. We study phase separation in mixed monolayers of neutral and highly negatively charged lipids, induced by the addition of divalent positively charged counterions, both experimentally and numerically. We find good agreement between experiments on mixtures of PIP(2) and 1-stearoyl-2-oleoyl phosphatidylcholine and simulations of a simplified model in which only the essential electrostatic interactions are retained. In addition, we find numerically that under certain conditions the effective attractions can rigidify the resulting clusters. Our results support an interpretation of PIP(2) clustering as governed primarily by electrostatic interactions. At physiological pH, the simulations suggest that the effective attractions are strong enough to give nearly pure clusters of PIP(2) even at small overall concentrations of PIP(2).

Polyelectrolyte properties of filamentous biopolymers and their consequences in biological fluids

Anionic polyelectrolyte filaments are common in biological cells. DNA, RNA, the cytoskeletal filaments F-actin, microtubules, and intermediate filaments, and polysaccharides such as hyaluronan that form the pericellular matrix all have large net negative charge densities distributed over their surfaces. Several filamentous viruses with diameters and stiffnesses similar to those of cytoskeletal polymers also have similar negative charge densities. Extracellular protein filaments such collagen, fibrin and elastin, in contrast, have notably smaller charge densities and do not behave as highly charged polyelectrolytes in solution. This review summarizes data that demonstrate generic counterion-mediated effects on four structurally unrelated biopolymers of similar charge density: F-actin, vimentin, Pf1 virus, and DNA, and explores the possible biological and pathophysiological consequences of the polyelectrolyte properties of biological filaments.

Counterion-mediated cluster formation by polyphosphoinositides.

Polyphosphoinositides (PPI) and in particular PI(4,5)P2, are among the most highly charged molecules in cell membranes, are important in many cellular signaling pathways, and are frequently targeted by peripheral polybasic proteins for anchoring through electrostatic interactions. Such interactions between PIP2 and proteins containing polybasic stretches depend on the physical state and the lateral distribution of PIP2 within the inner leaflet of the cells lipid bilayer. The physical and chemical properties of PIP2 such as pH-dependent changes in headgroup ionization and area per molecule as determined by experiments together with molecular simulations that predict headgroup conformations at various ionization states have revealed the electrostatic properties and phase behavior of PIP2-containing membranes. This review focuses on recent experimental and computational developments in defining the physical chemistry of PIP2 and its interactions with counterions. Ca(2+)-induced changes in PIP2 charge, conformation, and lateral structure within the membrane are documented by numerous experimental and computational studies. A simplified electrostatic model successfully predicts the Ca(2+)-driven formation of PIP2 clusters but cannot account for the different effects of Ca(2+) and Mg(2+) on PIP2-containing membranes. A more recent computational study is able to see the difference between Ca(2+) and Mg(2+) binding to PIP2 in the absence of a membrane and without cluster formation. Spectroscopic studies suggest that divalent cation- and multivalent polyamine-induced changes in the PIP2 lateral distribution in model membrane are also different, and not simply related to the net charge of the counterion. Among these differences is the capacity of Ca(2+) but not other polycations to induce nm scale clusters of PIP2 in fluid membranes. Recent super resolution optical studies show that PIP2 forms nanoclusters in the inner leaflet of a plasma membrane with a similar size distribution as those induced by Ca(2+) in model membranes. The mechanisms by which PIP2 forms nanoclusters and other structures inside a cell remain to be determined, but the unique electrostatic properties of PIP2 and its interactions with multivalent counterions might have particular physiological relevance.

Counterion-mediated pattern formation in membranes containing anionic lipids

Most lipid components of cell membranes are either neutral, like cholesterol, or zwitterionic, like phosphatidylcholine and sphingomyelin. Very few lipids, such as sphingosine, are cationic at physiological pH. These generally interact only transiently with the lipid bilayer, and their synthetic analogs are often designed to destabilize the membrane for drug or DNA delivery. However, anionic lipids are common in both eukaryotic and prokaryotic cell membranes. The net charge per anionic phospholipid ranges from -1 for the most abundant anionic lipids such as phosphatidylserine, to near -7 for phosphatidylinositol 3,4,5 trisphosphate, although the effective charge depends on many environmental factors. Anionic phospholipids and other negatively charged lipids such as lipopolysaccharides are not randomly distributed in the lipid bilayer, but are highly restricted to specific leaflets of the bilayer and to regions near transmembrane proteins or other organized structures within the plane of the membrane. This review highlights some recent evidence that counterions, in the form of monovalent or divalent metal ions, polyamines, or cationic protein domains, have a large influence on the lateral distribution of anionic lipids within the membrane, and that lateral demixing of anionic lipids has effects on membrane curvature and protein function that are important for biological control.

Structural Basis for PI(4)P-Specific Membrane Recruitment of the Legionella pneumophila Effector DrrA/SidM.

Recruitment of the Legionella pneumophila effector DrrA to the Legionella-containing vacuole, where it activates and AMPylates Rab1, is mediated by a P4M domain that binds phosphatidylinositol 4-phosphate [PI(4)P] with high affinity and specificity. Despite the importance of PI(4)P in Golgi trafficking and its manipulation by pathogens, the structural bases for PI(4)P-dependent membrane recruitment remain poorly defined. Here, we determined the crystal structure of a DrrA fragment including the P4M domain in complex with dibutyl PI(4)P and investigated the determinants of phosphoinositide recognition and membrane targeting. Headgroup recognition involves an elaborate network of direct and water-mediated interactions with basic and polar residues in the context of a deep, constrictive binding pocket. An adjacent hydrophobic helical element packs against the acyl chains and inserts robustly into PI(4)P-containing monolayers. The structural, biochemical, and biophysical data reported here support a detailed structural mechanism for PI(4)P-dependent membrane targeting by DrrA.

Gelation of semiflexible polyelectrolytes by multivalent counterions

Filamentous polyelectrolytes in aqueous solution aggregate into bundles by interactions with multivalent counterions. These effects are well documented by experiment and theory. Theories also predict a gel phase in isotropic rodlike polyelectrolyte solutions caused by multivalent counterion concentrations much lower than those required for filament bundling. We report here the gelation of Pf1 virus, a model semiflexible polyelectrolyte, by the counterions Mg(2+), Mn(2+) and spermine(4+). Gelation can occur at 0.04% Pf1 volume fraction, which is far below the isotropic-nematic transition of 0.7% for Pf1 in monovalent salt. Unlike strongly crosslinked gels of semiflexible polymers, which stiffen at large strains, Pf1 gels reversibly soften at high strain. The onset strain for softening depends on the strength of interaction between counterions and the polyelectrolyte. Simulations show that the elasticity of counterion crosslinked gels is consistent with a model of semiflexible filaments held by weak crosslinks that reversibly rupture at a critical force.

Cholesterol-Dependent Phase-Demixing in Lipid Bilayers as a Switch for the Activity of the Phosphoinositide-Binding Cytoskeletal Protein Gelsolin

The lateral distribution of phosphatidylinositol 4,5-bisphosphate (PIP2) in lipid bilayers is affected both by divalent cation-mediated attractions and cholesterol-dependent phase demixing. The effects of lateral redistribution of PIP2 within a membrane on PIP2-protein interactions are explored with an N-terminal fragment of gelsolin (NtGSN) that severs actin in a Ca(2+)-insensitive manner. The extent of NtGSN inhibition by PIP2-containing large unilamellar vesicles (LUVs) depends on the lateral organization of the membrane as quantified by an actin-severing assay. At a fixed PIP2 mole fraction, the inhibition is largely enhanced by the segregation of liquid ordered/liquid disordered (Lo/Ld) phases that is induced by altering either cholesterol content or temperature, whereas the presence of Ca(2+) only slightly improves the inhibition. Inhibition of gelsolin induced by demixed LUVs is more effective with decreasing temperature, coincident with increasing membrane order as determined by Laurdan generalized polarization and is reversible as the temperature increases. This result suggests that PIP2-mediated inhibition of gelsolin function depends not only on changes in global concentration but also on lateral distribution of PIP2. These observations imply that gelsolin, and perhaps other PIP2-regulated proteins, can be activated or inactivated by the formation of nanodomains or clusters without changing PIP2 bulk concentration in the cell membrane.

The Competitive Binding of Ca2+ vs. Mg2+ to PIP2-Containing Lipid Monolayers and the Comparison of Pi(4,5)P2 and its Isomers

Interactions between divalent cations and PtdIns(4,5)2-containing lipid model membrane are investigated. Surface pressure measurements of lipid monolayers formed by binary lipid mixtures containing 25mol% PIP2 were used to quantify divalent cation binding. Direct titration and competitive binding assays show that divalent cations including Mg2+, Ca2+, Sr2+, Ba2+, Co2+, Ni2+, Cu2+, Zn2+ and some multivalent polyamines bind PIP2 with Kd ranges from 200nM-50uM. Some of these cations, which are considered physiologically important, were examined more closely and evidence for divalent cation-induced lateral segregation of PIP2 is presented. Such studies are based on fluorescence and atomic force microscopic studies of Langmuir-Schaeffer lipid monolayers, and are coupled with numerical studies. Ca2+ and Mg2+ have similar binding affinity to PIP2-containing monolayers, but Ca2+ induces PIP2 lateral segregation more efficiently than compared to Mg2+. Ca2+ vs. Mg2+ competitive binding assays are also carried out on PtdIns(3,4)P2- and PtdIns(3,5)P2-containing monolayers. Significant differences in the response of these three PIP2 isomers to divalent cations are found.Besides monolayer studies, PIP2-cation interactions are also examined on a bilayer system. Fluorescence correlation spectroscopy (FCS) and laser scanning confocal microscopy (LSCM) are used following divalent cation titration on asymmetric labeled PIP2-containing giant unilamellar vesicles (GUVs). The diffusion coefficient and the fluorescence intensity variance of fluorescent PIP2 change with increasing divalent cation concentration. Forster resonance energy transfer (FRET) studies using bi-color labeled PIP2 are also carried out in a large unilamellar vesicle (LUV) system. These results together reveal that the lateral inhomogeneity of PIP2 changes with divalent cation concentration on bilayer model membranes. These results may provide insight into divalent cation-induced PIP2 microdomain formation in the cell membrane.