Phosphatidylinositol 4,5-bisphosphate functions as a second messenger that regulates cytoskeleton-plasma membrane adhesion

Binding interactions between the plasma membrane and the cytoskeleton define cell functions such as cell shape, formation of cell processes, cell movement, and endocytosis. Here we use optical tweezers tether force measurements and show that plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) acts as a second messenger that regulates the adhesion energy between the cytoskeleton and the plasma membrane. Receptor stimuli that hydrolyze PIP2 lowered adhesion energy, a process that could be mimicked by expressing PH domains that sequester PIP2 or by targeting a 5'-PIP2-phosphatase to the plasma membrane to selectively lower plasma membrane PIP2 concentration. Our study suggests that plasma membrane PIP2 controls dynamic membrane functions and cell shape by locally increasing and decreasing the adhesion between the actin-based cortical cytoskeleton and the plasma membrane.     

Agonist-induced PIP(2) hydrolysis inhibits cortical actin dynamics: regulation at a global but not at a micrometer scale

Phosphatidylinositol 4, 5-bisphosphate (PIP(2)) at the inner leaflet of the plasma membrane has been proposed to locally regulate the actin cytoskeleton. Indeed, recent studies that use GFP-tagged pleckstrin homology domains (GFP-PH) as fluorescent PIP(2) sensors suggest that this lipid is enriched in membrane microdomains. Here we report that this concept needs revision. Using three distinct fluorescent GFP-tagged pleckstrin homology domains, we show that highly mobile GFP-PH patches colocalize perfectly with various lipophilic membrane dyes and, hence, represent increased lipid content rather than PIP(2)-enriched microdomains. We show that bright patches are caused by submicroscopical folds and ruffles in the membrane that can be directly visualized at approximately 15 nm axial resolution with a novel numerically enhanced imaging method. F-actin motility is inhibited significantly by agonist-induced PIP(2) breakdown, and it resumes as soon as PIP(2) levels are back to normal. Thus, our data support a role for PIP(2) in the regulation of cortical actin, but they challenge a model in which spatial differences in PIP(2) regulation of the cytoskeleton exist at a micrometer scale.     

Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells

Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2).     

Rho and Rho-kinase mediate thrombin-induced phosphatidylinositol 4-phosphate 5-kinase trafficking in platelets

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) catalyzes the rate-limiting step in the production of phosphatidylinositol 4,5-bisphosphate (PIP(2)), a signaling phospholipid that contributes to actin dynamics. We have shown in transfected tissue culture cells that PIP5K translocates from the cytosol to the plasma membrane following agonist-induced stimulation of Rho family GTPases. Nonetheless, it is unclear whether Rho GTPases induce PIP5K relocalization in platelets. We used PIP5K isoform-specific immunoblotting and lipid kinase assays to examine the intracellular localization of PIP5K in resting and activated platelets. Using differential centrifugation to separate the membrane skeleton, actin filaments and associated proteins, and cytoplasmic fractions, we found that PIP5K isoforms were translocated from cytosol to actin-rich fractions following stimulation of the thrombin receptor. PIP5K translocation was detectable within 30 s of stimulation and was complete by 2-5 min. This agonist-induced relocalization and activation of PIP5K was inhibited by 8-(4-parachlorophenylthio)-cAMP, a cAMP analogue that inhibits Rho and Rac. In contrast, 8-(4-parachlorophenylthio)-cGMP, a cGMP analogue that inhibits Rac but not Rho, did not affect PIP5K translocation and activation. This suggests that Rho GTPase may be an essential regulator of PIP5K in platelets. Consistent with this hypothesis, we found that C3 exotoxin (a Rho-specific inhibitor) and HA1077 (an inhibitor of the Rho effector, Rho-kinase) also eliminated PIP5K activation and trafficking into the membrane cytoskeleton. Thus, these data indicate that Rho GTPase and its effector Rho-kinase have an intimate relationship with the trafficking and activation of platelet PIP5K. Moreover, these data suggest that relocalization of platelet PIP5K following agonist stimulation may play an important role in regulating the assembly of the platelet cytoskeleton.     

PIP5K-dependent production of PIP2 sustains microtubule organization to establish polarized transport in the Drosophila oocyte

The attachment of the cytoskeleton to the plasma membrane is crucial in controlling the polarized transport of cell-fate-determining molecules. Attachment involves adaptor molecules, which have the capacity to bind to both the plasma membrane and elements of the cytoskeleton, such as microtubules and actin filaments. Using the Drosophila oocyte as a model system, we show that the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), Skittles, is necessary to sustain the organization of microtubules and actin cytoskeleton required for the asymmetric transport of oskar, bicoid and gurken mRNAs and thereby controls the establishment of cell polarity. We show that Skittles function is crucial to synthesize and maintain phosphatidylinositol 4,5 bisphosphate (PIP2) at the plasma membrane in the oocyte. Reduction of Skittles activity impairs activation at the plasma membrane of Moesin, a member of the ERM family known to link the plasma membrane to the actin-based cytoskeleton. Furthermore, we provide evidence that Skittles, by controlling the localization of Bazooka, Par-1 and Lgl, but not Lkb1, to the cell membrane, regulates PAR polarity proteins and the maintenance of specific cortical domains along the anteroposterior axis.     

Actin polymerization serves as a membrane domain switch in model lipid bilayers

The ability of cells to mount localized responses to external or internal stimuli is critically dependent on organization of lipids and proteins in the plasma membrane. Involvement of the actin cytoskeleton in membrane organization has been documented, but an active role for actin networks that directly links internal organization of the cytoskeleton with membrane organization has not yet been identified. Here we show that branched actin networks formed on model lipid membranes enriched with the lipid second messenger PIP(2) trigger both temporal and spatial rearrangement of membrane components. Using giant unilamellar vesicles able to separate into two coexisting liquid phases, we demonstrate that polymerization of dendritic actin networks on the membrane induces phase separation of initially homogenous vesicles. This switch-like behavior depends only on the PIP(2)-N-WASP link between the membrane and actin network, and we find that the presence of a preexisting actin network spatially biases the location of phase separation. These results show that dynamic, membrane-bound actin networks alone can control when and where membrane domains form and may actively contribute to membrane organization during cell signaling.     

Phosphoinositide binding and phosphorylation act sequentially in the activation mechanism of ezrin

Ezrin, a membrane-actin cytoskeleton linker, which participates in epithelial cell morphogenesis, is held inactive in the cytoplasm through an intramolecular interaction. Phosphatidylinositol 4,5-bisphosphate (PIP2) binding and the phosphorylation of threonine 567 (T567) are involved in the activation process that unmasks both membrane and actin binding sites. Here, we demonstrate that ezrin binding to PIP2, through its NH2-terminal domain, is required for T567 phosphorylation and thus for the conformational activation of ezrin in vivo. Furthermore, we found that the T567D mutation mimicking T567 phosphorylation bypasses the need for PIP2 binding for unmasking both membrane and actin binding sites. However, PIP2 binding and T567 phosphorylation are both necessary for the correct apical localization of ezrin and for its role in epithelial cell morphogenesis. These results establish that PIP2 binding and T567 phosphorylation act sequentially to allow ezrin to exert its cellular functions.     

Phosphatidylinositol-4,5-bisphosphate influences Nt-Rac5-mediated cell expansion in pollen tubes of Nicotiana tabacum

The regulation of pollen tube growth by the phospholipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2) ) is not well understood. The Arabidopsis genome encodes two type A phosphatidylinositol-4-phosphate (PI4P) 5-kinases, PIP5K10 and PIP5K11, which are exclusively expressed in pollen and produce PtdIns(4,5)P(2) in vitro. Fluorescence-tagged PIP5K10 and PIP5K11 localized to lateral subapical plasma membrane microdomains in tobacco pollen tubes in a pattern closely resembling the distribution of PtdIns(4,5)P(2,) with the exception of notably weaker association at the extreme apex. Overexpression of PIP5K10 or PIP5K11 in tobacco pollen tubes resulted in severe tip swelling and altered actin fine structure similar to that reported for overexpression of tobacco Nt-Rac5, a monomeric GTPase known to regulate the actin cytoskeleton. Increased sensitivity of Arabidopsis pip5k10 pip5k11 double mutant pollen tubes to Latrunculin B (LatB) further supports a role for type A PI4P 5-kinases in controlling the actin cytoskeleton. Despite the disruption of both its type A PI4P 5-kinases, the pip5k10 pip5k11 double mutant was fertile, indicating that one of the remaining type B PI4P 5-kinase isoforms might be functionally redundant with PIP5K10 and PIP5K11. Antagonistic effects of PIP5K11 and the Nt-Rac5-specific guanine nucleotide dissociation inhibitor, Nt-RhoGDI2, on tip swelling observed in coexpression-titration experiments indicate a link between PtdIns(4,5)P(2) and Rac-signaling in pollen tubes. The data suggest that type A PI4P 5-kinases influence the actin cytoskeleton in pollen tubes in part by counteracting Nt-RhoGDI2, possibly contributing to the control of the pool of plasma membrane-associated Nt-Rac5.     

Activation of moesin, a protein that links actin cytoskeleton to the plasma membrane, occurs by phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequentially to two sites and releasing an autoinhibitory linker

Many cellular processes depend on ERM (ezrin, moesin, and radixin) proteins mediating regulated linkage between plasma membrane and actin cytoskeleton. Although conformational activation of the ERM protein is mediated by the membrane PIP2, the known properties of the two described PIP2-binding sites do not explain activation. To elucidate the structural basis of possible mechanisms, we generated informative moesin mutations and tested three attributes: membrane localization of the expressed moesin, moesin binding to PIP2, and PIP2-induced release of moesin autoinhibition. The results demonstrate for the first time that the POCKET containing inositol 1,4,5-trisphosphate on crystal structure (the "POCKET" Lys-63, Lys-278 residues) mediates all three functions. Furthermore the second described PIP2-binding site (the "PATCH," Lys-253/Lys-254, Lys-262/Lys-263) is also essential for all three functions. In native autoinhibited ERM proteins, the POCKET is a cavity masked by an acidic linker, which we designate the "FLAP." Analysis of three mutant moesin constructs predicted to influence FLAP function demonstrated that the FLAP is a functional autoinhibitory region. Moreover, analysis of the cooperativity and stoichiometry demonstrate that the PATCH and POCKET do not bind PIP2 simultaneously. Based on our data and supporting published data, we propose a model of progressive activation of autoinhibited moesin by a single PIP2 molecule in the membrane. Initial transient binding of PIP2 to the PATCH initiates release of the FLAP, which enables transition of the same PIP2 molecule into the newly exposed POCKET where it binds stably and completes the conformational activation.     

Molecular Dynamics of the Association of L-Selectin and FERM Regulated by PIP2

Phosphatidylinositol 4,5-bisphosphate (PIP2) acts as a signaling lipid, mediating membrane trafficking and recruitment of proteins to membranes. A key example is the PIP2-dependent regulation of the adhesion of L-selectin to the cytoskeleton adaptors of the N-terminal subdomain of ezrin-radixin-moesin (FERM). The molecular details of the mediating behavior of multivalent anionic PIP2 lipids in this process, however, remain unclear. Here, we use coarse-grained molecular dynamics simulation to explore the mechanistic details of PIP2 in the transformation, translocation, and association of the FERM/L-selectin complex. We compare membranes of different compositions and find that anionic phospholipids are necessary for both FERM and the cytoplasmic domain of L-selectin to absorb on the membrane surface. The subsequent formation of the FERM/L-selectin complex is strongly favored by the presence of PIP2, which clusters around both proteins and triggers a conformational transition in the cytoplasmic domain of L-selectin. We are able to quantify the effect of PIP2 on the association free energy of the complex by means of a potential of mean force. We conclude that PIP2 behaves as an adhesive agent to enhance the stability of the FERM/L-selectin complex and identify key residues involved. The molecular information revealed in this study highlights the specific role of membrane lipids such as PIP2 in protein translocation and potential signaling.     

磷脂酰肌醇-4,5-二磷酸调控细胞迁移的研究进展

磷脂酰肌醇-4,5-二磷酸(phosphatidylinositol-4,5-bisphosphate,PIP2)是细胞膜上一种重要的磷脂酰肌醇,通过作为第二信使前体及自身信号分子的作用,控制其效应物的靶向定位和活性从而调节细胞迁移、囊泡运输、细胞形态发生、信号传导等过程.细胞迁移异常会导致人类多种疾病包括神经发育异常、阿尔茨海默病、癌症和纤毛疾病等.作为细胞骨架的调节剂,PIP2在细胞迁移的关键作用已经被广泛证实,本文将从由PIP5KIs介导的PIP2产生与踝蛋白、Rho家族小GTP酶等效应物关联调节黏附作用和肌动蛋白聚合的角度,讨论PIP2在细胞迁移中发挥作用的具体机制.     

Hypertonic stress increases phosphatidylinositol 4,5-bisphosphate levels by activating PIP5KIbeta

Hyperosmotic stress increases phosphoinositide levels, reorganizes the actin cytoskeleton, and induces multiple acute and adaptive physiological responses. Here we showed that phosphatidylinositol 4,5-bisphosphate (PIP(2)) level increased rapidly in HeLa cells during hypertonic treatment. Depletion of the human type I phosphatidylinositol 4-phosphate 5-kinase beta isoform (PIP5KIbeta) by RNA interference impaired both the PIP(2) and actin cytoskeletal responses. PIP5KIbeta was recruited to membranes and was activated by hypertonic stress through Ser/Thr dephosphorylation. Calyculin A, a protein phosphatase 1 inhibitor, blocked the hypertonicity-induced PIP5KIbeta dephosphorylation/activation as well as PIP(2) increase in cells. Urea, which raises osmolarity without inducing cell shrinkage, did not promote dephosphorylation nor increase PIP(2) levels. Disruption or stabilization of the actin cytoskeleton, or inhibition of the Rho kinase, did not block the PIP(2) increase nor PIP5KIbeta dephosphorylation. Therefore, PIP5KIbeta is dephosphorylated in a volume-dependent manner by a calyculin A-sensitive protein phosphatase, which is activated upstream of actin remodeling and independently of Rho kinase activation. Our results establish a cause-and-effect relation between PIP5KIbeta dephosphorylation, lipid kinase activation, and PIP(2) increase in cells. This PIP(2) increase can orchestrate multiple downstream responses, including the reorganization of the actin cytoskeleton.     

Receptor-mediated hydrolysis of plasma membrane messenger PIP2 leads to K+-current desensitization

Phosphatidylinositol bisphosphate (PIP2) directly regulates functions as diverse as the organization of the cytoskeleton, vesicular transport and ion channel activity. It is not known, however, whether dynamic changes in PIP2 levels have a regulatory role of physiological importance in such functions. Here, we show in both native cardiac cells and heterologous expression systems that receptor-regulated PIP2 hydrolysis results in desensitization of a GTP-binding protein-stimulated potassium current. Two receptor-regulated pathways in the plasma membrane cross-talk at the level of these channels to modulate potassium currents. One pathway signals through the betagamma subunits of G proteins, which bind directly to the channel. Gbetagamma subunits stabilize interactions with PIP2 and lead to persistent channel activation. The second pathway activates phospholipase C (PLC) which hydrolyses PIP2 and limits Gbetagamma-stimulated activity. Our results provide evidence that PIP2 itself is a receptor-regulated second messenger, downregulation of which accounts for a new form of desensitization.     

Dibasic amino acid residues at the carboxy-terminal end of kinase homology domain participate in the plasma membrane localization and function of phosphatidylinositol 5-kinase gamma

Type I phosphatidylinositol 4-phosphate (PI(4)P) 5-kinases (PIP5Ks) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), an essential lipid molecule involved in various cellular processes such as regulation of actin cytoskeleton and membrane traffic. The protein localizes to the plasma membrane where its activity has been shown to be regulated by small GTPase ARFs and/or phosphatidic acid. Deletion analysis of amino- or carboxy-terminal sequences of PIP5Kgamma fused with EGFP demonstrated that the presence of central kinase homology domain (KHD), a 380 amino acid-long region highly conserved among PIP5K family, was necessary and sufficient for the plasma membrane localization of PIP5Kgamma. Particularly, the dibasic Arg-Lys sequence located at the carboxy-terminal end of KHD was shown to be crucial for the plasma membrane targeting of PIP5Kgamma, since the deletion or charge-reversal mutation of this dibasic sequence resulted in the mislocalization of the protein to the cytoplasm. Mislocalized mutants also failed to complement the temperature-sensitive growth of Saccharomyces cerevisiae mss4-1 mutant defective in PIP5K function. The presence of dibasic residues at the C-terminal end of KHD was conserved among mammalian as well as invertebrate PIP5K family members, but not in the type II PIPKs that are not targeted to the plasma membrane, suggesting that the conserved dibasic motif provides a mechanism essential for the proper localization and cellular function of PIP5Ks.     

Phosphatidylinositol-4,5-biphosphate (PIP2) differentially regulates the interaction of human erythrocyte protein 4.1 (4.1R) with membrane proteins

Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton. In the present study, we characterized the interaction of 4.1R with phosphatidylinositol-4,5-bisphosphate (PIP2) and assessed the effect of PIP2 on the interaction of 4.1R with membrane proteins. We found that 4.1R bound to PIP2-containing liposomes through its N-terminal 30 kDa membrane-binding domain and PIP2 binding induced a conformational change in this domain. Phosphatidylinositol-4-phosphate (PIP) was a less effective inducer of this conformational change, and phosphatidylinositol (PI) and inositol-1,4,5-phosphate (IP3) induced no change. Replacement of amino acids K63,64 and K265,266 by alanine abolished the interaction of the membrane-binding domain with PIP2. Importantly, binding of PIP2 to 4.1R selectively modulated the ability of 4.1R to interact with its different binding partners. While PIP2 significantly enhanced the binding of 4.1R to glycophorin C (GPC), it inhibited the binding of 4.1R to band 3 in vitro. PIP2 had no effect on 4.1R binding to p55. Furthermore, GPC was more readily extracted by Triton X-100 from adenosine triphosphate (ATP)-depleted erythrocytes, implying that the GPC-4.1R interaction may be regulated by PIP2 in situ. These findings define an important role for PIP2 in regulating the function of 4.1R. Because 4.1R and its family members (4.1R, 4.1B, 4.1G, and 4.1N) are widely expressed and the PIP2-binding motifs are highly conserved, it is likely that the functions of other 4.1 proteins are similarly regulated by PIP2 in many different cell types.     

Phospholipase C–mediated hydrolysis of PIP2 releases ERM proteins from lymphocyte membrane

Mechanisms controlling the disassembly of ezrin/radixin/moesin (ERM) proteins, which link the cytoskeleton to the plasma membrane, are incompletely understood. In lymphocytes, chemokine (e.g., SDF-1) stimulation inactivates ERM proteins, causing their release from the plasma membrane and dephosphorylation. SDF-1–mediated inactivation of ERM proteins is blocked by phospholipase C (PLC) inhibitors. Conversely, reduction of phosphatidylinositol 4,5-bisphosphate (PIP2) levels by activation of PLC, expression of active PLC mutants, or acute targeting of phosphoinositide 5-phosphatase to the plasma membrane promotes release and dephosphorylation of moesin and ezrin. Although expression of phosphomimetic moesin (T558D) or ezrin (T567D) mutants enhances membrane association, activation of PLC still relocalizes them to the cytosol. Similarly, in vitro binding of ERM proteins to the cytoplasmic tail of CD44 is also dependent on PIP2. These results demonstrate a new role of PLCs in rapid cytoskeletal remodeling and an additional key role of PIP2 in ERM protein biology, namely hydrolysis-mediated ERM inactivation.     

Distribution of gelsolin and phosphoinositol 4,5-bisphosphate in lamellipodia during EGF-induced motility

During induced cell motility the actin cytoskeleton at the leading edge must undergo constant reorganization. Recently, phosphoinositides have been shown to be central to cytoskeleton-membrane linkages and actin organization and turnover. Epidermal growth factor (EGF) receptor (EGFR)-mediated cell motility requires phospholipase C-gamma (PLCgamma), hydrolysis of phosphoinsotide 4,5-bisphosphate (PIP(2)) and subsequent release of gelsolin. We hypothesized this led to the mobilization of PIP(2)-binding proteins which modify the actin cytoskeleton and thus sought to determine whether the leading edge was a site of active PIP(2) hydrolysis and gelsolin redistribution to cytoskeleton. Herein, we report that during EGF-induced motility, the leading edge's submembranous region constitutes a distinct subcellular locale. The relevant phosphoinositide composition of this space was determined by probing with an antibody to PIP(2) and a green fluorescence protein (GFP)-tagged pleckstrin homology (PH) domain of PLCdelta (GFP-PH) that recognizes both PIP(2) and inositol 1,4,5-trisphosphate (IP(3)). PIP(2) was absent from leading lamellipodia despite an increase in IP(3) generation, suggesting an increase in PIP(2) hydrolysis at the leading edge. Visualized with immunofluorescence, gelsolin preferentially concentrated near the leading edge in a punctate fashion. Examining the Triton X-insoluble actin cytoskeleton fractions, we observe a PLCgamma-dependent increase of gelsolin incorporation upon EGF stimulation. At a molecular level, field emission scanning electron microscopy (FE-SEM) shows that gelsolin incorporates preferentially into the submembranous actin arcs at the leading edge of the lamellipodia. Together these data suggest a model of PIP(2) hydrolysis at the leading edge causing a localized release of PIP(2)-binding proteins-particularly gelsolin-that drives cytoskeletal rearrangement and protrusion.     

Binding of moesin and ezrin to membranes containing phosphatidylinositol (4,5) bisphosphate: a comparative study of the affinity constants and conformational changes

The plasma membrane-cytoskeleton interface is a dynamic structure participating in a variety of cellular events. Moesin and ezrin, proteins from the ezrin/radixin/moesin (ERM) family, provide a direct linkage between the cytoskeleton and the membrane via their interaction with phosphatidylinositol 4,5-bisphosphate (PIP(2)). PIP(2) binding is considered as a prerequisite step in ERM activation. The main objective of this work was to compare moesin and ezrin interaction with PIP(2)-containing membranes in terms of affinity and to analyze secondary structure modifications leading eventually to ERM activation. For this purpose, we used two types of biomimetic model membranes, large and giant unilamellar vesicles. The dissociation constant between moesin and PIP(2)-containing large unilamellar vesicles or PIP(2)-containing giant unilamellar vesicles was found to be very similar to that between ezrin and PIP(2)-containing large unilamellar vesicles or PIP(2)-containing giant unilamellar vesicles. In addition, both proteins were found to undergo conformational changes after binding to PIP(2)-containing large unilamellar vesicles. Changes were evidenced by an increased sensitivity to proteolysis, modifications in the fluorescence intensity of the probe attached to the C-terminus and in the proportion of secondary structure elements.     

Phosphatidylinositol 4,5-bisphosphate regulates activation-induced platelet microparticle formation

While the role of the cytoskeleton in microparticle formation is well-described, the role of membrane phospholipids in regulating this process is poorly defined. PIP(2) binds many cytoskeletal proteins and may oppose microparticle formation through associations with these proteins. To determine whether PIP(2) effects microparticle formation, PIP(2) was incorporated into platelet membranes prior to activation-induced microparticle formation. Incorporation of PIP(2) into platelet membranes inhibited activation-induced microparticle formation by >or=90%. Inhibition was dose-dependent with an IC(50) of 12-18 microM. A permeabilized platelet system was next used to assess the effect of modulation of endogenous PIP(2) levels on microparticle formation. Infusion of type IIbeta PIP kinase into permeabilized platelets inhibited microparticle formation by 75 +/- 8%. In contrast, incubation of permeabilized platelets with PI-specific phospholipase C augmented microparticle formation by greater than 3-fold. Evaluation of PIP kinases following platelet activation demonstrated that they were lost from platelets in a calpain-dependent manner during microparticle formation. Purified mu-calpain cleaved recombinant type IIbeta PIP kinase and inhibited its ability to phosphorylate PI(5)P. In permeabilized platelets, incubation of purified mu-calpain reduced PIP(2) levels, while exposure to calpeptin increased PIP(2) levels. Calpain has previously been implicated in platelet microparticle formation. These studies show that calpain may help limit PIP(2) formation following platelet activation and that PIP(2) content is an important determinant of platelet microparticle formation.     

Phosphatidylinositol 4,5-bisphosphate induces actin-based movement of raft-enriched vesicles through WASP-Arp2/3

BACKGROUND: Phosphatidylinositol 4,5-bisphosphate (PIP(2)) has been implicated in the regulation of the actin cytoskeleton and vesicle trafficking. It stimulates de novo actin polymerization by activating the pathway involving the Wiskott-Aldrich syndrome protein (WASP) and the actin-related protein complex Arp2/3. Other studies show that actin polymerizes from cholesterol-sphingolipid-rich membrane microdomains called 'rafts', in a manner dependent on tyrosine phosphorylation. Although actin has been implicated in vesicle trafficking, and rafts are sites of active phosphoinositide and tyrosine kinase signaling that mediate apically directed vesicle trafficking, it is not known whether phosphoinositide regulation of actin dynamics occurs in rafts, or if it is linked to vesicle movements. RESULTS: Overexpression of type I phosphatidylinositol phosphate 5-kinase (PIP5KI), which synthesizes PIP(2), promoted actin polymerization from membrane-bound vesicles to form motile actin comets. Pervanadate (PV), a tyrosine phosphatase inhibitor, induced comets even in the absence of PIP5KI overexpression. PV increased PIP(2) levels, suggesting that it induces comets by changing PIP(2) homeostasis and by increasing tyrosine phosphorylation. Platelet-derived growth factor (PDGF) enhanced PV-induced comet formation, and these stimuli together potentiated the PIP5KI effect. The vesicles at the heads of comets were enriched in PIP5KIs and tyrosine phosphoproteins. WASP-Arp2/3 involvement was established using dominant-negative WASP constructs. Endocytic and exocytic markers identified vesicles enriched in lipid rafts as preferential sites of comet generation. Extraction of cholesterol with methyl-beta-cyclodextrin reduced comets, establishing that rafts promote comet formation. CONCLUSIONS: Sphingolipid-cholesterol rafts are preferred platforms for membrane-linked actin polymerization. This is mediated by in situ PIP(2) synthesis and tyrosine kinase signaling through the WASP-Arp2/3 pathway. Actin comets may provide a novel mechanism for raft-dependent vesicle transport and apical membrane trafficking.