Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC).In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the beta-subunit, possibly in, or in close proximity of, the ankyrin-binding site.In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.     

Brain spectrin exerts much stronger effect on anionic phospholipid monolayers than erythroid spectrin

Red blood cell spectrin and its nonerythroid analogues are linked to integral proteins of the membrane by several skeletal protein receptors, such as ankyrin and protein 4.1 together with p55. However, there are also many reasons for believing that they are insufficient to engender all the properties that characterise the native membrane. Therefore, we are concerned with the mechanism by which brain spectrin interacts with phospholipids of the membrane bilayer. Brain and erythrocyte spectrin were shown previously to bind phospholipid vesicles as well as monolayers prepared from aminophospholipids: phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (PC).In the present study, it is shown that brain spectrin binds to monolayers prepared from anionic phospholipids, such as phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidyl glycerol, diphosphatidylglycerol, and their mixtures with PC. Brain spectrin injected into the subphase to reach nanomolar concentration induced a substantial increase in the surface pressure of monolayers prepared from the phospholipids and their mixtures mentioned above, possibly by penetrating them. This effect is stronger in the case of monolayers prepared from anionic phospholipids alone and weaker when monolayers were prepared from mixtures with PC. The weakest effect was observed in the case of phosphatidylinositol-4,5-bisphosphate monolayers. An interaction of brain spectrin with monolayers prepared from anionic phospholipids (PI/PC 7:3 and PA/PC 7:3) was inhibited (PI/PC much stronger than PA/PC) by purified erythrocyte ankyrin, which indicates that the binding site for those lipids is located in the β-subunit, possibly in, or in close proximity of, the ankyrin-binding site.In contrast, erythrocyte spectrin injected into the subphase induced a change in the surface pressure of monolayers prepared from anionic phospholipids, which was equal or smaller than the value of surface pressure change induced by protein without a monolayer. This effect was different from what had been observed previously for monolayers prepared from aminophospholipids and their mixtures with PC, and from the data for nonerythroid spectrin presented here.     

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P₂ and other acidic phospholipids in a Ca²⁺-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P₂, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P₂ clustering are also capable of forming interconnections between PI(4,5)P₂-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P₂ and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P₂-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.     

Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase.

Pleckstrin homology (PH) domains may act as membrane localization modules through specific interactions with phosphoinositide phospholipids. These interactions could represent responses to second messengers, with scope for regulation by soluble inositol polyphosphates. A biosensor-based assay was used here to probe interactions between PH domains and unilamellar liposomes containing different phospholipids and to demonstrate specificity for distinct phosphoinositides. The dynamin PH domain specifically interacted with liposomes containing phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] and, more weakly, with liposomes containing phosphatidylinositol-4-phosphate [PI(4)P]. This correlates with phosphoinositide activation of the dynamin GTPase. The functional GTPase of a dynamin mutant lacking the PH domain, however, cannot be activated by PI(4,5)P2. The phosphoinositide-PH domain interaction can be abolished selectively by point mutations in the putative binding pocket predicted by molecular modelling and NMR spectroscopy. In contrast, the Bruton's tyrosine kinase (Btk)PH domain specifically bound liposomes containing phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]: an interaction requiring Arg28, a residue found to be mutated in some X-linked agammaglobulinaemia patients. A rational explanation for these different specificities is proposed through modelling of candidate binding pockets and is supported by NMR spectroscopy.     

PIP3, PIP2, and cell movement--similar messages, different meanings?

The inositol lipids PI(4,5)P(2) and PI(3,4,5)P(3) are important regulators of actin polymerization, but their different temporal and spatial dynamics suggest that they perform separate roles. PI(3,4,5)P(3) seems to act as an instructive second messenger, inducing local actin polymerization. PI(4,5)P(2) appears to be present at too high a concentration and homogeneous a distribution to fulfil a similar role. Instead, we suggest that PI(4,5)P(2) acts permissively, restricting new actin polymerization to the region of the plasma membrane.     

Anionic lipids activate group IVA cytosolic phospholipase A2 via distinct and separate mechanisms

Previously, ceramide-1-phosphate (C1P) and phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] were demonstrated to be potent and specific activators of group IVA cytosolic phospholipase A2 (cPLA2alpha). In this study, we hypothesized that these anionic lipids functionally activated the enzyme by distinctly different mechanisms. Indeed, surface plasmon resonance and surface dilution kinetics demonstrated that C1P was a more potent effector than PI(4,5)P2 in decreasing the dissociation constant of the cPLA2alpha-phosphatidylcholine (PC) interaction and increasing the residence time of the enzyme on the vesicles/micelles. PI(4,5)P2, in contrast to C1P, decreased the Michaelis-Menten constant, increasing the catalytic efficiency of the enzyme. Furthermore, PI(4,5)P2 activated cPLA2alpha with a stoichiometry of 1:1 versus C1P at 2.4:1. Lastly, PI(4,5)P2, but not C1P, increased the penetration ability of cPLA2alpha into PC-rich membranes. Therefore, this study demonstrates two distinct mechanisms for the activation of cPLA2alpha by anionic lipids. First, C1P activates cPLA2alpha by increasing the residence time of the enzyme on membranes. Second, PI(4,5)P2 activates the enzyme by increasing catalytic efficiency through increased membrane penetration.     

PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells

A phosphatidylinositol (PI) kinase activity associated with certain protein tyrosine kinases important in cell proliferation phosphorylates the 3' hydroxyl position of PI to produce phosphatidylinositol-3-phosphate (PI-3-P). Here we report that, in addition to PI-3' kinase activity, anti-phosphotyrosine (alpha-P-tyr) immunoprecipitates from platelet-derived growth factor (PDGF)-stimulated smooth muscle cells (SMC) contain lipid kinase activities that utilize the substrates phosphatidylinositol-4-phosphate (PI-4-P) and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). These activities are absent in alpha-P-tyr immunoprecipitates from quiescent SMC. The product of PI-4-P phosphorylation appears to be phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2), a lipid not previously reported. The product of PI-4,5-P2 phosphorylation is phosphatidylinositol-trisphosphate (PIP3). PI-3-P was detected in quiescent SMC and increased only slightly in response to PDGF. PIP3 and the putative PI-3,4-P2 appeared only after the addition of mitogen. Both the temporal production of these novel phospholipids after PDGF stimulation and the observation of the enzymatic activities that produce them in alpha-P-tyr immunoprecipitates suggest that these phospholipids are excellent candidates for mediators of the PDGF mitogenic response.     

Transition between conformational states of the TREK-1 K2P channel promoted by interaction with PIP2

Members of the TREK family of two-pore domain potassium channels are highly sensitive to regulation by membrane lipids, including phosphatidylinositol-4,5-bisphosphate (PIP₂). Previous studies have demonstrated that PIP₂ increases TREK-1 channel activity; however, the mechanistic understanding of the conformational transitions induced by PIP₂ remain unclear. Here, we used coarse-grained molecular dynamics and atomistic molecular dynamics simulations to model the PIP₂-binding site on both the up and down state conformations of TREK-1. We also calculated the free energy of PIP₂ binding relative to other anionic phospholipids in both conformational states using potential of mean force and free-energy-perturbation calculations. Our results identify state-dependent binding of PIP₂ to sites involving the proximal C-terminus, and we show that PIP₂ promotes a conformational transition from a down state toward an intermediate that resembles the up state. These results are consistent with functional data for PIP₂ regulation, and together provide evidence for a structural mechanism of TREK-1 channel activation by phosphoinositides.     

A map of the subcellular distribution of phosphoinositides in the erythrocytic cycle of the malaria parasite Plasmodium falciparum

Despite representing a small percentage of the cellular lipids of eukaryotic cells, phosphoinositides (PIPs) are critical in various processes such as intracellular trafficking and signal transduction. Central to their various functions is the differential distribution of PIP species to specific membrane compartments through the actions of kinases, phosphatases and lipases. Despite their importance in the malaria parasite lifecycle, the subcellular distribution of most PIP species in this organism is still unknown. We here localise several species of PIPs throughout the erythrocytic cycle of Plasmodium falciparum. We show that PI3P is mostly found at the apicoplast and the membrane of the food vacuole, that PI4P associates with the Golgi apparatus and the plasma membrane and that PI(4,5)P2, in addition to being detected at the plasma membrane, labels some cavity-like spherical structures. Finally, we show that the elusive PI5P localises to the plasma membrane, the nucleus and potentially to the transitional endoplasmic reticulum (ER). Our map of the subcellular distribution of PIP species in P. falciparum will be a useful tool to shed light on the dynamics of these lipids in this deadly parasite.     

Membrane Lipid Co-Aggregation with α-Synuclein Fibrils

Amyloid deposits from several human diseases have been found to contain membrane lipids. Co-aggregation of lipids and amyloid proteins in amyloid aggregates, and the related extraction of lipids from cellular membranes, can influence structure and function in both the membrane and the formed amyloid deposit. Co-aggregation can therefore have important implications for the pathological consequences of amyloid formation. Still, very little is known about the mechanism behind co-aggregation and molecular structure in the formed aggregates. To address this, we study in vitro co-aggregation by incubating phospholipid model membranes with the Parkinson’s disease-associated protein, α-synuclein, in monomeric form. After aggregation, we find spontaneous uptake of phospholipids from anionic model membranes into the amyloid fibrils. Phospholipid quantification, polarization transfer solid-state NMR and cryo-TEM together reveal co-aggregation of phospholipids and α-synuclein in a saturable manner with a strong dependence on lipid composition. At low lipid to protein ratios, there is a close association of phospholipids to the fibril structure, which is apparent from reduced phospholipid mobility and morphological changes in fibril bundling. At higher lipid to protein ratios, additional vesicles adsorb along the fibrils. While interactions between lipids and amyloid-protein are generally discussed within the perspective of different protein species adsorbing to and perturbing the lipid membrane, the current work reveals amyloid formation in the presence of lipids as a co-aggregation process. The interaction leads to the formation of lipid-protein co-aggregates with distinct structure, dynamics and morphology compared to assemblies formed by either lipid or protein alone.     

A steep phosphoinositide bis-phosphate gradient forms during fungal filamentous growth

Membrane lipids have been implicated in many critical cellular processes, yet little is known about the role of asymmetric lipid distribution in cell morphogenesis. The phosphoinositide bis-phosphate PI(4,5)P(2) is essential for polarized growth in a range of organisms. Although an asymmetric distribution of this phospholipid has been observed in some cells, long-range gradients of PI(4,5)P(2) have not been observed. Here, we show that in the human pathogenic fungus Candida albicans a steep, long-range gradient of PI(4,5)P(2) occurs concomitant with emergence of the hyphal filament. Both sufficient PI(4)P synthesis and the actin cytoskeleton are necessary for this steep PI(4,5)P(2) gradient. In contrast, neither microtubules nor asymmetrically localized mRNAs are critical. Our results indicate that a gradient of PI(4,5)P(2), crucial for filamentous growth, is generated and maintained by the filament tip-localized PI(4)P-5-kinase Mss4 and clearing of this lipid at the back of the cell. Furthermore, we propose that slow membrane diffusion of PI(4,5)P(2) contributes to the maintenance of such a gradient.     

Courier service for phosphatidylinositol: PITPs deliver on demand

Phosphatidylinositol is the parent lipid for the synthesis of seven phosphorylated inositol lipids and each of them play specific roles in numerous processes including receptor-mediated signalling, actin cytoskeleton dynamics and membrane trafficking. PI synthesis is localised to the endoplasmic reticulum (ER) whilst its phosphorylated derivatives are found in other organelles where the lipid kinases also reside. Phosphorylation of PI to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P₂) at the plasma membrane and to phosphatidylinositol 4-phosphate (PI4P) at the Golgi are key events in lipid signalling and Golgi function respectively. Here we review a family of proteins, phosphatidylinositol transfer proteins (PITPs), that can mobilise PI from the ER to provide the substrate to the resident kinases for phosphorylation. Recent studies identify specific and overlapping functions for the three soluble PITPs (PITPα, PITPβ and PITPNC1) in phospholipase C signalling, neuronal function, membrane trafficking, viral replication and in cancer metastases.     

Is DRM lipid composition relevant in cell-extracellular matrix adhesion structures?

Focal adhesions mediate cell-extracellular matrix adhesion. They are inserted in detergent-resistant membrane microdomains enriched in phosphatidylinositol-4,5-bisphosphate. In spite of the relevance that membrane lipids appear to have on cell adhesion structures, to our knowledge, there are no previous reports on the membrane lipid composition where focal adhesions are located in vivo or on how changes in local membrane composition contribute to focal adhesion maintenance. This may be due to the fact that the explosion of information in the fields of genomics and proteomics has not been matched by a corresponding advancement of knowledge in the field of lipids. The physiological importance of lipids is illustrated by the numerous diseases to which lipid abnormalities contribute. To gain insight into the role of membrane lipid composition in the preservation of epithelial cell adhesion to the substratum, how specific changes in the membrane lipid composition in vivo affect the maintenance of focal adhesions in renal papillae collecting duct cells has been previously studied. It is currently considered that phosphatidylinositol-4,5-bisphosphate plays a crucial role in the maintenance of assembled focal adhesion. However, such pool of polyphosphoinositides has to be part of a domain of a specific lipid composition to serve as a membrane lipid stabilizing the focal adhesion plaque.     

Finding a Needle in a Haystack: The Role of Electrostatics in Target Lipid Recognition by PH Domains

Interactions between protein domains and lipid molecules play key roles in controlling cell membrane signalling and trafficking. The pleckstrin homology (PH) domain is one of the most widespread, binding specifically to phosphatidylinositol phosphates (PIPs) in cell membranes. PH domains must locate specific PIPs in the presence of a background of approximately 20% anionic lipids within the cytoplasmic leaflet of the plasma membrane. We investigate the mechanism of such recognition via a multiscale procedure combining Brownian dynamics (BD) and molecular dynamics (MD) simulations of the GRP1 PH domain interacting with phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)). The interaction of GRP1-PH with PI(3,4,5)P(3) in a zwitterionic bilayer is compared with the interaction in bilayers containing different levels of anionic 'decoy' lipids. BD simulations reveal both translational and orientational electrostatic steering of the PH domain towards the PI(3,4,5)P(3)-containing anionic bilayer surface. There is a payoff between non-PIP anionic lipids attracting the PH domain to the bilayer surface in a favourable orientation and their role as 'decoys', disrupting the interaction of GRP1-PH with the PI(3,4,5)P(3) molecule. Significantly, approximately 20% anionic lipid in the cytoplasmic leaflet of the bilayer is nearly optimal to both enhance orientational steering and to localise GRP1-PH proximal to the surface of the membrane without sacrificing its ability to locate PI(3,4,5)P(3) within the bilayer plane. Subsequent MD simulations reveal binding to PI(3,4,5)P(3), forming protein-phosphate contacts comparable to those in X-ray structures. These studies demonstrate a computational framework which addresses lipid recognition within a cell membrane environment, offering a link between structural and cell biological characterisation.     

Salt-stress-induced association of phosphatidylinositol 4,5-bisphosphate with clathrin-coated vesicles in plants

Plants exposed to hyperosmotic stress undergo changes in membrane dynamics and lipid composition to maintain cellular integrity and avoid membrane leakage. Various plant species respond to hyperosmotic stress with transient increases in PtdIns(4,5)P(2); however, the physiological role of such increases is unresolved. The plasma membrane represents the outermost barrier between the symplast of plant cells and its apoplastic surroundings. In the present study, the spatio-temporal dynamics of stress-induced changes in phosphoinositides were analysed in subcellular fractions of Arabidopsis leaves to delineate possible physiological roles. Unlabelled lipids were separated by TLC and quantified by gas-chromatographic detection of associated fatty acids. Transient PtdIns(4,5)P(2) increases upon exposure to hyperosmotic stress were detected first in enriched plasmamembrane fractions, however, at later time points, PtdIns(4,5)P(2) was increased in the endomembrane fractions of the corresponding two-phase systems. When major endomembranes were enriched from rosette leaves prior to hyperosmotic stress and during stimulation for 60 min, no stress-induced increases in the levels of PtdIns(4,5)P(2) were found in fractions enriched for endoplasmic reticulum, nuclei or plastidial membranes. Instead, increased PtdIns(4,5)P(2) was found in CCVs (clathrin-coated vesicles), which proliferated several-fold in mass within 60 min of hyperosmotic stress, according to the abundance of CCV-associated proteins and lipids. Monitoring the subcellular distribution of fluorescence-tagged reporters for clathrin and PtdIns(4,5)P(2) during transient co-expression in onion epidermal cells indicates rapid stress-induced co-localization of clathrin with PtdIns(4,5)P(2) at the plasma membrane. The results indicate that PtdIns(4,5)P(2) may act in stress-induced formation of CCVs in plant cells, highlighting the evolutionary conservation of the phosphoinositide system between organismic kingdoms.     

A role for diacylglycerol in annexin A7-mediated fusion of lung lamellar bodies

Lung surfactant secretion in alveolar type II cells occurs following lamellar body fusion with plasma membrane. Annexin A7 is a Ca2+-dependent membrane-binding protein that is postulated to promote membrane fusion during exocytosis in some cell types including type II cells. Since annexin A7 preferably binds to lamellar body membranes, we postulated that specific lipids could modify the mode of annexin A7 interaction with membranes and its membrane fusion activity. Initial studies with phospholipid vesicles containing phosphatidylserine and other lipids showed that certain lipids affected protein interaction with vesicle membranes as determined by change in protein tryptophan fluorescence, protein interaction with trans membranes, and by protein sensitivity to limited proteolysis. The presence of signaling lipids, diacylglycerol or phosphatidylinositol-4,5-bisphosphate, as minor components also modified the lipid vesicle effect on these characteristics and membrane fusion activity of annexin A7. In vitro incubation of lamellar bodies with diacylglycerol or phosphatidylinositol-4,5-bisphosphate caused their enrichment with either lipid, and increased the annexin A7 and Ca2+-mediated fusion of lamellar bodies. Treatment of isolated lung lamellar bodies with phosphatidylinositol- or phosphatidylcholine phospholipase C to increase diacylglycerol, without or with preincubation with phosphatidylinositol-4,5-bisphosphate, augmented the fusion activity of annexin A7. Thus, increased diacylglycerol in lamellar bodies following cell stimulation with secretagogues may enhance membrane fusion activity of annexin A7.     

Effects of cholesterol and PIP2 on interactions between glycophorin A and Band 3 in lipid bilayers

In the erythrocyte membrane, the interactions between glycophorin A (GPA) and Band 3 are associated strongly with the biological function of the membrane and several blood disorders. In this work, using coarse-grained molecular-dynamics simulations, we systematically investigate the effects of cholesterol and phosphatidylinositol-4,5-bisphosphate (PIP2) on the interactions of GPA with Band 3 in the model erythrocyte membranes. We examine the dynamics of the interactions of GPA with Band 3 in different lipid bilayers on the microsecond time scale and calculate the binding free energy between GPA and Band 3. The results indicate that cholesterols thermodynamically favor the binding of GPA to Band 3 by increasing the thickness of the lipid bilayer and by producing an effective attraction between the proteins due to the depletion effect. Cholesterols also slow the kinetics of the binding of GPA to Band 3 by reducing the lateral mobility of the lipids and proteins and may influence the binding sites between the proteins. The anionic PIP2 lipids prefer binding to the surface of the proteins through electrostatic attraction between the PIP2 headgroup and the positively charged residues on the protein surface. Ions in the solvent facilitate PIP2 aggregation, which promotes the binding of GPA to Band 3.     

Regulation of TRPV1 Ion Channel by Phosphoinositide (4,5)-Bisphosphate: THE ROLE OF MEMBRANE ASYMMETRY*

Membrane asymmetry is essential for generating second messengers that act in the cytosol and for trafficking of membrane proteins and membrane lipids, but the role of asymmetry in regulating membrane protein function remains unclear. Here we show that the signaling lipid phosphoinositide 4,5-bisphosphate (PI(4,5)P₂) has opposite effects on the function of TRPV1 ion channels depending on which leaflet of the cell membrane it resides in. We observed potentiation of capsaicin-activated TRPV1 currents by PI(4,5)P₂ in the intracellular leaflet of the plasma membrane but inhibition of capsaicin-activated currents when PI(4,5)P₂ was in both leaflets of the membrane, although much higher concentrations of PI(4,5)P₂ in the extracellular leaflet were required for inhibition compared with the concentrations of PI(4,5)P₂ in the intracellular leaflet that produced activation. Patch clamp fluorometry using a synthetic PI(4,5)P₂ whose fluorescence reports its concentration in the membrane indicates that PI(4,5)P₂ must incorporate into the extracellular leaflet for its inhibitory effects to be observed. The asymmetry-dependent effect of PI(4,5)P₂ may resolve the long standing controversy about whether PI(4,5)P₂ is an activator or inhibitor of TRPV1. Our results also underscore the importance of membrane asymmetry and the need to consider its influence when studying membrane proteins reconstituted into synthetic bilayers.     

Membrane binding properties of IRSp53-missing in metastasis domain (IMD) protein

The 53-kDa insulin receptor substrate protein (IRSp53) organizes the actin cytoskeleton in response to stimulation of small GTPases, promoting the formation of cell protrusions such as filopodia and lamellipodia. IMD is the N-terminal 250 amino acid domain (IRSp53/MIM Homology Domain) of IRSp53 (also called I-BAR), which can bind to negatively charged lipid molecules. Overexpression of IMD induces filopodia formation in cells and purified IMD assembles finger-like protrusions in reconstituted lipid membranes. IMD was shown by several groups to bundle actin filaments, but other groups showed that it also binds to membranes. IMD binds to negatively charged lipid molecules with preference to clusters of PI(4,5)P₂. Here, we performed a range of different in vitro fluorescence experiments to determine the binding properties of the IMD to phospholipids. We used different constructs of large unilamellar vesicles (LUVETs), containing neutral or negatively charged phospholipids. We found that IMD has a stronger binding interaction with negatively charged PI(4,5)P₂ or PS lipids than PS/PC or neutral PC lipids. The equilibrium dissociation constant for the IMD–lipid interaction falls into the 78–170 μM range for all the lipids tested. The solvent accessibility of the fluorescence labels on the IMD during its binding to lipids is also reduced as the lipids become more negatively charged. Actin affects the IMD–lipid interaction, depending on its polymerization state. Monomeric actin partially disrupts the binding, while filamentous actin can further stabilize the IMD–lipid interaction.