The distribution of polyphosphoinositides in lipid films
Introduction
Phosphorylated derivatives of phosphatidylinositol, also called polyphosphoinositides (PPIs), are essential components of many cellular signal transduction pathways [1], [2], [3]. In addition to the function of PIP2 (phosphatidylinositol-4,5-bisphosphate) as a substrate for phospholipase C (PLC) to produce diacylglycerol (DAG) and inositol trisphosphate (IP3) [4], phosphorylated inositol lipids themselves function to activate, inactivate, or localize a large number of proteins involved in cytoskeletal organization, cell adhesion, ion transport, vesicle trafficking, and cell division. In a few cases, binding of a cytosolic protein to PIP2 involves sole interaction with the headgroup of a single lipid molecule [5], but in most cases the PPI must be contained within a multimolecular complex of lipids to bind its ligand, even if the resulting stoichiometry of binding is almost 1:1 [6].
In biological membranes, the sum of PPIs (including all isomers of PIP, PIP2 and PIP3) constitute no more than a few percent of total lipid; but in vitro, several proteins do not bind PIP2 unless it is present in bilayers at approximately 10 mol% [6], [7]. This result and the finding that specialized regions of the plasma membrane such as caveoli or lipid rafts may be highly enriched (>20%) in PPI [8] suggest that clustering of inositol lipids in specialized regions of a lipid bilayer may be an important aspect of their ability to activate specific ligands. Moreover, transitions in the packing of PPI induced by one protein may alter the lipid's reactivity with other proteins.
While a great number of studies have characterized binding of various PPIs to many proteins or implicated these interactions in specific cellular functions, there is very little known about how PPIs are distributed in a lipid bilayer, and the assumption that these lipids randomly diffuse through the membrane is at odds with its apparent ability to stimulate activities that require its concentration or clustering. Neither PIP2 (phosphatidylinositol-4,5-bisphosphate or phosphatidylinositol-3,4-bisphosphate) nor PIP3 (phosphatidylinositol-3,4,5-trisphosphate) are stable in a bilayer vesicle and instead, form spherical or rod-like micelles in water [9], [10], and ribbon-like lamellar aggregates in the presence of divalent cations [11]. The large size of the inositol rings relative to other lipid headgroups, together with the large net negative charge of PPIs suggest that these lipids may strongly perturb the distribution of other lipids that are stable in flat bilayers and so, might influence or initiate changes in lipid texture or curvature that could affect cellular functions.
In this paper, it is shown that the effect of PPIs on the texture of reconstituted Langmuir films is dramatic and can be explained using electrostatic arguments. The effect of the polyphosphoinositides on the structure of lipid films was explored using Langmuir monolayers as a model system with the goal of isolating the effect of charged lipids on membrane domains. A large change in the shape of domains in the gas/liquid-expanded region was observed as the ratio of phosphatidylinositol monophosphate (PIP) to phosphatidylcholine (PC) was increased. This effect is reversed by screening electrostatic interactions using a monovalent salt, confirming that the effect is due to electrostatics and not steric interactions. This result can be explained using a framework that is, at a fundamental level, similar to the theory of the effect of cholesterol on lipid films. This earlier theory has been elegantly tested in a series of experiments conducted by McConnell et al. [12], [13], [14], [15].
Section snippets
Materials and techniques
l-α-Phosphatidylcholine and DTPC (Ditridecanoylphosphatidylcholine) were purchased from Avanti (Alabaster, AL, USA), l-α-phosphatidylinositol 4-monophosphate, and sodium salt was purchased from Sigma (St. Louis, MO, USA). These two lipids are natural products containing a variety of acyl chains with a significant fraction of unsaturated chains preventing the formation of gel phases. N-(Texas Red sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine (referred to here as Texas
Theory
Taking into account both dipole–dipole and charge–charge interactions, the free energy of a mixed lipid monolayer system is
This can be re-written in an alternative form using the following steps:
Green's theoremresults in the identitywhere and Δ is small.
Thus,Where λ is the line tension, μ is the dipole density, ρ is the
The effect of PIP on PC monolayer texture
Lipid films were produced on a HEPES-buffered (pH 7.0) solution containing 10 mM HEPES, 0.1 mM EDTA underlying PC and PIP with PC:PIP ratios of 100:0, 90:10, 50:50 and 0:100, respectively. Under these conditions the headgroup of PIP carries a nominal charge of approximately −2.5. In most experiments, 1% Texas Red-labeled PE was added to the mixtures to allow observation of the films using florescence microscopy. As the concentration of PIP was increased, the texture of the film changed, from
Discussion
The phosphorylated inositol phospholipids are key components of signal transduction pathways as well as being important in the regulation of numerous enzymes. For example, the actin binding protein gelsolin is regulated by its interaction with phosphorylated PI. Also, specific inositol lipid-binding structures, such as the pH and FYVE domains, are thought to have a role in localizing proteins that contain these domains to membranes. Overexpression of either the kinases that produce cellular PIP2
Acknowledgements
This work was supported by a grant from the US National Institutes of Health AR38910. PAJ wishes to thank Paavo Kinnunen and Juha Holopainen for their helpful discussions and the Sigrid Juselius Foundation for additional support.
References (21)
- et al.
Phospholipid signaling
Cell
(1995) - et al.
Phosphatidylinositol 4,5-bisphosphate (PIP2)-enhanced G protein-coupled receptor kinase (GRK) activity. Location, structure, and regulation of the PIP2 binding site distinguishes the GRK subfamilies
J. Biol. Chem.
(1996) Structure of molecular aggregates of 1-(3-sn-phosphatidyl)-l-myo-inositol 3,4-bis(phosphate) in water
Biochim Biophys Acta
(1981)- et al.
The structure of divalent cation-induced aggregates of PIP2 and their alteration by gelsolin and tau
Biophys. J.
(1997) - et al.
Identification of a polyphosphoinositide-binding sequence in an actin monomer-binding domain of gelsolin
J. Biol. Chem.
(1992) - et al.
Massive actin polymerization-induced by phosphatidylinositol-4-phosphate 5-kinase in vivo
J. Biol. Chem.
(1997) - et al.
Phosphoinositides as regulators in membrane traffic
Science
(1996) Phosphoinositides and calcium as regulators of cellular actin assembly and disassembly
Ann. Rev. Physiol.
(1994)Inositol trisphosphate and diacylglycerol as second messengers
Biochem.
(1984)- et al.
Structure of the binding site for inositol phosphates in a pH domain
Embo J.
(1995)
Cited by (12)
Lipid Monolayers at the Air-Water Interface. A Tool for Understanding Electrostatic Interactions and Rheology in Biomembranes
2014, Advances in Planar Lipid Bilayers and LiposomesCitation Excerpt :Compared to a neutral domain, the free energy of the domain has an additional term that takes into account the effect of charge–charge repulsion on the domain shape. Then, as expected, they found that at a high charge density, a noncircular shape minimizes the domain energy and the critical size at which instability occurs, increases with the ionic strength [109,110]. In addition, Loverde et al. [111] explored the asphericity of charged domains using molecular dynamic simulations and also found that increasing the electrostatic contribution influences the shape of the domains and strongly increases correlation and ordering between domains.
Structural investigation on the adsorption of the MARCKS peptide on anionic lipid monolayers - Effects beyond electrostatic
2011, Chemistry and Physics of LipidsCitation Excerpt :Both acidic lipids show a complete different behavior in the simple lipid mixture depending on their charge. It was found by Dietrich et al. (2009), that the polyvalent PIP2 molecules were arranged in a uniform distribution in the disordered phase of the mixed monolayer, which is consistent with the literature (DeWolf et al., 1999; Foster and Janmey, 2001; Fernandes et al., 2006). This lateral distribution was presumably caused by the perpendicular orientation of the PI(4,5) group to the membrane interface (Zhou et al., 1997; Bradshaw et al., 1999) and their repulsive interaction (Leventhal et al., 2008).
Interaction of the MARCKS peptide with PIP<inf>2</inf> in phospholipid monolayers
2009, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :A schematic depiction of these changes is illustrated in Fig. 7. We were able to show the presence of a uniform distribution of the PIP2 molecules in the disordered phase of the mixed monolayer, which is consistent with literature [51–53]. This lateral distribution is obviously caused by the perpendicular orientation of the PI(4,5) group to the membrane interface [47,48].
Protein kinase C mediates translocation of type II phosphatidylinositol 5-phosphate 4-kinase required for platelet α-granule secretion
2003, Journal of Biological ChemistryCitation Excerpt :We, therefore, sought to assess the hypothesis that type II PIPK mediates the recruitment of PtdIns(4,5)P2-binding proteins in platelets in an agonist-dependent manner. For these experiments, BAPTA-AM-treated platelets were incubated in the presence or absence of SFLLRN and subsequently permeabilized in the presence of a FITC-labeled PtdIns(4,5)P2-binding peptide, FITC-QRLFQVKGRR, derived from segment 2 of gelsolin (amino acids 160–169) (30, 44, 45). The rhodamine B-conjugated form of this peptide, termed PBP10, binds PtdIns(4,5)P2 preferentially to PtdIns(4)P or PtdIns and fails to interact significantly with phosphatidylserine or phosphatidylcholine (30).
Phosphoinositides and Actin Cytoskeletal Rearrangement
2003, Handbook of Cell Signaling: Volume 1-3Cell Permeant Polyphosphoinositide-binding Peptides that Block Cell Motility and Actin Assembly
2001, Journal of Biological ChemistryCitation Excerpt :In addition, binding of these different ligands may have different effects on lipid distribution in complex bilayers. Recent studies of lipid monolayers show that peptides similar to PBP10 localize to domains enriched in PIP when such domains form at moderate surface pressures (47), and the binding of peptides can further reorganize the lipid packing. If, asin vitro studies suggest, different classes of PPI-binding peptides have different degrees of interaction with the hydrophobic domain of lipid bilayers, they would likewise have very different effects on surface pressure of monolayers and therefore on such features as local curvature or lipid demixing in bilayers.