Involvement of phosphatidylinositol 4,5-bisphosphate in phosphatidylserine exposure in platelets: use of a permeant phosphoinositide-binding peptide.

During platelet activation, phosphatidylserine (PS) exposure on the extracellular face of the plasma membrane is associated with increased procoagulant activity. PS externalization is generally attributed to an increase in intracellular Ca(2+). Various phospholipid transporters, such as specific scramblases or proteins from the family of multidrug resistance proteins, and cofactors such as phosphatidylinositol 4,5-bisphosphate (PIP2) have been proposed to participate in this process. In this study, we used a membrane-permeant polycationic peptide (RhB-QRLFQVKGRR), derived from the PIP2-binding site of gelsolin (GS 160-169) and linked to rhodamine B, to investigate the role of PIP2 in PS externalization in whole platelets. The peptide penetrated rapidly into the platelets, specifically bound to PIP2, and induced PS exposure to a similar extent as thrombin or collagen, but independently of changes in intracellular Ca(2+) or phosphoinositide 3-kinase activity. A pretreatment of platelets with quercetin, an inhibitor of phosphoinositide metabolism, drastically decreased PS exposure induced by agonists or peptide. In large unilamellar vesicles (LUVs), the presence of PIP2 was strictly required for the induction of scrambling of NBD-labeled phospholipids (PC and PS) by the peptide. In inside-out vesicles from erythrocytes (IOVs), the peptide also induced redistribution of PC and PS. Our data suggest that, in intact platelets, PIP2 acts as a target of polycationic effectors, including Ca(2+), to promote PS exposure. The use of a membrane-permeant and fluorescent peptide which binds to PIP2 is a promising tool to investigate the role of PIP2 in various cellular processes.

Cell permeant polyphosphoinositide-binding peptides that block cell motility and actin assembly.

Polyphosphoinositides (PPIs) affect the localization and activities of many cellular constituents, including actin-modulating proteins. Several classes of polypeptide sequences, including pleckstrin homology domains, FYVE domains, and short linear sequences containing predominantly hydrophobic and cationic residues account for phosphoinositide binding by most such proteins. We report that a ten-residue peptide derived from the phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding region in segment 2 of gelsolin, when coupled to rhodamine B has potent PIP(2) binding activity in vitro; crosses the cell membrane of fibroblasts, platelets, melanoma cells, and neutrophils by a process not involving endocytosis; and blocks cell motility. This peptide derivative transiently disassembles actin filament structures in GFP-actin-expressing NIH3T3 fibroblasts and prevents thrombin- or chemotactic peptide-stimulated actin assembly in platelets and neutrophils, respectively, but does not block the initial [Ca(2+)] increase caused by these agonists. The blockage of actin assembly and motility is transient, and cells recover motility within an hour after their immobilization by 5-20 microm peptide. This class of reagents confirms the critical relation between inositol lipids and cytoskeletal structure and may be useful to probe the location and function of polyphosphoinositides in vivo.

Enhancement of phosphoinositide 3-kinase (PI 3-kinase) activity by membrane curvature and inositol-phospholipid-binding peptides.

The phosphorylation of phosphatidylinositol (PtdIns) on the 3' position of the inositol ring by phosphoinositide 3-kinase (PI 3-kinase) is shown to depend strongly on the curvature of liposomes containing a mixture of phosphatidylcholine (PtdCho) and PtdIns. Vesicles with an average diameter of 50 nm are phosphorylated 100 times faster than chemically identical vesicles with an average diameter greater than 300 nm. The low reactivity of large vesicles is not due to the difference in vesicle number for large and small vesicles at constant total lipid, nor to occlusion of lipid surfaces in multilammelar structures, and can be reversed by addition of low (< 1:100) molar ratios of either the PtdIns transfer protein sec14p or a ten-residue peptide derived from the inositol-phospholipid-binding site of gelsolin. Similar measurements using PI 4-kinase showed a weak dependence on vesicle size. The strong dependence of PI 3-kinase function on membrane curvature suggests possible localization of PI 3-kinase activity at sites where clustering of receptors, for example, may locally deform the membrane, and suggests that once PI 3-kinase is localized and activated at surface sites, the reaction may become self-accelerating.

Mechanical effects of neurofilament cross-bridges. Modulation by phosphorylation, lipids, and interactions with F-actin.

The structure of gels formed by bovine spinal cord neurofilaments was determined by fluorescence and electron microscopy and compared to mechanical properties measured by their elastic and viscous response to shear forces. Neurofilaments formed gels of high elastic modulus (>100 Pa) after addition of millimolar Mg2+. Gelation caused a slow increase in shear moduli to levels similar to those of vimentin intermediate filament networks, followed by a rapid rise due to formation of links between neurofilaments, mediated by cross-bridging structures that vimentin filaments lack. Neurofilament gels are more resistant to large deformations than are vimentin networks, suggesting the importance of cross-bridges for neurofilament mechanical properties. Fluorescence imaging of single neurofilaments showed flexible filaments that became straighter when they adhered to glass or were incorporated into filament bundles. Electron microscopy of neurofilament gels showed a system of bundles intertwined within a more isotropic network of individual filaments. Neurofilament gel formation was stimulated in vitro by acid phosphatase treatment or by inositol phospholipids. In contrast, addition of actin filaments reduced the resistance of neurofilament gels to large stresses. These results suggest that dynamic and regulated interactions occur between neurofilaments to form viscoelastic networks with properties distinct from other cytoskeletal structures.

Spectroscopic studies of a phosphoinositide-binding peptide from gelsolin: behavior in solutions of mixed solvent and anionic micelles.

The peptide G(150-169) corresponds to a phosphatidylinositol 4,5-bisphosphate (PIP2) and filamentous actin (F-actin) binding site on gelsolin (residues 150-169, with the sequence KHVVPNEVVVQRLFQVKGRR). The conformation of this peptide in trifluoroethanol (TFE) aqueous solution was determined by 1H nuclear magnetic resonance as the first step toward understanding the structural aspects of the interaction of G(150-169) and PIP2. The circular dichroism experiments show that G(150-169) adopts a predominantly alpha-helical form in both 50% TFE aqueous solution and in the presence of PIP2 micelles, therefore establishing a connection between the two conformations. 1H nuclear magnetic resonance experiments of G(150-169) in TFE co-solvent show that the helical region extends from Pro-154 to Lys-166. The amphiphilic nature of this helical structure may be the key to understanding the binding of the peptide to lipids. Sodium dodecyl sulfate micelle solution is used as a model for anionic lipid environments. Preliminary studies of the conformation of G(150-169) in sodium dodecyl sulfate micelle solution show that the peptide forms an alpha-helix similar to but with some structural differences from that in TFE co-solvent. Fluorescence experiments provide evidence of peptide clustering over a narrow range of peptide/PIP2 ratios, which is potentially relevant to the biological function of PIP2.

Use of a gel-forming dipeptide derivative as a carrier for antigen presentation.

A dipeptide of the formula Fmoc-Leu-Asp and some other related dipeptides were synthesized in solution by standard methods. When such peptides are dissolved in water at concentrations below 1% at 100 degrees C and cooled below 60 degrees C they form turbid solutions and eventually viscoelastic gels at lower temperatures. Such gels are thermoreversible and can also be disrupted by mechanical agitation. At a concentration of 2 mg/ml the peptide Fmoc-Leu-Asp forms an aqueous gel at 60 degrees C with a shear modulus of 80 Pa measured at a frequency of 1 rad/s. Peptide solutions undergo an abrupt increase in light scattering between 1 and 1.5 mg/ml at both 23 and 60 degrees C. By analogy with previous observations of other systems, this increase appears to be due to the formation of filamentous micelles and the aggregation of filaments into a three-dimensional network. When low molecular weight adamantanamine derivatives, which are inherently non-antigenic antiviral drugs, were incorporated into the Fmoc-Leu-Asp gel and injected into rabbits, high titre specific antibodies were efficiently produced without the need for additional adjuvant. Both the physical properties of the gel and its effect on the antigenicity of low molecular weight compounds suggest a number of practical applications.

Optical properties of colloidal carbon and colloidal silica films.

Colloidal silica films on glass produced by withdrawal from colloidal baths are of value as antireflection coatings. The effect of particle size on the refractive index and density of these films was determined, and the optimum particle diameter for maximum transmittance of solar radiation through glass was estimated at 45 nm. Mixed colloidal carbon and silica films produced by the same means were also studied. These films are useful as solar selective absorbing coatings when deposited on copper. The optical constants of the mixed colloidal films were determined and the results compared with the predictions of the Maxwell Garnett theory. The effect on the thermal efficiency of solar absorbing coatings of a variation in the carbon-to-silica weight ratio was determined.