Elsevier

Biophysical Chemistry

Volume 91, Issue 3, 24 July 2001, Pages 211-218
Biophysical Chemistry

The distribution of polyphosphoinositides in lipid films

https://doi.org/10.1016/S0301-4622(01)00171-5Get rights and content

Abstract

Fluorescence microscopy of Langmuir films is used to determine the effect of polyphosphoinositides (PPIs) on the structure of phosphatidylcholine-containing monolayers. Dramatic alterations in the texture of these films occur as the fraction of PPI in the film is altered. These changes depend on the ionic strength of the underlying subphase and can be accounted for by considering the electrostatic interactions among PPIs. Surface adsorption of a fluorescent peptide derivative based on the PPI binding site of the protein gelsolin co-localizes with PPI-rich domains. Localization of polyphosphoinositides in domains within the inner leaflet of the plasma membrane is proposed to be a key element in some aspects of intracellular signaling, and these data have implications for explaining the cause of restructuring of such domains.

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 isF=λ∮dr+μ22∫∫dAdAr−r′3+ρ22∫∫dAdAr−r′

This can be re-written in an alternative form using the following steps:

Green's theorem∫∫2Q∂x′∂x+2P∂y′∂ydAdA′=∯Pddx′+Qddy′results in the identity∫∫ξm+2dAdA′=−m2∯ξ−mddr′+Δm+2m∫∫ξm+4dAdA′where ξ=r−r′2+Δ212 and Δ is small.

Thus,F=Aε+λ−μ2dr−μ22ddr′r−r′ρ22r−r′ddr′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)

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