The plasma membrane inner leaflet PI(4,5)P2 is essential for the activation of proton-activated chloride channels.

Proton-activated chloride (PAC) channels, ubiquitously expressed in tissues, regulate intracellular Cl- levels and cell death following acidosis. However, molecular mechanisms and signaling pathways involved in PAC channel modulation are largely unknown. Herein, we determine that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] of the plasma membrane inner leaflet is essential for the proton activation of PAC channels. PI(4,5)P2 depletion by activating phosphatidylinositol 5-phosphatases or Gq protein-coupled muscarinic receptors substantially inhibits human PAC currents. In excised inside-out patches, PI(4,5)P2 application to the cytoplasmic side increases the currents. Structural simulation reveals that the putative PI(4,5)P2-binding site is localized within the cytosol in resting state but shifts to the cell membrane's inner surface in an activated state and interacts with inner leaflet PI(4,5)P2. Alanine neutralization of basic residues near the membrane-cytosol interface of the transmembrane helice 2 significantly attenuates PAC currents. Overall, our study uncovers a modulatory mechanism of PAC channel through inner membrane PI(4,5)P2.

PTEN and the PTEN-like phosphatase CnrN have both distinct and overlapping roles in a Dictyostelium chemorepulsion pathway.

Little is known about eukaryotic chemorepulsion. The enzymes phosphatase and tensin homolog (PTEN) and CnrN dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Dictyostelium discoideum cells require both PTEN and CnrN to induce chemorepulsion of cells away from the secreted chemorepellent protein AprA. How D. discoideum cells utilize two proteins with redundant phosphatase activities in response to AprA is unclear. Here, we show that D. discoideum cells require both PTEN and CnrN to locally inhibit Ras activation, decrease basal levels of PI(3,4,5)P3 and increase basal numbers of macropinosomes, and AprA prevents this increase. AprA requires both PTEN and CnrN to increase PI(4,5)P2 levels, decrease PI(3,4,5)P3 levels, inhibit proliferation, decrease myosin II phosphorylation and increase filopod sizes. PTEN, but not CnrN, decreases basal levels of PI(4,5)P2, and AprA requires PTEN, but not CnrN, to induce cell roundness. Together, our results suggest that CnrN and PTEN play unique roles in AprA-induced chemorepulsion.

Differentiating human phospholipase A2's activity toward phosphatidylinositol, phosphatidylinositol phosphate and phosphatidylinositol bisphosphate.

Phospholipase A2's (PLA2's) constitute a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain on glycerophospholipids. We have previously reported that each PLA2 Type shows a unique substrate specificity for the molecular species it hydrolyzes, especially the acyl chain that is cleaved from the sn-2 position and to some extent the polar group. However, phosphatidylinositol (PI) and PI phosphates (PIPs) have not been as well studied as substrates as other phospholipids because the PIPs require adaptation of the standard analysis methods, but they are important in vivo. We determined the in vitro activity of the three major types of human PLA2's, namely the cytosolic (c), calcium-independent (i), and secreted (s) PLA2's toward PI, PI-4-phosphate (PI(4)P), and PI-4,5-bisphosphate (PI(4,5)P2). The in vitro assay revealed that Group IVA cPLA2 (GIVA cPLA2) showed relatively high activity toward PI and PI(4)P among the tested PLA2's; nevertheless, the highly hydrophilic headgroup disrupted the interaction between the lipid surface and the enzyme. GIVA cPLA2 and GVIA iPLA2 showed detectable activity toward PI(4,5)P2, but it appeared to be a poorer substrate for all of the PLA2's tested. Furthermore, molecular dynamics (MD) simulations demonstrated that Thr416 and Glu418 of GIVA cPLA2 contribute significantly to accommodating the hydrophilic head groups of PI and PI(4)P, which could explain some selectivity for PI and PI(4)P. These results indicated that GIVA cPLA2 can accommodate PI and PI(4)P in its active site and hydrolyze them, suggesting that the GIVA cPLA2 may best account for the PI and PIP hydrolysis in living cells.

Membrane-induced 2D phase separation of the focal adhesion protein talin.

Focal adhesions form liquid-like assemblies around activated integrin receptors at the plasma membrane. How they achieve their flexible properties is not well understood. Here, we use recombinant focal adhesion proteins to reconstitute the core structural machinery in vitro. We observe liquid-liquid phase separation of the core focal adhesion proteins talin and vinculin for a spectrum of conditions and interaction partners. Intriguingly, we show that binding to PI(4,5)P2-containing membranes triggers phase separation of these proteins on the membrane surface, which in turn induces the enrichment of integrin in the clusters. We suggest a mechanism by which 2-dimensional biomolecular condensates assemble on membranes from soluble proteins in the cytoplasm: lipid-binding triggers protein activation and thus, liquid-liquid phase separation of these membrane-bound proteins. This could explain how early focal adhesions maintain a structured and force-resistant organization into the cytoplasm, while still being highly dynamic and able to quickly assemble and disassemble.

PIP2 modulates TRPC3 activity via TRP helix and S4-S5 linker.

The transient receptor potential canonical type 3 (TRPC3) channel plays a pivotal role in regulating neuronal excitability in the brain via its constitutive activity. The channel is intricately regulated by lipids and has previously been demonstrated to be positively modulated by PIP2. Using molecular dynamics simulations and patch clamp techniques, we reveal that PIP2 predominantly interacts with TRPC3 at the L3 lipid binding site, located at the intersection of pre-S1 and S1 helices. We demonstrate that PIP2 sensing involves a multistep mechanism that propagates from L3 to the pore domain via a salt bridge between the TRP helix and S4-S5 linker. Notably, we find that both stimulated and constitutive TRPC3 activity require PIP2. These structural insights into the function of TRPC3 are invaluable for understanding the role of the TRPC subfamily in health and disease, in particular for cardiovascular diseases, in which TRPC3 channels play a major role.

Sur7 mediates a novel pathway for PI4,5P2 regulation in C. albicans that promotes stress resistance and cell wall morphogenesis.

The human fungal pathogen Candida albicans can cause lethal systemic infections due to its ability to resist stress from the host and to undergo invasive hyphal growth. Previous studies showed that plasma membrane MCC/eisosome domains were important for virulence by promoting the ability of Sur7 to mediate normal cell wall morphogenesis and stress resistance. The sur7Δ mutant displayed abnormal clusters of PI4,5P2, suggesting that misregulation of this lipid underlies the sur7Δ phenotype. To test this, we increased PI4,5P2 levels by deleting combinations of the three PI4,5P2 5' phosphatase genes (INP51, INP52, and INP54) and found that some combinations, such as inp51Δ inp52Δ, gave phenotypes similar the sur7Δ mutant. In contrast, deleting one copy of MSS4, the gene that encodes the 5' kinase needed to create PI4,5P2, reduced the abnormal PI4,5P2 clusters and also decreased the abnormal cell wall and stress sensitive phenotypes of the sur7Δ mutant. Additional studies support a model that the abnormal PI4,5P2 patches recruit septin proteins, which in turn promote aberrant cell wall growth. These results identify Sur7 as a novel regulator of PI4,5P2 and highlight the critical role of PI4,5P2 in the regulation of C. albicans virulence properties.

Decoding how receptor tyrosine kinases (RTKs) mediate nuclear calcium signaling.

Calcium (Ca2+) is a highly versatile intracellular messenger that regulates several cellular processes. Although it is unclear how a single-second messenger coordinates various effects within a cell, there is growing evidence that spatial patterns of Ca2+ signals play an essential role in determining their specificity. Ca2+ signaling patterns can differ in various cell regions, and Ca2+ signals in the nuclear and cytoplasmic compartments have been observed to occur independently. The initiation and function of Ca2+ signaling within the nucleus are not yet fully understood. Receptor tyrosine kinases (RTKs) induce Ca2+ signaling resulting from phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and inositol 1,4,5-trisphosphate (InsP3) formation within the nucleus. This signaling mechanism may be responsible for the effects of specific growth factors on cell proliferation and gene transcription. This review highlights the recent advances in RTK trafficking to the nucleus and explains how these receptors initiate nuclear calcium signaling.

PIP2 Is An Electrostatic Catalyst for Vesicle Fusion by Lowering the Hydration Energy: Arresting Vesicle Fusion by Masking PIP2.

Lipids are key factors in regulating membrane fusion. Lipids are not only structural components to form membranes but also active catalysts for vesicle fusion and neurotransmitter release, which are driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins seem to be partially assembled before fusion, but the mechanisms that arrest vesicle fusion before Ca2+ influx are still not clear. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) electrostatically triggers vesicle fusion as an electrostatic catalyst by lowering the hydration energy and that a myristoylated alanine-rich C-kinase substrate (MARCKS), a PIP2-binding protein, arrests vesicle fusion in a vesicle docking state where the SNARE complex is partially assembled. Vesicle-mimicking liposomes fail to reproduce vesicle fusion arrest by masking PIP2, indicating that native vesicles are essential for the reconstitution of physiological vesicle fusion. PIP2 attracts cations to repel water molecules from membranes, thus lowering the hydration energy barrier.

The impact of volatile anesthetics and propofol on phosphatidylinositol 4,5-bisphosphate signaling.

Phosphatidylinositol 4,5-bisphosphate (PIP2), as well as other anionic phospholipids, play a pivotal role in various cellular processes, including ion channel regulation, receptor trafficking, and intracellular signaling pathways. The binding of volatile anesthetics and propofol to PIP2 leads to alterations in PIP2-mediated signaling causing modulation of ion channels such as ɣ-aminobutyric acid type A (GABAA) receptors, voltage-gated calcium channels, and potassium channels through various mechanisms. Additionally, the interaction between anionic phospholipids and G protein-coupled receptors plays a critical role in various anesthetic pathways, with these anesthetic-induced changes impacting PIP2 levels which cause cascading effects on receptor trafficking, including GABAA receptor internalization. This comprehensive review of various mechanisms of interaction provides insights into the intricate interplay between PIP2 signaling and anesthetic-induced changes, shedding light on the molecular mechanisms underlying anesthesia.

PLCß4 driven by cadmium-exposure during gestation and lactation contributes to cognitive deficits by suppressing PIP2/PLCγ1/CREB/BDNF signaling pathway in male offspring.

The fetus and infants are particularly vulnerable to Cadmium (Cd) due to the immaturity of the blood-brain barrier. In utero and early life exposure to Cd is associated with cognitive deficits. Although such exposure has attracted widespread attention, its gender-specificity remains controversial, and there are no reports disclosing the underlying mechanism of gender­specific neurotoxicity. We extensively evaluated the learning and cognitive functions and synaptic plasticity of male and female rats exposed to maternal Cd. Maternal Cd exposure induced learning and memory deficits in male offspring rats, but not in female offspring rats. PLCß4 was identified as a critical protein, which might be related to the gender­specific cognitive deficits in male rats. The up-regulated PLCß4 competed with PLCγ1 to bind to PIP2, which counteracted the hydrolysis of PIP2 by PLCγ1. The decreased activation of PLCγ1 inhibited the phosphorylation of CREB to reduce BDNF transcription, which consequently resulted in the damage of hippocampal neurons and cognitive deficiency. Moreover, the low level of BDNF promoted AEP activation to induce Aß deposition in the hippocampus. These findings highlight that PLCß4 might be a potential target for the therapy of learning and cognitive deficits caused by Cd exposure in early life.

Defects in integrin complex formation promote CHKB-mediated muscular dystrophy.

Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in CHKB, encoding the first step in the synthesis of PC, is the cause of a rostrocaudal muscular dystrophy in both humans and mice. Loss of sarcolemma integrity is a hallmark of muscular dystrophies; however, how this occurs in the absence of choline kinase function is not known. We determine that in Chkb -/- mice there is a failure of the α7ß1 integrin complex that is specific to affected muscle. We observed that in Chkb -/- hindlimb muscles there is a decrease in sarcolemma association/abundance of the PI(4,5)P2 binding integrin complex proteins vinculin, and α-actinin, and a decrease in actin association with the sarcolemma. In cells, pharmacological inhibition of choline kinase activity results in internalization of a fluorescent PI(4,5)P2 reporter from discrete plasma membrane clusters at the cell surface membrane to cytosol, this corresponds with a decreased vinculin localization at plasma membrane focal adhesions that was rescued by overexpression of CHKB.

Phosphoinositide detection at synapses of fixed murine hippocampal neurons.

The minor phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is crucial for neurotransmission and has been implicated in Parkinson's disease. Here, we present a staining protocol for the analysis of activity-dependent changes of PI(4,5)P2 at synapses. We describe steps for stimulating and fixing murine hippocampal neurons, staining with probes for PI(4,5)P2 and a synaptic marker, and analysis by high-resolution microscopy. Our approach gives insights into local PI(4,5)P2 synthesis and turnover at synapses and can be extended to phosphoinositide lipids other than PI(4,5)P2. For complete details on the use and execution of this protocol, please refer to Bolz et al.1.

Structural determinants for membrane binding of the EGFR juxtamembrane domain.

Overactivation of the epidermal growth factor receptor (EGFR) is critical for the development of multiple cancers. Previous studies have shown that the cell membrane is a key regulator of EGFR kinase activity through its interaction with the EGFR juxtamembrane domain (JM). However, the lipid recognition specificity of EGFR-JM and its interaction details remain unclear. Using lipid strip and liposome pulldown assays, we showed that EGFR-JM could specifically interact with PI(4,5)P2-or phosphatidylserine-containing membranes. We further characterized the JM-membrane interaction using NMR-titration-based chemical shift perturbation and paramagnetic relaxation enhancement analyses, and found that residues I649 - L659 comprised the membrane-binding site. Furthermore, the membrane-binding region contains the predicted dimerization motif of JM, 655LRRLL659, suggesting that membrane binding may affect JM dimerization and, therefore, regulate kinase activation.

Nuclear phosphoinositide signaling promotes YAP/TAZ-TEAD transcriptional activity in breast cancer.

The Hippo pathway effectors Yes-associated protein 1 (YAP) and its homolog TAZ are transcriptional coactivators that control gene expression by binding to TEA domain (TEAD) family transcription factors. The YAP/TAZ-TEAD complex is a key regulator of cancer-specific transcriptional programs, which promote tumor progression in diverse types of cancer, including breast cancer. Despite intensive efforts, the YAP/TAZ-TEAD complex in cancer has remained largely undruggable due to an incomplete mechanistic understanding. Here, we report that nuclear phosphoinositides function as cofactors that mediate the binding of YAP/TAZ to TEADs. The enzymatic products of phosphoinositide kinases PIPKIα and IPMK, including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P3), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P2 and PI(3,4,5)P3 attenuates YAP/TAZ interaction with the TEADs, the expression of YAP/TAZ target genes, and breast cancer cell motility. Although we could not conclusively exclude the possibility that other enzymatic products of IPMK such as inositol phosphates play a role in the mechanism, our results point to a previously unrecognized role of nuclear phosphoinositide signaling in control of YAP/TAZ activity and implicate this pathway as a potential therapeutic target in YAP/TAZ-driven breast cancer.

Minor electrostatic changes robustly increase VP40 membrane binding, assembly, and budding of Ebola virus matrix protein derived virus-like particles.

Ebola virus (EBOV) is a filamentous negative-sense RNA virus, which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work, we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane-binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine and phosphatidylinositol 4,5-bisphosphate in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles. In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together, our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

Control of the signaling role of PtdIns(4)P at the plasma membrane through H2O2-dependent inactivation of synaptojanin 2 during endocytosis.

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is implicated in various processes, including hormone-induced signal transduction, endocytosis, and exocytosis in the plasma membrane. However, how H2O2 accumulation regulates the levels of PtdIns(4,5)P2 in the plasma membrane in cells stimulated with epidermal growth factors (EGFs) is not known. We show that a plasma membrane PtdIns(4,5)P2-degrading enzyme, synaptojanin (Synj) phosphatase, is inactivated through oxidation by H2O2. Intriguingly, H2O2 inhibits the 4-phosphatase activity of Synj but not the 5-phosphatase activity. In EGF-activated cells, the oxidation of Synj dual phosphatase is required for the transient increase in the plasma membrane levels of phosphatidylinositol 4-phosphate [PtdIns(4)P], which can control EGF receptor-mediated endocytosis. These results indicate that intracellular H2O2 molecules act as signaling mediators to fine-tune endocytosis by controlling the stability of plasma membrane PtdIns(4)P, an intermediate product of Synj phosphoinositide dual phosphatase.

INPP5E Regulates the Distribution of Phospholipids on Cilia in RPE1 Cells.

BACKGROUND: Primary cilia are static microtubule-based structures protruding from the cell surface and present on most vertebrate cells. The appropriate localization of phospholipids is essential for cilia formation and stability. INPP5E is a cilia-localized inositol 5-phosphatase; its deletion alters the phosphoinositide composition in the ciliary membrane, disrupting ciliary function. METHODS: The EGFP-2xP4MSidM, PHPLCδ1-EGFP, and SMO-tRFP plasmids were constructed by the Gateway system to establish a stable RPE1 cell line. The INPP5E KO RPE1 cell line was constructed with the CRISPR/Cas9 system. The localization of INPP5E and the distribution of PI(4,5)P2 and PI4P were examined by immunofluorescence microscopy. The fluorescence intensity co-localized with cilia was quantified by ImageJ. RESULTS: In RPE1 cells, PI4P is localized at the ciliary membrane, whereas PI(4,5)P2 is localized at the base of cilia. Knocking down or knocking out INPP5E alters this distribution, resulting in the distribution of PI(4,5)P2 along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P2 is located in the ciliary membrane labeled by SMO-tRFP. CONCLUSIONS: INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P2 localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P2, and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane.

Ion-Induced PIP2 Clustering with Martini3: Modification of Phosphate-Ion Interactions and Comparison with CHARMM36.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is a critical lipid for cellular signaling. The specific phosphorylation of the inositol ring controls protein binding as well as clustering behavior. Two popular models to describe ion-mediated clustering of PIP2 are Martini3 (M3) and CHARMM36 (C36). Molecular dynamics simulations of PIP2-containing bilayers in solutions of potassium chloride, sodium chloride, and calcium chloride, and at two different resolutions are performed to understand the aggregation and the model parameters that drive it. The average M3 clusters of PIP2 in bilayers of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine and PIP2 bilayers in the presence of K+, Na+, or Ca2+ contained 2.2, 2.6, and 6.4 times more PIP2 than C36 clusters, respectively. Indeed, the Ca2+-containing systems often formed a single large aggregate. Reparametrization of the M3 ion-phosphate Lennard-Jones interaction energies to reproduce experimental osmotic pressure of sodium dimethyl phosphate (DMP), K[DMP], and Ca[DMP]2 solutions, the same experimental target as C36, yielded comparably sized PIP2 clusters for the two models. Furthermore, C36 and the modified M3 predict similar saturation of the phosphate groups with increasing Ca2+, although the coarse-grained model does not capture the cooperativity between K+ and Ca2+. This characterization of the M3 behavior in the presence of monovalent and divalent ions lays a foundation to study cation/protein/PIP2 clustering.

Effect of hormone-induced plasma membrane phosphatidylinositol 4,5-bisphosphate depletion on receptor endocytosis suggests the importance of local regulation in phosphoinositide signaling.

Phosphatidylinositol 4,5-bisphosphate (PIP2) has been shown to be critical for the endocytosis of G protein-coupled receptors (GPCRs). We have previously demonstrated that depletion of PIP2 by chemically induced plasma membrane (PM) recruitment of a 5-phosphatase domain prevents the internalization of the ß2 adrenergic receptor (ß2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on ß2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of ß2ARs in HEK 293T cells using bioluminescence resonance energy transfer between the receptor and endosome marker Rab5. To compare the effect of lipid depletion by different means, we created and tested an AT1R fusion protein that is capable of both recruitment-based and hormone-induced depletion methods. The rate of PM PIP2 depletion was measured using a biosensor based on the PH domain of phospholipase Cδ1. As expected, ß2AR internalization was inhibited when PIP2 depletion was evoked by recruiting 5-phosphatase to PM-anchored AT1R. A similar inhibition occurred when wild-type AT1R was activated by adding angiotensin II. However, stimulation of the desensitization/internalization-impaired mutant AT1R (TSTS/4A) caused very little inhibition of ß2AR internalization, despite the higher rate of measurable PIP2 depletion. Interestingly, inhibition of PIP2 resynthesis with the selective PI4KA inhibitor GSK-A1 had little effect on the change in PH-domain-measured PM PIP2 levels but did significantly decrease ß2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process.

The expanding roles of PI4P and PI(4,5)P2 at the plasma membrane: Role of phosphatidylinositol transfer proteins.

Phosphoinositides are phosphorylated derivatives of phosphatidylinositol, a phospholipid that is synthesised at the endoplasmic reticulum. The plasma membrane contains the enzymes to phosphorylate phosphatidylinositol and is therefore rich in the phosphorylated derivatives, PI4P and PI(4,5)P2. PI(4,5)P2 is a substrate for phospholipase C and during cell signaling, PI(4,5)P2 levels are reduced. Here I discuss a family of proteins, phosphatidylinositol transfer proteins (PITPs) that can restore PI(4,5)P2 levels.