Phosphoinositide-binding Motif Research Articles

Calcium influx promotes PLEKHG4B localization to cell-cell junctions and regulates the integrity of junctional actin filaments.

PLEKHG4B is a Cdc42-targeting guanine-nucleotide exchange factor implicated in forming epithelial cell-cell junctions. Here we explored the mechanism regulating PLEKHG4B localization. PLEKHG4B localized to the basal membrane in normal Ca2+ medium but accumulated at cell-cell junctions upon ionomycin treatment. Ionomycin-induced junctional localization of PLEKHG4B was suppressed upon disrupting its annexin-A2 (ANXA2)-binding ability. Thus, Ca2+ influx and ANXA2 binding are crucial for PLEKHG4B localization to cell-cell junctions. Treatments with low Ca2+ or BAPTA-AM (an intracellular Ca2+ chelator) suppressed PLEKHG4B localization to the basal membrane. Mutations of the phosphoinositide-binding motif in the pleckstrin homology (PH) domain of PLEKHG4B or masking of membrane phosphatidylinositol-4,5-biphosphate [PI(4,5)P2] suppressed PLEKHG4B localization to the basal membrane, indicating that basal membrane localization of PLEKHG4B requires suitable intracellular Ca2+ levels and PI(4,5)P2 binding of the PH domain. Activation of mechanosensitive ion channels (MSCs) promoted PLEKHG4B localization to cell-cell junctions, and their inhibition suppressed it. Moreover, similar to the PLEKHG4B knockdown phenotypes, inhibition of MSCs or treatment with BAPTA-AM disturbed the integrity of actin filaments at cell-cell junctions. Taken together, our results suggest that Ca2+ influx plays crucial roles in PLEKHG4B localization to cell-cell junctions and the integrity of junctional actin organization, with MSCs contributing to this process.

Stepwise phosphorylation of BLT1 defines complex assemblies with β-arrestin serving distinct functions.

G protein-coupled receptors (GPCRs) utilize complex cellular systems to respond to diverse ligand concentrations. By taking BLT1, a GPCR for leukotriene B4 (LTB4 ), as a model, our previous work elucidated that this system functions through the modulation of phosphorylation status on two specific residues: Thr308 and Ser310 . Ser310 phosphorylation occurs at a lower LTB4 concentration than Thr308 , leading to a shift in ligand affinity from a high-to-low state. However, the implications of BLT1 phosphorylation in signal transduction processes or the underlying mechanisms have remained unclear. Here, we identify the sequential BLT1-engaged conformations of β-arrestin and subsequent alterations in signal transduction. Stimulation of the high-affinity BLT1 with LTB4 induces phosphorylation at Ser310 via the ERK1/2-GRK pathway, resulting in a β-arrestin-bound low-affinity state. This configuration, referred to as the "low-LTB4 -induced complex," necessitates the finger loop region and the phosphoinositide-binding motif of β-arrestins to interact with BLT1 and deactivates the ERK1/2 signaling. Under high LTB4 concentrations, the low-affinity BLT1 again binds to the ligand and triggers the generation of the low-LTB4 -induced complex into a different form termed "high-LTB4 -induced complex." This change is propelled by The308 -phosphorylation-dependent basal phosphorylation by PKCs. Within the high-LTB4 -induced complex, β-arrestin adapts a unique configuration that involves additional N domain interaction to the low-affinity BLT1 and stimulates the PI3K/AKT pathway. We propose that the stepwise phosphorylation of BLT1 defines the formation of complex assemblies, wherein β-arrestins perform distinct functions.

Human BIN1 isoforms grow, maintain, and regenerate excitation-contraction couplons in adult rat and human stem cell-derived cardiomyocytes.

In ventricular myocytes, transverse-tubules (T-tubules) are instrumental for excitation-contraction (EC)coupling and their disarray is a hallmark of cardiac diseases. BIN1 is a key contributor to their biogenesis. Our study set out to investigate the role of human BIN1 splice variants in the maintenance and regeneration of EC-coupling in rat adult ventricular myocytes and human-induced pluripotent stem cell-derived cardiac myocytes (hiPS-CMs). In heart samples from healthy human donors expression patterns of five BIN1 splice variants were identified. Following viral transduction of human BIN1 splice variants in cellular models of T-tubular disarray, we employed high-speed confocal calcium imaging and CaCLEAN analysis to identify functional EC-coupling sites (couplons) and T-tubular architecture. Adult rat ventricular myocytes were used to investigate the regeneration after loss and maintenance of EC-coupling while we studied the enhancement of EC-coupling in hiPS-CMs. All five human BIN1 splice variants induced de-novo generation of T-tubules in both cell types. Isoforms with the phosphoinositide-binding motif (PI) were most potent in maintenance and regeneration of T-tubules and functional EC-coupling in adult rat myocytes. In hiPSC-CMs, BIN1 variants with PI-motif-induced de novo generation of T-tubules, functional couplons and enhanced calcium handling. BIN1 is essential for the maintenance, regeneration, and de novo generation of functional T-tubules. Isoforms with PI-motifs appeared as particulalrly potent. These T-tubules trigger the development of functional couplons resulting in enhanced calcium handling.

Nanobar Array Assay Revealed Complementary Roles of BIN1 Splice Isoforms in Cardiac T-Tubule Morphogenesis.

Bridging integrator-1 (BIN1) is a family of banana-shaped molecules implicated in cell membrane tubulation. To understand the curvature sensitivity and functional roles of BIN1 splicing isoforms, we engineered vertical nanobars on a cell culture substrate to create high and low curvatures. When expressed individually, BIN1 isoforms with phosphoinositide-binding motifs (pBIN1) appeared preferentially at high-curvature nanobar ends, agreeing well with their membrane tubulation in cardiomyocytes. In contrast, the ubiquitous BIN1 isoform without phosphoinositide-binding motif (uBIN1) exhibited no affinity to membranes around nanobars but accumulated along Z-lines in cardiomyocytes. Importantly, in pBIN1-uBIN1 coexpression, pBIN1 recruited uBIN1 to high-curvature membranes at nanobar ends, and uBIN1 attached the otherwise messy pBIN1 tubules to Z-lines. The complementary cooperation of BIN1 isoforms (comboBIN1) represents a novel mechanism of T-tubule formation along Z-lines in cardiomyocytes. Dysregulation of BIN1 splicing, e.g., during myocardial infarction, underlied T-tubule disorganization, and correction of uBIN1/pBIN1 stoichiometry rescued T-tubule morphology in heart disease.

Optically sensing phospholipid induced coil-helix transitions in the phosphoinositide-binding motif of gelsolin.

We present a systematic experimental and computational study of phospholipid induced peptide coil-helix transitions which are relevant in the context of proteins mediating cytoskeletal rearrangement via membrane binding. We developed a sensitive Förster resonance energy transfer (FRET) based assay to address whether coil-helix transitions in phospholipid binding motifs of actin-binding proteins can be induced by physiologically-relevant concentrations (1-20 μM) of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) phospholipids. Based on inter-residue distance constraints obtained from Molecular Dynamics (MD) simulations of a 20 residue peptide (Gel 150-169) from the actin-severing protein gelsolin, we synthetized and labeled the peptide with a tryptophan donor and IAEDANS acceptor pair. Upon addition of PI(4,5)P2 micelles and mixed vesicles containing PI(4,5)P2 and phosphatidylcholine to the peptide, we observed a decrease in the tryptophan emission intensity with increasing concentrations of PI(4,5)P2. The IAEDANS emission spectra showed a more complex profile exhibiting a blue shift of the emission peak and non-monotonic changes in the intensity profile with increasing concentrations of PI(4,5)P2. We showed that the IAEDANS acceptor emission response is a result of both intrinsic polarity sensitivity of the acceptor in the vicinity of the membrane surface and fluorescence energy transfer from the donor. Importantly, the fluorescence lifetime of the donor (tryptophan) showed a monotonous decrease with increasing mol% of PI(4,5)P2 from 1.13 ± 0.10 ns in the absence of phospholipids to 0.25 ± 0.03 ns in the presence of 100% PI(4,5)P2 micelles. We also showed a concomitant increase in FRET efficiency with increasing PI(4,5)P2 levels indicating a PI(4,5)P2 concentration dependent coil-helix transition. Our studies demonstrate that membrane PI(4,5)P2 concentrations as low as 2.5-5 μM can trigger helix-coil conformational changes in gelsolin relevant for triggering regulatory processes in the cell.

Membrane targeting of TIRAP is negatively regulated by phosphorylation in its phosphoinositide-binding motif

Pathogen-activated Toll-like receptors (TLRs), such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide (PI)-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed PIs. TIRAP PBM bound PIs through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing PI interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses.

Membrane Targeting of TIRAP is Negatively Regulated by Phosphorylation in its Phosphoinositide-Binding Motif

Toll-like receptors (TLRs) trigger early immune system recognition and response to infection. Pathogen-activated TLRs, such as TLR2 and TLR4, dimerize and move laterally across the plasma membrane to phosphatidylinositol (4,5)-bisphosphate-enriched domains. At these sites, TLRs interact with the TIR domain-containing adaptor protein (TIRAP), triggering a signaling cascade that leads to innate immune responses. Membrane recruitment of TIRAP is mediated by its phosphoinositide-binding motif (PBM). We show that TIRAP PBM transitions from a disordered to a helical conformation in the presence of either zwitterionic micelles or monodispersed phosphoinositides. TIRAP PBM bound phosphoinositides through basic and nonpolar residues with high affinity, favoring a more ordered structure. TIRAP is phosphorylated at Thr28 within its PBM, which leads to its ubiquitination and degradation. We demonstrate that phosphorylation distorts the helical structure of TIRAP PBM, reducing phosphoinositide interactions and cell membrane targeting. Our study provides the basis for TIRAP membrane insertion and the mechanism by which it is removed from membranes to avoid sustained innate immune responses.

Phosphoinositides in the nucleus and myogenic differentiation: how a nuclear turtle with a PHD builds muscle.

Phosphoinositides are a family of phospholipid messenger molecules that control various aspects of cell biology in part by interacting with and regulating downstream protein partners. Importantly, phosphoinositides are present in the nucleus. They form part of the nuclear envelope and are present within the nucleus in nuclear speckles, intra nuclear chromatin domains, the nuclear matrix and in chromatin. What their exact role is within these compartments is not completely clear, but the identification of nuclear specific proteins that contain phosphoinositide interaction domains suggest that they are important regulators of DNA topology, chromatin conformation and RNA maturation and export. The plant homeo domain (PHD) finger is a phosphoinositide binding motif that is largely present in nuclear proteins that regulate chromatin conformation. In the present study I outline how changes in the levels of the nuclear phosphoinositide PtdIns5P impact on muscle cell differentiation through the PHD finger of TAF3 (TAF, TATA box binding protein (TBP)-associated factor), which is a core component of a number of different basal transcription complexes.

BIN1/M-Amphiphysin2 induces clustering of phosphoinositides to recruit its downstream partner dynamin.

Phosphoinositides play a central role in many physiological processes by assisting the recruitment of proteins to membranes through specific phosphoinositide-binding motifs. How this recruitment is coordinated in space and time is not well understood. Here we show that BIN1/M-Amphiphysin2, a protein involved in T-tubule biogenesis in muscle cells and frequently mutated in centronuclear myopathies, clusters PtdIns(4,5)P2 to recruit its downstream partner dynamin. By using several mutants associated with centronuclear myopathies, we find that the N-BAR and the SH3 domains of BIN1 control the kinetics and the accumulation of dynamin on membranes, respectively. We show that phosphoinositide clustering is a mechanism shared by other proteins that interact with PtdIns(4,5)P2, but do not contain a BAR domain. Our numerical simulations point out that clustering is a diffusion-driven process in which phosphoinositide molecules are not sequestered. We propose that this mechanism plays a key role in the recruitment of downstream phosphoinositide-binding proteins.

IQGAP1 is a novel phosphatidylinositol 4,5 bisphosphate effector in regulation of directional cell migration

Phosphatidylinositol 4,5 bisphosphate (PIP₂) is a key lipid messenger for regulation of cell migration. PIP₂ modulates many effectors, but the specificity of PIP₂ signalling can be defined by interactions of PIP₂-generating enzymes with PIP₂ effectors. Here, we show that type Iγ phosphatidylinositol 4-phosphate 5-kinase (PIPKIγ) interacts with the cytoskeleton regulator, IQGAP1, and modulates IQGAP1 function in migration. We reveal that PIPKIγ is required for IQGAP1 recruitment to the leading edge membrane in response to integrin or growth factor receptor activation. Moreover, IQGAP1 is a PIP₂ effector that directly binds PIP₂ through a polybasic motif and PIP₂ binding activates IQGAP1, facilitating actin polymerization. IQGAP1 mutants that lack PIPKIγ or PIP₂ binding lose the ability to control directional cell migration. Collectively, these data reveal a synergy between PIPKIγ and IQGAP1 in the control of cell migration.

Synergistic Activation of p21-activated Kinase 1 by Phosphatidylinositol 4,5-Bisphosphate and Rho GTPases

Autoinhibited p21-activated kinase 1 (Pak1) can be activated in vitro by the plasma membrane-bound Rho GTPases Rac1 and Cdc42 as well as by the lipid phosphatidylinositol (4,5)-bisphosphate (PIP2). Activator binding is mediated by a GTPase-binding motif and an adjacent phosphoinositide-binding motif. Whether these two classes of activators play alternative, additive, or synergistic roles in Pak1 activation is unknown, as is their contributions to Pak1 activation in vivo. To address these questions, we developed a system to mimic the membrane anchoring of Rho GTPases by creating liposomes containing both PIP2 and a Ni(2+)-NTA modified lipid capable of binding hexahistidine-tagged Cdc42. We find that among all biologically relevant phosphoinositides, only PIP2 is able to synergistically activate Pak1 in concert with Cdc42. Membrane binding of the kinase was highly sensitive to the spatial density of PIP2 and Pak1 demonstrated dramatically enhanced affinity for Cdc42 anchored in a PIP2 environment. To validate these findings in vivo, we utilized an inducible recruitment system to drive the ectopic synthesis of PIP2 on Golgi membranes, which normally have active Cdc42 but lack significant concentrations of PIP2. Pak1 was recruited to PIP2-containing membranes in a manner dependent on the ability of Pak1 to bind to both PIP2 and Cdc42. These findings provide a mechanistic explanation for the essential role of both phosphoinositides and GTPases in Pak1 recruitment and activation. In contrast, Ack, another Cdc42 effector kinase that lacks an analogous phosphoinositide-binding motif, fails to show the same enhancement of membrane binding and activation by PIP2, thus indicating that regulation by PIP2 and Cdc42 could provide a combinatorial code for activation of different GTPase effectors in different subcellular locations.

A human phospholipid phosphatase activated by a transmembrane control module

In voltage-sensitive phosphatases (VSPs), a transmembrane voltage sensor domain (VSD) controls an intracellular phosphoinositide phosphatase domain, thereby enabling immediate initiation of intracellular signals by membrane depolarization. The existence of such a mechanism in mammals has remained elusive, despite the presence of VSP-homologous proteins in mammalian cells, in particular in sperm precursor cells. Here we demonstrate activation of a human VSP (hVSP1/TPIP) by an intramolecular switch. By engineering a chimeric hVSP1 with enhanced plasma membrane targeting containing the VSD of a prototypic invertebrate VSP, we show that hVSP1 is a phosphoinositide-5-phosphatase whose predominant substrate is PI(4,5)P(2). In the chimera, enzymatic activity is controlled by membrane potential via hVSP1's endogenous phosphoinositide binding motif. These findings suggest that the endogenous VSD of hVSP1 is a control module that initiates signaling through the phosphatase domain and indicate a role for VSP-mediated phosphoinositide signaling in mammals.

A Dual Polybasic Motif Determines Phosphoinositide Binding and Regulation in the P2X Channel Family

Phosphoinositides modulate the function of several ion channels, including most ATP-gated P2X receptor channels in neurons and glia, but little is known about the underlying molecular mechanism. We identified a phosphoinositide-binding motif formed of two clusters of positively charged amino acids located on the P2X cytosolic C-terminal domain, proximal to the second transmembrane domain. For all known P2X subtypes, the specific arrangement of basic residues in these semi-conserved clusters determines their sensitivity to membrane phospholipids. Neutralization of these positive charges disrupts the functional properties of the prototypical phosphoinositide-binding P2X4 subtype, mimicking wortmannin-induced phosphoinositide depletion, whereas adding basic residues at homologous positions to the natively insensitive P2X5 subtype establishes de novo phosphoinositide-mediated regulation. Moreover, biochemical evidence of in vitro P2X subunit-phospholipid interaction and functional intracellular phosphoinositide-binding assays demonstrate that the dual polybasic cluster is necessary and sufficient for regulation of P2X signaling by phospholipids.

The CD3 zeta subunit contains a phosphoinositide-binding motif that is required for the stable accumulation of TCR-CD3 complex at the immunological synapse.

T cell activation involves a cascade of TCR-mediated signals that are regulated by three distinct intracellular signaling motifs located within the cytoplasmic tails of the CD3 chains. Whereas all the CD3 subunits possess at least one ITAM, the CD3 ε subunit also contains a proline-rich sequence and a basic-rich stretch (BRS). The CD3 ε BRS complexes selected phosphoinositides, interactions that are required for normal cell surface expression of the TCR. The cytoplasmic domain of CD3 ζ also contains several clusters of arginine and lysine residues. In this study, we report that these basic amino acids enable CD3 ζ to complex the phosphoinositides PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, PtdIns(3,5)P(2), and PtdIns(3,4,5)P(3) with high affinity. Early TCR signaling pathways were unaffected by the targeted loss of the phosphoinositide-binding functions of CD3 ζ. Instead, the elimination of the phosphoinositide-binding function of CD3 ζ significantly impaired the ability of this invariant chain to accumulate stably at the immunological synapse during T cell-APC interactions. Without its phosphoinositide-binding functions, CD3 ζ was concentrated in intracellular structures after T cell activation. Such findings demonstrate a novel functional role for CD3 ζ BRS-phosphoinositide interactions in supporting T cell activation.

Controlling the Activity of a Phosphatase and Tensin Homolog (PTEN) by Membrane Potential

The recently discovered voltage-sensitive phosphatases (VSPs) hydrolyze phosphoinositides upon depolarization of the membrane potential, thus representing a novel principle for the transduction of electrical activity into biochemical signals. Here, we demonstrate the possibility to confer voltage sensitivity to cytosolic enzymes. By fusing the tumor suppressor PTEN to the voltage sensor of the prototypic VSP from Ciona intestinalis, Ci-VSP, we generated chimeric proteins that are voltage-sensitive and display PTEN-like enzymatic activity in a strictly depolarization-dependent manner in vivo. Functional coupling of the exogenous enzymatic activity to the voltage sensor is mediated by a phospholipid-binding motif at the interface between voltage sensor and catalytic domains. Our findings reveal that the main domains of VSPs and related phosphoinositide phosphatases are intrinsically modular and define structural requirements for coupling of enzymatic activity to a voltage sensor domain. A key feature of this prototype of novel engineered voltage-sensitive enzymes, termed Ci-VSPTEN, is the novel ability to switch enzymatic activity of PTEN rapidly and reversibly. We demonstrate that experimental control of Ci-VSPTEN can be obtained either by electrophysiological techniques or more general techniques, using potassium-induced depolarization of intact cells. Thus, Ci-VSPTEN provides a novel approach for studying the complex mechanism of activation, cellular control, and pharmacology of this important tumor suppressor. Moreover, by inducing temporally precise perturbation of phosphoinositide concentrations, Ci-VSPTEN will be useful for probing the role and specificity of these messengers in many cellular processes and to analyze the timing of phosphoinositide signaling.

Roles of the Lipid-binding Motifs of Atg18 and Atg21 in the Cytoplasm to Vacuole Targeting Pathway and Autophagy

Atg18 and Atg21 are homologous WD-40 repeat proteins that bind phosphoinositides via a novel conserved Phe-Arg-Arg-Gly motif and function in autophagy-related pathways. Atg18 is required for the cytoplasm to vacuole targeting (Cvt) pathway and autophagy, whereas Atg21 is only required for the Cvt pathway. Currently, the functions of both proteins are poorly understood. Here, we examined the relationship between the phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding abilities of Atg18 and Atg21 and autophagy by expressing variants of these proteins that have mutations in their phosphoinositide-binding motifs. Cells expressing PtdIns(3)P-binding mutants of both these proteins showed highly reduced autophagy. Furthermore, the localization of components of two related ubiquitin-like protein conjugation systems, Atg8 and Atg16, to the phagophore assembly site is affected. Consistent with the aberrant localization of the above Atg proteins, precursor Ape1, a cargo of the Cvt pathway and autophagy, is partially protease-sensitive in starvation conditions. This finding suggests a requirement for the PtdIns(3)P binding capability of Atg18 and Atg21 in efficient completion of the sequestering autophagic vesicles. Finally, using a multiple knock-out strain, we found that Atg18 and Atg21 facilitate the recruitment of Atg8-PE to the site of autophagosome formation and protect it from premature cleavage by Atg4, which represents a key aspect of post-translational autophagy regulation. Taken together, our results suggest that PtdIns(3)P binding by at least Atg18 or Atg21 is required for robust autophagic activity and that the PtdIns(3)P-binding motifs of Atg18 and Atg21 can compensate for one another in the recruitment of Atg components that are dependent on PtdIns(3)P for their phagophore assembly site association.

PTEN and Ci-VSP Show Similar Phosphoinositide Binding Preferences

The Ciona intestinalis voltage sensor containing phosphatase (Ci-VSP) is a voltage dependent phosphatidylinositol phosphatase with two main domains, the voltage sensing domain (VSD) and the phosphatase domain (PD). Ci-VSP's PD bears homology with the phosphatase domain of the tumor suppressor protein PTEN (phosphatase and tensin homolog deleted chromosome 10). Recently, it has been proposed that the linker between the VSD and PD Ci-VSP constitute a Phosphoinositide Binding Motif (PBM). The PBM of Ci-VSP shares high homology with PTEN's N-terminal, which, in turn, is known to bind PI(4,5)P2 leading to an allosteric activation of PTEN. Similarly to the PBM of PTEN, we have found that a peptide representing the Ci-VSP's PBM (Ci-VSP240-271) binds PI(4,5)P2 significantly more strongly than other phosphatidyinositol bisphosphates. A Ci-VSP chimera created by replacing Arg 257 and Lys 258 with the amino acids found at the corresponding positions in the PTEN sequence, Gln 16 and Glu17, lead to a peptide that showed significantly reduced binding to PI(4,5)P2. While Ci-VSP240-271 exhibited a mixture of random and a-helical secondary structural elements, it was found that the chimeric Ci-VSP showed an increased b-sheet content. Molecular dynamics simulations were performed using the package NAMD and showed that the peptide does not form a helical structure and its charged residues interact in a pairwise fashion with PI(4,5)P2. Those pairs are form by R245 and R246, K252 and R253, and R254 and R257. Experimentally, the binding of the PBM has a great influence on the rate of return of the sensing charges. However, in a deletion mutant lacking the PD we observed that the PBM alone is not enough to limit the rate of return of the sensing currents. These observations strongly suggest that the binding of the PBM might be stabilized by the PD.

The BAR Domain Superfamily: Membrane-Molding Macromolecules

Membrane-shaping proteins of the BAR domain superfamily are determinants of organelle biogenesis, membrane trafficking, cell division, and cell migration. An upsurge of research now reveals new principles of BAR domain-mediated membrane remodeling, enhancing our understanding of membrane curvature-mediated information processing.

A Trypanosoma cruzi Phosphatidylinositol 3-Kinase (TcVps34) Is Involved in Osmoregulation and Receptor-mediated Endocytosis

Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking.

Neuralized Contains a Phosphoinositide-Binding Motif Required Downstream of Ubiquitination for Delta Endocytosis and Notch Signaling

The Notch signaling pathway, which plays a critical role in cell-fate decisions throughout development, is regulated by endocytosis of both the ligand and receptor. Endocytosis of the Drosophila ligands, Delta and Serrate, is required in the signaling cell for signal initiation and requires one of two ubiquitin ligases, Neuralized or Mind bomb. Through in vitro binding assays we have identified an interaction between Neuralized and phosphoinositides, modified membrane lipids that mediate membrane trafficking and signaling. We show that interactions between phosphoinositides and Neuralized contribute to the membrane localization of Neuralized in the absence of Delta, and that the phosphoinositide-binding motif is required for Neuralized to endocytose Delta downstream of Delta ubiquitination. Lastly, we provide evidence that this interaction may also be important for vertebrate Neuralized function. These results demonstrate that, through interactions with Neuralized, phosphoinositides may regulate Delta endocytosis and, by extension, Notch signal transduction.