PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics during focal adhesions assembly and disassembly in a cancer cell line

Focal adhesions (FAs) are large assemblies of proteins that mediate intracellular signals between the cytoskeleton and the extracellular matrix (ECM). The turnover of FA proteins plays a critical regulatory role in cancer cell migration. Plasma membrane lipids locally generated or broken down by different inositide kinases and phosphatase enzymes to activate and recruit proteins to specific regions in the plasma membrane. Presently, little attention has been given to the use of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) fluorescent biosensors in order to determine the spatiotemporal organisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 within and around or during assembly and disassembly of FAs. In this study, specific biosensors were used to detect PtdIns(4,5)P2, PtdIns(3,4,5)P3, and FAs proteins conjugated to RFP/GFP in order to monitor changes of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels within FAs. We demonstrated that the localisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were moderately correlated with that of FA proteins. Furthermore, we demonstrate that local levels of PtdIns(4,5)P2 increased within FA assembly and declined within FA disassembly. However, PtdIns(3,4,5)P3 levels remained constant within FAs assembly and disassembly. In conclusion, this study shows that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 localised in FAs may be regulated differently during FA assembly and disassembly.     

PtdIns(4,5)P2 is not required for secretory granule docking

Phosphoinositides (PtdIns) play important roles in exocytosis and are thought to regulate secretory granule docking by co‐clustering with the SNARE protein syntaxin to form a docking receptor in the plasma membrane. Here we tested this idea by high‐resolution total internal reflection imaging of EGFP‐labeled PtdIns markers or syntaxin‐1 at secretory granule release sites in live insulin‐secreting cells. In intact cells, PtdIns markers distributed evenly across the plasma membrane with no preference for granule docking sites. In contrast, syntaxin‐1 was found clustered in the plasma membrane, mostly beneath docked granules. We also observed rapid accumulation of syntaxin‐1 at sites where granules arrived to dock. Acute depletion of plasma membrane phosphatidylinositol (4,5) bisphosphate (PtdIns(4,5)P₂) by recruitment of a 5′‐phosphatase strongly inhibited Ca²⁺‐dependent exocytosis, but had no effect on docked granules or the distribution and clustering of syntaxin‐1. Cell permeabilization by α‐toxin or formaldehyde‐fixation caused PtdIns marker to slowly cluster, in part near docked granules. In summary, our data indicate that PtdIns(4,5)P₂ accelerates granule priming, but challenge a role of PtdIns in secretory granule docking or clustering of syntaxin‐1 at the release site.      

Novel regulatory roles of PtdIns(4,5)P2 generating enzyme EhPIPKI in actin dynamics and phagocytosis of Entamoeba histolytica

Motility and phagocytosis are the two important processes that are intricately linked to survival and virulence potential of the protist parasite Entamoeba histolytica. These processes primarily rely on actin‐dependent pathways, and regulation of these pathways is critical for understanding the pathology of E. histolytica. Generally, phosphoinositides dynamics have not been explored in amoebic actin dynamics and particularly during phagocytosis in E. histolytica. We have explored the roles of PtdIns(4,5)P₂ as well as the enzyme that produces this metabolite, EhPIPKI during phagocytosis. Immunofluorescence and live cell images showed enrichment of EhPIPKI in different stages of phagocytosis from initiation till the cups progressed towards closure. However, the enzyme was absent after phagosomes are pinched off from the membrane. Overexpression of a dominant negative mutant revealed a reduction in the formation of phagocytic cups and inhibition in the rate of engulfment of erythrocytes. Moreover, EhPIPKI binds directly to F and G‐actin unlike PIPKs from other organisms. PtdIns(4,5)P₂, the product of the enzyme, also followed a similar distribution pattern during phagocytosis as determined by a GFP‐tagged PH‐domain from PLCδ, which specifically binds PtdIns(4,5)P₂ in trophozoites. In summary, EhPIPKI regulates initiation of phagocytosis by regulating actin dynamics.     

Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation

Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P(2)-binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P(2). Amer1 translocates to the plasma membrane in a PtdIns(4,5)P(2)-dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P(2). Amer1 binds CK1γ, recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/β-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P(2) leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6.     

Molecular control of PtdIns(3,4,5)P3 signaling in neutrophils

Neutrophils play critical roles in innate immunity and host defense. However, excessive neutrophil accumulation or hyper-responsiveness of neutrophils can be detrimental to the host system. Thus, the response of neutrophils to inflammatory stimuli needs to be tightly controlled. Many cellular processes in neutrophils are mediated by localized formation of an inositol phospholipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), at the plasma membrane. The PtdIns(3,4,5)P3 signaling pathway is negatively regulated by lipid phosphatases and inositol phosphates, which consequently play a critical role in controlling neutrophil function and would be expected to act as ideal therapeutic targets for enhancing or suppressing innate immune responses. Here, we comprehensively review current understanding about the action of lipid phosphatases and inositol phosphates in the control of neutrophil function in infection and inflammation.     

CRAC channel is inhibited by neomycin in a Ptdlns(4,5)P2‐independent manner

Depletion of intracellular Ca²⁺ stores evokes store‐operated Ca²⁺ entry through the Ca²⁺ release‐activated Ca²⁺ (CRAC) channels. In this study, we found that the store‐operated Ca²⁺ entry was inhibited by neomycin, an aminoglycoside that strongly binds phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2). Patch clamp recordings revealed that neomycin blocked the CRAC currents reconstituted by co‐expression of Orai1 and Stim1 in HEK293 cells. Using a rapamycin‐inducible PtdIns(4,5)P2‐specific phosphatase (Inp54p) system to manipulate the PtdIns(4,5)P2 in the plasma membrane, we found that the CRAC current was not altered by PtdIns(4,5)P2 depletion. This result suggests that PtdIns(4,5)P2 is not required for CRAC channel activity, and thereby, neomycin inhibits CRAC channels in a manner that is independent of neomycin–PtdIns(4,5)P2 binding. Copyright © 2015 John Wiley & Sons, Ltd.     

The ER-Localized Transmembrane Protein TMEM39A/SUSR2 Regulates Autophagy by Controlling the Trafficking of the PtdIns(4)P Phosphatase SAC1

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A Pitfall of the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) Assay due to Evaporation in wells on the Edge of a 96 well Plate

The 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) calorimetric assay is replacing the traditional 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a fast, one-step assay of cell viability. We have observed that evaporation of the outer wells of a 96 well plate increases the absorbancy by 52% compared to the inner wells. Filling the outer 2 rows of wells with media and replacement of the media prior to addition of the MTS reagent will, however, correct this inaccuracy.     

The C2 and PH domains of CAPS constitute an effective PI(4,5)P2-binding unit essential for Ca2+-regulated exocytosis

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Modulation of synaptic function by VAC14, a protein that regulates the phosphoinositides PI(3,5)P(2) and PI(5)P

Normal steady-state levels of the signalling lipids PI(3,5)P(2) and PI(5)P require the lipid kinase FAB1/PIKfyve and its regulators, VAC14 and FIG4. Mutations in the PIKfyve/VAC14/FIG4 pathway are associated with Charcot-Marie-Tooth syndrome and amyotrophic lateral sclerosis in humans, and profound neurodegeneration in mice. Hence, tight regulation of this pathway is critical for neural function. Here, we examine the localization and physiological role of VAC14 in neurons. We report that endogenous VAC14 localizes to endocytic organelles in fibroblasts and neurons. Unexpectedly, VAC14 exhibits a pronounced synaptic localization in hippocampal neurons, suggesting a role in regulating synaptic function. Indeed, the amplitude of miniature excitatory postsynaptic currents is enhanced in both Vac14(-/-) and Fig4(-/-) neurons. Re-introduction of VAC14 in postsynaptic Vac14(-/-) cells reverses this effect. These changes in synaptic strength in Vac14(-/-) neurons are associated with enhanced surface levels of the AMPA-type glutamate receptor subunit GluA2, an effect that is due to diminished regulated endocytosis of AMPA receptors. Thus, VAC14, PI(3,5)P(2) and/or PI(5)P play a role in controlling postsynaptic function via regulation of endocytic cycling of AMPA receptors.     

EGFR kinase activity is required for TLR4 signaling and the septic shock response

Mammalian Toll-like receptors (TLR) recognize microbial products and elicit transient immune responses that protect the infected host from disease. TLR4—which signals from both plasma and endosomal membranes—is activated by bacterial lipopolysaccharides (LPS) and induces many cytokine genes, the prolonged expression of which causes septic shock in mice. We report here that the expression of some TLR4-induced genes in myeloid cells requires the protein kinase activity of the epidermal growth factor receptor (EGFR). EGFR inhibition affects TLR4-induced responses differently depending on the target gene. The induction of interferon-β (IFN-β) and IFN-inducible genes is strongly inhibited, whereas TNF-α induction is enhanced. Inhibition is specific to the IFN-regulatory factor (IRF)-driven genes because EGFR is required for IRF activation downstream of TLR—as is IRF co-activator β-catenin—through the PI3 kinase/AKT pathway. Administration of an EGFR inhibitor to mice protects them from LPS-induced septic shock and death by selectively blocking the IFN branch of TLR4 signaling. These results demonstrate a selective regulation of TLR4 signaling by EGFR and highlight the potential use of EGFR inhibitors to treat septic shock syndrome.     

Identification of P-Rex1 as a novel Rac1-guanine nucleotide exchange factor (GEF) that promotes actin remodeling and GLUT4 protein trafficking in adipocytes

Phosphoinositide 3-kinase (PI3K) signaling promotes the translocation of the glucose transporter, GLUT4, to the plasma membrane in insulin-sensitive tissues to facilitate glucose uptake. In adipocytes, insulin-stimulated reorganization of the actin cytoskeleton has been proposed to play a role in promoting GLUT4 translocation and glucose uptake, in a PI3K-dependent manner. However, the PI3K effectors that promote GLUT4 translocation via regulation of the actin cytoskeleton in adipocytes remain to be fully elucidated. Here we demonstrate that the PI3K-dependent Rac exchange factor, P-Rex1, enhances membrane ruffling in 3T3-L1 adipocytes and promotes GLUT4 trafficking to the plasma membrane at submaximal insulin concentrations. P-Rex1-facilitated GLUT4 trafficking requires a functional actin network and membrane ruffle formation and occurs in a PI3K- and Rac1-dependent manner. In contrast, expression of other Rho GTPases, such as Cdc42 or Rho, did not affect insulin-stimulated P-Rex1-mediated GLUT4 trafficking. P-Rex1 siRNA knockdown or expression of a P-Rex1 dominant negative mutant reduced but did not completely inhibit glucose uptake in response to insulin. Collectively, these studies identify a novel RacGEF in adipocytes as P-Rex1 that, at physiological insulin concentrations, functions as an insulin-dependent regulator of the actin cytoskeleton that contributes to GLUT4 trafficking to the plasma membrane.     

Phosphatidylinositol (4, 5)‐bisphosphate targets double C2 domain protein B to the plasma membrane

Double C2 domain protein B (DOC2B) is a high‐affinity Ca²⁺ sensor that translocates from the cytosol to the plasma membrane (PM) and promotes vesicle priming and fusion. However, the molecular mechanism underlying its translocation and targeting to the PM in living cells is not completely understood. DOC2B interacts in vitro with the PM components phosphatidylserine, phosphatidylinositol (4, 5)‐bisphosphate [PI(4, 5)P₂] and target SNAREs (t‐SNAREs). Here, we show that PI(4, 5)P₂ hydrolysis at the PM of living cells abolishes DOC2B translocation, whereas manipulations of t‐SNAREs and other phosphoinositides have no effect. Moreover, we were able to redirect DOC2B to intracellular membranes by synthesizing PI(4, 5)P₂ in those membranes. Molecular dynamics simulations and mutagenesis in the calcium and PI(4, 5)P₂‐binding sites strengthened our findings, demonstrating that both calcium and PI(4, 5)P₂ are required for the DOC2B–PM association and revealing multiple PI(4, 5)P₂–C2B interactions. In addition, we show that DOC2B translocation to the PM is ATP‐independent and occurs in a diffusion‐like manner. Our data suggest that the Ca²⁺‐triggered translocation of DOC2B is diffusion‐driven and aimed at PI(4, 5)P₂‐containing membranes.      

The C2 domains of classical PKCs are specific PtdIns(4,5)P2-sensing domains with different affinities for membrane binding

C2 domains are conserved protein modules in many eukaryotic signaling proteins, including the protein kinase (PKCs). The C2 domains of classical PKCs bind to membranes in a Ca(2+)-dependent manner and thereby act as cellular Ca(2+) effectors. Recent findings suggest that the C2 domain of PKCalpha interacts specifically with phosphatidylinositols 4,5-bisphosphate (PtdIns(4,5)P(2)) through its lysine rich cluster, for which it shows higher affinity than for POPS. In this work, we compared the three C2 domains of classical PKCs. Isothermal titration calorimetry revealed that the C2 domains of PKCalpha and beta display a greater capacity to bind to PtdIns(4,5)P(2)-containing vesicles than the C2 domain of PKCgamma. Comparative studies using lipid vesicles containing both POPS and PtdIns(4,5)P(2) as ligands revealed that the domains behave as PtdIns(4,5)P(2)-binding modules rather than as POPS-binding modules, suggesting that the presence of the phosphoinositide in membranes increases the affinity of each domain. When the magnitude of PtdIns(4,5)P(2) binding was compared with that of other polyphosphate phosphatidylinositols, it was seen to be greater in both PKCbeta- and PKCgamma-C2 domains. The concentration of Ca(2+) required to bind to membranes was seen to be lower in the presence of PtdIns(4,5)P(2) for all C2 domains, especially PKCalpha. In vivo experiments using differentiated PC12 cells transfected with each C2 domain fused to ECFP and stimulated with ATP demonstrated that, at limiting intracellular concentration of Ca(2+), the three C2 domains translocate to the plasma membrane at very similar rates. However, the plasma membrane dissociation event differed in each case, PKCalpha persisting for the longest time in the plasma membrane, followed by PKCgamma and, finally, PKCbeta, which probably reflects the different levels of Ca(2+) needed by each domain and their different affinities for PtdIns(4,5)P(2).     

Inhibition of lipid kinase PIKfyve reveals a role for phosphatase Inpp4b in the regulation of PI(3)P-mediated lysosome dynamics through VPS34 activity

Lysosome membranes contain diverse phosphoinositide (PtdIns) lipids that coordinate lysosome function and dynamics. The PtdIns repertoire on lysosomes is tightly regulated by the actions of diverse PtdIns kinases and phosphatases; however, specific roles for PtdIns in lysosomal functions and dynamics are currently unclear and require further investigation. It was previously shown that PIKfyve, a lipid kinase that synthesizes PtdIns(3,5)P₂ from PtdIns(3)P, controls lysosome “fusion-fission” cycle dynamics, autophagosome turnover, and endocytic cargo delivery. Furthermore, INPP4B, a PtdIns 4-phosphatase that hydrolyzes PtdIns(3,4)P₂ to form PtdIns(3)P, is emerging as a cancer-associated protein with roles in lysosomal biogenesis and other lysosomal functions. Here, we investigated the consequences of disrupting PIKfyve function in Inpp4b-deficient mouse embryonic fibroblasts. Through confocal fluorescence imaging, we observed the formation of massively enlarged lysosomes, accompanied by exacerbated reduction of endocytic trafficking, disrupted lysosome fusion-fission dynamics, and inhibition of autophagy. Finally, HPLC scintillation quantification of ³H-myo-inositol labeled PtdIns and PtdIns immunofluorescence staining, we observed that lysosomal PtdIns(3)P levels were significantly elevated in Inpp4b-deficient cells due to the hyperactivation of phosphatidylinositol 3-kinase catalytic subunit VPS34 enzymatic activity. In conclusion, our study identifies a novel signaling axis that maintains normal lysosomal homeostasis and dynamics, which includes the catalytic functions of Inpp4b, PIKfyve, and VPS34.     

PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2‐dependent actin remodeling

Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLCβ1 enzymatically reduce plasma membrane PI(4,5)P₂ levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P₂ abundance by these enzymes releases the PI(4,5)P₂‐binding protein cofilin from its inactive membrane‐associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid‐dependent sequestration of an actin‐remodeling factor.     

Molecular Determinants of Phosphatidylinositol 4,5-Bisphosphate (PI(4,5)P2) Binding to Transient Receptor Potential V1 (TRPV1) Channels

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P₂ is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P₂ binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P₂ interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P₂ behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P₂ binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P₂ in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P₂ binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.