Search

Transient receptor potential canonical 3 (TRPC3) is a calcium-permeable, non-selective cation channel known to be regulated by components of the phospholipase C (PLC)-mediated signaling pathway, such as Ca 2+ , diacylglycerol (DAG) and phosphatidylinositol 4,5-biphosphate (PI(4,5)P 2 ). However, the molecular gating mechanism by these regulators is not yet fully understood, especially its regulation by PI(4,5)P 2 , despite the importance of this channel in cardiovascular pathophysiology. Recently, Clarke et al. (2024) have reported that PI(4,5)P 2 is a positive modulator for TRPC3 using molecular dynamics simulations and patch-clamp techniques. They have demonstrated a multistep gating mechanism of TRPC3 with the binding of PI(4,5)P 2 to the lipid binding site located at the pre-S1/S1 nexus, and the propagation of PI(4,5)P 2 sensing to the pore domain via a salt bridge between the TRP helix and the S4-S5 linker., Competing Interests: Declaration of competing interest Authors have no competing financial interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The spatial organization of inositol 1,4,5-trisphosphate (IP 3 )-evoked Ca 2+ signals underlies their versatility. Low stimulus intensities evoke Ca 2+ puffs, localized Ca 2+ signals arising from a few IP 3 receptors (IP 3 Rs) within a cluster tethered beneath the plasma membrane. More intense stimulation evokes global Ca 2+ signals. Ca 2+ signals propagate regeneratively as the Ca 2+ released stimulates more IP 3 Rs. How is this potentially explosive mechanism constrained to allow local Ca 2+ signaling? We developed methods that allow IP 3 produced after G-protein coupled receptor (GPCR) activation to be intercepted and replaced by flash photolysis of a caged analog of IP 3 . We find that phosphatidylinositol 4,5-bisphosphate (PIP 2 ) primes IP 3 Rs to respond by partially occupying their IP 3 -binding sites. As GPCRs stimulate IP 3 formation, they also deplete PIP 2 , relieving the priming stimulus. Loss of PIP 2 resets IP 3 R sensitivity and delays the transition from local to global Ca 2+ signals. Dual regulation of IP 3 Rs by PIP 2 and IP 3 through GPCRs controls the transition from local to global Ca 2+ signals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Phosphorylated residues of G protein-coupled receptors bind to the N-domain of arrestin, resulting in the release of its C-terminus. This induces further allosteric conformational changes, such as polar core disruption, alteration of interdomain loops, and domain rotation, which transform arrestins into the receptor-activated state. It is widely accepted that arrestin activation occurs by conformational changes propagated from the N- to the C-domain. However, recent studies have revealed that binding of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) to the C-domain transforms arrestins into a pre-active state. Here, we aimed to elucidate the mechanisms underlying PIP 2 -induced arrestin pre-activation. We compare the conformational changes of β-arrestin-2 upon binding of PIP 2 or phosphorylated C-tail peptide of vasopressin receptor type 2 using hydrogen/deuterium exchange mass spectrometry (HDX-MS). Introducing point mutations on the potential routes of the allosteric conformational changes and analyzing these mutant constructs with HDX-MS reveals that PIP 2 -binding at the C-domain affects the back loop, which destabilizes the gate loop and βXX to transform β-arrestin-2 into the pre-active state., (© 2024. The Author(s).)

Phosphatidylinositol phosphates are powerful signaling molecules that orchestrate signaling and direct membrane trafficking in the cytosol. Interestingly, phosphatidylinositol phosphates also localize within the membrane-less compartments of the cell nucleus, where they participate in the regulation of gene expression. Nevertheless, current models of gene expression, which include condensates of proteins and nucleic acids, do not include nuclear phosphatidylinositol phosphates. This gap is partly a result of the missing detailed analysis of the subnuclear distribution of phosphatidylinositol phosphates and their relationships with gene expression. Here, we used quantitative dual-color direct stochastic optical reconstruction microscopy to analyze the nanoscale co-patterning between RNA polymerase II transcription initiation and elongation markers with respect to phosphatidylinositol 4,5- or 3,4-bisphosphate in the nucleoplasm and nuclear speckles and compared it with randomized data and cells with inhibited transcription. We found specific co-patterning of the transcription initiation marker P-S5 with phosphatidylinositol 4,5-bisphosphate in the nucleoplasm and with phosphatidylinositol 3,4-bisphosphate at the periphery of nuclear speckles. We showed the specific accumulation of the transcription elongation marker PS-2 and of nascent RNA in the proximity of phosphatidylinositol 3,4-bisphosphate associated with nuclear speckles. Taken together, this shows that the distinct spatial associations between the consecutive stages of RNA polymerase II transcription and nuclear phosphatidylinositol phosphates exhibit specificity within the gene expression compartments. Thus, in analogy to the cellular membranes, where phospholipid composition orchestrates signaling pathways and directs membrane trafficking, we propose a model in which the phospholipid identity of gene expression compartments orchestrates RNA polymerase II transcription., (© 2024 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Phospholipase A 2 's (PLA 2 's) constitute a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain on glycerophospholipids. We have previously reported that each PLA 2 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 PLA 2 's, namely the cytosolic (c), calcium-independent (i), and secreted (s) PLA 2 ' s toward PI, PI-4-phosphate (PI(4)P), and PI-4,5-bisphosphate (PI(4,5)P 2 ). The in vitro assay revealed that Group IVA cPLA 2 (GIVA cPLA 2 ) showed relatively high activity toward PI and PI(4)P among the tested PLA 2 's; nevertheless, the highly hydrophilic headgroup disrupted the interaction between the lipid surface and the enzyme. GIVA cPLA 2 and GVIA iPLA 2 showed detectable activity toward PI(4,5)P 2 , but it appeared to be a poorer substrate for all of the PLA 2 's tested. Furthermore, molecular dynamics (MD) simulations demonstrated that Thr416 and Glu418 of GIVA cPLA 2 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 cPLA 2 can accommodate PI and PI(4)P in its active site and hydrolyze them, suggesting that the GIVA cPLA 2 may best account for the PI and PIP hydrolysis in living cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Small-conductance Ca 2+ -activated K + channels (SK, K Ca 2) are gated solely by intracellular microdomain Ca 2+ . The channel has emerged as a therapeutic target for cardiac arrhythmias. Calmodulin (CaM) interacts with the CaM binding domain (CaMBD) of the SK channels, serving as the obligatory Ca 2+ sensor to gate the channels. In heterologous expression systems, phosphatidylinositol 4,5-bisphosphate (PIP2) coordinates with CaM in regulating SK channels. However, the roles and mechanisms of PIP2 in regulating SK channels in cardiomyocytes remain unknown. Here, optogenetics, magnetic nanoparticles, combined with Rosetta structural modeling, and molecular dynamics (MD) simulations revealed the atomistic mechanisms of how PIP2 works in concert with Ca 2+ -CaM in the SK channel activation. Our computational study affords evidence for the critical role of the amino acid residue R395 in the S6 transmembrane segment, which is localized in propinquity to the intracellular hydrophobic gate. This residue forms a salt bridge with residue E398 in the S6 transmembrane segment from the adjacent subunit. Both R395 and E398 are conserved in all known isoforms of SK channels. Our findings suggest that the binding of PIP2 to R395 residue disrupts the R395:E398 salt bridge, increasing the flexibility of the transmembrane segment S6 and the activation of the channel. Importantly, our findings serve as a platform for testing of structural-based drug designs for therapeutic inhibitors and activators of the SK channel family. The study is timely since inhibitors of SK channels are currently in clinical trials to treat atrial arrhythmias., Competing Interests: Competing interests statement:The authors declare no competing interest.

The signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) regulates many ion channels. It inhibits eukaryotic cyclic nucleotide-gated (CNG) channels while activating their relatives, the hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels. The prokaryotic SthK channel from Spirochaeta thermophila shares features with CNG and HCN channels and is an established model for this channel family. Here, we show SthK activity is inhibited by PIP2. A cryo-EM structure of SthK in nanodiscs reveals a PIP2-fitting density coordinated by arginine and lysine residues from the S4 helix and the C-linker, located between voltage-sensing and pore domains of adjacent subunits. Mutation of two arginine residues weakens PIP2 inhibition with the double mutant displaying insensitivity to PIP2. We propose that PIP2 inhibits SthK by gluing S4 and S6 together, stabilizing a resting channel conformation. The PIP2 binding site is partially conserved in CNG channels suggesting the possibility of a similar inhibition mechanism in the eukaryotic homologs., (© 2024. The Author(s).)

Phosphatidylinositol is a precursor of various phosphoinositides, which play crucial roles in intracellular signaling and membrane dynamics and have impact on diverse aspects of cell physiology. Phosphoinositide synthesis and turnover occur in the cytoplasmic leaflet of the organellar and plasma membranes. P4-ATPases (lipid flippases) are responsible for translocating membrane lipids from the exoplasmic (luminal) to the cytoplasmic leaflet, thereby regulating membrane asymmetry. However, the mechanism underlying phosphatidylinositol translocation across cellular membranes remains elusive. Here, we discovered that the phosphatidylcholine flippases ATP8B1, ATP8B2, and ATP10A can also translocate phosphatidylinositol at the plasma membrane. To explore the function of these phosphatidylinositol flippases, we used cells depleted of CDC50A, a protein necessary for P4-ATPase function and ATP8B1 and ATP8B2, which express in HeLa cells. Upon activation of the Gq-coupled receptor, depletion of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] was accelerated in CDC50A knockout (KO) and ATP8B1/8B2 double KO cells compared with control cells, suggesting a decrease in PtdIns(4,5)P2 levels within the plasma membrane of the KO cells upon stimulation. These findings highlight the important role of P4-ATPases in maintaining phosphoinositide homeostasis and suggest a mechanism for asymmetry of phosphatidylinositol in the cytoplasmic leaflet of the plasma membrane., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Human immunodeficiency virus (HIV)-1 assembly is initiated by Gag binding to the inner leaflet of the plasma membrane (PM). Gag targeting is mediated by its N-terminally myristoylated matrix (MA) domain and PM phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ]. Upon Gag assembly, envelope (Env) glycoproteins are recruited to assembly sites; this process depends on the MA domain of Gag and the Env cytoplasmic tail. To investigate the dynamics of Env recruitment, we applied a chemical dimerizer system to manipulate HIV-1 assembly by reversible PI(4,5)P 2 depletion in combination with super resolution and live-cell microscopy. This approach enabled us to control and synchronize HIV-1 assembly and track Env recruitment to individual nascent assembly sites in real time. Single virion tracking revealed that Gag and Env are accumulating at HIV-1 assembly sites with similar kinetics. PI(4,5)P 2 depletion prevented Gag PM targeting and Env cluster formation, confirming Gag dependence of Env recruitment. In cells displaying pre-assembled Gag lattices, PI(4,5)P 2 depletion resulted in the disintegration of the complete assembly domain, as not only Gag but also Env clusters were rapidly lost from the PM. These results argue for the existence of a Gag-induced and -maintained membrane micro-environment, which attracts Env. Gag cluster dissociation by PI(4,5)P 2 depletion apparently disrupts this micro-environment, resulting in the loss of Env from the former assembly domain.IMPORTANCEHuman immunodeficiency virus (HIV)-1 assembles at the plasma membrane of infected cells, resulting in the budding of membrane-enveloped virions. HIV-1 assembly is a complex process initiated by the main structural protein of HIV-1, Gag. Interestingly, HIV-1 incorporates only a few envelope (Env) glycoproteins into budding virions, although large Env accumulations surrounding nascent Gag assemblies are detected at the plasma membrane of HIV-expressing cells. The matrix domain of Gag and the Env cytoplasmatic tail play a role in Env recruitment to HIV-1 assembly sites and its incorporation into nascent virions. However, the regulation of these processes is incompletely understood. By combining a chemical dimerizer system to manipulate HIV-1 assembly with super resolution and live-cell microscopy, our study provides new insights into the interplay between Gag, Env, and host cell membranes during viral assembly and into Env incorporation into HIV-1 virions., Competing Interests: The authors declare no conflict of interest.

Cytokinesis is the final stage of the cell cycle that results in the physical separation of daughter cells. To accomplish cytokinesis, many organisms build an actin- and myosin-based cytokinetic ring (CR) that is anchored to the plasma membrane (PM). Defects in CR-PM anchoring can arise when the PM lipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] is depleted. In Schizosaccharomyces pombe, reduced PM PI(4,5)P2 results in a CR that cannot maintain a medial position and slides toward one cell end, resulting in two differently sized daughter cells. S. pombe PM PI(4,5)P2 is synthesized by the phosphatidylinositol 4-phosphate 5-kinase (PI5-kinase) Its3, but what regulates this enzyme to maintain appropriate PM PI(4,5)P2 levels in S. pombe is not known. To identify Its3 regulators, we used proximity-based biotinylation, and the uncharacterized protein Duc1 was specifically detected. We discovered that Duc1 decorates the PM except at the cell division site and that its unique localization pattern is dictated by binding to the endoplasmic reticulum (ER)-PM contact site proteins Scs2 and Scs22. Our evidence suggests that Duc1 also binds PI(4,5)P2 and helps enrich Its3 at the lateral PM, thereby promoting PM PI(4,5)P2 synthesis and robust CR-PM anchoring., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The ability for cells to localize and activate peripheral membrane-binding proteins is critical for signal transduction. Ubiquitously important in these signaling processes are phosphatidylinositol phosphate (PIP) lipids, which are dynamically phosphorylated by PIP lipid kinases on intracellular membranes. Functioning primarily at the plasma membrane, phosphatidylinositol-4-phosphate 5-kinases (PIP5K) catalyzes the phosphorylation of PI(4)P to generate most of the PI(4,5)P 2 lipids found in eukaryotic plasma membranes. Recently, we determined that PIP5K displays a positive feedback loop based on membrane-mediated dimerization and cooperative binding to its product, PI(4,5)P 2 . Here, we examine how two motifs contribute to PI(4,5)P 2 recognition to control membrane association and catalysis of PIP5K. Using a combination of single molecule TIRF microscopy and kinetic analysis of PI(4)P lipid phosphorylation, we map the sequence of steps that allow PIP5K to cooperatively engage PI(4,5)P 2 . We find that the specificity loop regulates the rate of PIP5K membrane association and helps orient the kinase to more effectively bind PI(4,5)P 2 lipids. After correctly orienting on the membrane, PIP5K transitions to binding PI(4,5)P 2 lipids near the active site through a motif previously referred to as the substrate or PIP-binding motif (PIPBM). The PIPBM has broad specificity for anionic lipids and serves a role in regulating membrane association in vitro and in vivo. Overall, our data supports a two-step membrane-binding model where the specificity loop and PIPBM act in concert to help PIP5K orient and productively engage anionic lipids to drive the positive feedback during PI(4,5)P 2 production., Competing Interests: Conflicts of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is a phospholipid essential for plasma membrane functions, but its two-dimensional distribution is not clear. Here, we compared the result of sodium dodecyl sulfate-treated freeze-fracture replica labeling (SDS-FRL) of quick-frozen cells with the actual PtdIns(4,5)P2 content and the results obtained by fluorescence biosensor and by labeling of chemically-fixed membranes. In yeast, enrichment of PtdIns(4,5)P2 in the membrane compartment of Can1 (MCC)/eisosome, especially in the curved MCC/eisosome, was evident by SDS-FRL, but not by fluorescence biosensor, GFP-PLC1δ-PH. PtdIns(4,5)P2 remaining after acute ATP depletion and in the stationary phase, 30.0% and 56.6% of the control level, respectively, was not detectable by fluorescence biosensor, whereas the label intensity by SDS-FRL reflected the PtdIns(4,5)P2 amount. In PC12 cells, PtdIns(4,5)P2 was observed in a punctate pattern in the formaldehyde-fixed plasma membrane, whereas it was distributed randomly by SDS-FRL and showed clustering after formaldehyde fixation. The results indicate that the distribution of PtdIns(4,5)P2 can be defined most reliably by SDS-FRL of quick-frozen cells., (© 2024 Tsuji et al.)

The binding of the HIV-1 Gag polyprotein to the plasma membrane is a critical step in viral replication. The association with membranes depends on the lipid composition, but its mechanisms remain unclear. Here, we report the binding of non-myristoylated Gag to lipid membranes of different lipid compositions to dissect the influence of each component. We tested the contribution of phosphatidylserine, PI(4,5)P2, and cholesterol to membrane charge density and Gag affinity to membranes. Taking into account the influence of the membrane surface potential, we quantitatively characterized the adsorption of the protein onto model lipid membranes. The obtained Gag binding constants appeared to be the same regardless of the membrane charge. Furthermore, Gag adsorbed on uncharged membranes, suggesting a contribution of hydrophobic forces to the protein-lipid interaction. Charge-charge interactions resulted in an increase in protein concentration near the membrane surface. Lipid-specific interactions were observed in the presence of cholesterol, resulting in a two-fold increase in binding constants. The combination of cholesterol with PI(4,5)P2 showed cooperative effects on protein adsorption. Thus, we suggest that the affinity of Gag to lipid membranes results from a combination of electrostatic attraction to acidic lipids, providing different protein concentrations near the membrane surface, and specific hydrophobic interactions.

Annexin A2 (A2)-induced microdomain formation is a key step in biological processes such as Ca 2+ -mediated exocytosis in neuroendocrine cells. In this work, a total of 15 coarse-grained molecular dynamics simulations were performed on vesicle models having a diameter of approximately 250 Å for 15 μs each using the Martini2 force field. Five simulations were performed in the presence of 10 A2, 5 in the presence of A2 but absence of PIP 2 , and 5 simulations in the absence of A2 but presence of PIP 2 . Consistent results were generated among the simulations. A2-induced PIP 2 microdomain formation was observed and shown to occur in three phases: A2-vesicle association, localized A2-induced PIP 2 clustering, and A2 aggregation driving PIP 2 microdomain formation. The relationship between A2 aggregation and PIP 2 microdomain formation was quantitatively described using a novel method which calculated the variance among protein and lipid positions via the Fréchet mean. A large reduction in PIP 2 variance was observed in the presence of A2 but not in its absence. This reduction in PIP 2 variance was proportional to the reduction observed in A2 variance and demonstrates that the observed PIP 2 microdomain formation is dependent upon A2 aggregation. The three-phase model of A2-induced microdomain formation generated in this work will serve as a valuable guide for further experimental studies and the development of novel A2 inhibitors. No microdomain formation was observed in the absence of A2 and minimal A2-membrane interaction was observed in the absence of PIP 2 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

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)P 2 ] of the plasma membrane inner leaflet is essential for the proton activation of PAC channels. PI(4,5)P 2 depletion by activating phosphatidylinositol 5-phosphatases or G q protein-coupled muscarinic receptors substantially inhibits human PAC currents. In excised inside-out patches, PI(4,5)P 2 application to the cytoplasmic side increases the currents. Structural simulation reveals that the putative PI(4,5)P 2 -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)P 2 . 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)P 2 ., (© 2024. The Author(s).)

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 gender‑specific 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. PLCβ4 was identified as a critical protein, which might be related to the gender‑specific cognitive deficits in male rats. The up-regulated PLCβ4 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 Aβ deposition in the hippocampus. These findings highlight that PLCβ4 might be a potential target for the therapy of learning and cognitive deficits caused by Cd exposure in early life., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Qihao Zhang reports financial support was provided by the National Natural Science Foundation of China (No. 81973072) and the Natural Science Foundation of Guangdong province (No. 2023A1515012105). If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Cell signaling is an important process involving complex interactions between lipids and proteins. The myristoylated alanine-rich C-kinase substrate (MARCKS) has been established as a key signaling regulator, serving a range of biological roles. Its effector domain (ED), which anchors the protein to the plasma membrane, induces domain formation in membranes containing phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and phosphatidylserine (PS). The mechanisms governing the MARCKS-ED binding to membranes remain elusive. Here, we investigate the composition-dependent affinity and MARCKS-ED-binding-induced changes in interfacial environments using two-dimensional infrared spectroscopy and fluorescence anisotropy. Both negatively charged lipids facilitate the MARCKS-ED binding to lipid vesicles. Although the hydrogen-bonding structure at the lipid-water interface remains comparable across vesicles with varied lipid compositions, the dynamics of interfacial water show divergent patterns due to specific interactions between lipids and peptides. Our findings also reveal that PIP 2 becomes sequestered by bound peptides, while the distribution of PS exhibits no discernible change upon peptide binding. Interestingly, PIP 2 and PS become colocalized into domains both in the presence and absence of MARCKS-ED. More broadly, this work offers molecular insights into the effects of membrane composition on binding., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ), 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 PIP 2 leads to alterations in PIP 2 -mediated signaling causing modulation of ion channels such as ɣ-aminobutyric acid type A (GABA A ) 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 PIP 2 levels which cause cascading effects on receptor trafficking, including GABA A receptor internalization. This comprehensive review of various mechanisms of interaction provides insights into the intricate interplay between PIP 2 signaling and anesthetic-induced changes, shedding light on the molecular mechanisms underlying anesthesia., (Published by Elsevier Inc.)

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 PI 4,5 P 2 , suggesting that misregulation of this lipid underlies the sur7Δ phenotype. To test this, we increased PI 4,5 P 2 levels by deleting combinations of the three PI 4,5 P 2 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 PI 4,5 P 2 , reduced the abnormal PI 4,5 P 2 clusters and also decreased the abnormal cell wall and stress sensitive phenotypes of the sur7Δ mutant. Additional studies support a model that the abnormal PI 4,5 P 2 patches recruit septin proteins, which in turn promote aberrant cell wall growth. These results identify Sur7 as a novel regulator of PI 4,5 P 2 and highlight the critical role of PI 4,5 P 2 in the regulation of C. albicans virulence properties.

The minor phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] 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)P 2 at synapses. We describe steps for stimulating and fixing murine hippocampal neurons, staining with probes for PI(4,5)P 2 and a synaptic marker, and analysis by high-resolution microscopy. Our approach gives insights into local PI(4,5)P 2 synthesis and turnover at synapses and can be extended to phosphoinositide lipids other than PI(4,5)P 2 . For complete details on the use and execution of this protocol, please refer to Bolz et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 PIP 2 . Using molecular dynamics simulations and patch clamp techniques, we reveal that PIP 2 predominantly interacts with TRPC3 at the L3 lipid binding site, located at the intersection of pre-S1 and S1 helices. We demonstrate that PIP 2 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 PIP 2 . 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., (© 2024. The Author(s).)

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)P 2 -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., (© 2024. The Author(s).)

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)P 2 -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, 655 LRRLL 659 , suggesting that membrane binding may affect JM dimerization and, therefore, regulate kinase activation., (© 2024 Federation of European Biochemical Societies.)

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 α7β1 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)P 2 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)P 2 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 ., (© 2024 Tavasoli and McMaster.)

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P 2 ] is implicated in various processes, including hormone-induced signal transduction, endocytosis, and exocytosis in the plasma membrane. However, how H 2 O 2 accumulation regulates the levels of PtdIns(4,5)P 2 in the plasma membrane in cells stimulated with epidermal growth factors (EGFs) is not known. We show that a plasma membrane PtdIns(4,5)P 2 -degrading enzyme, synaptojanin (Synj) phosphatase, is inactivated through oxidation by H 2 O 2 . Intriguingly, H 2 O 2 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 H 2 O 2 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., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of interest The authors declare that they have not conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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)P 2 ) and phosphatidylinositol 3,4,5-trisphosphate (P(I3,4,5)P 3 ), bridge the binding of YAP/TAZ to TEAD. Inhibiting these kinases or the association of YAP/TAZ with PI(4,5)P 2 and PI(3,4,5)P 3 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., (© 2024. The Author(s).)

Calcium (Ca 2+ ) 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 Ca 2+ signals play an essential role in determining their specificity. Ca 2+ signaling patterns can differ in various cell regions, and Ca 2+ signals in the nuclear and cytoplasmic compartments have been observed to occur independently. The initiation and function of Ca 2+ signaling within the nucleus are not yet fully understood. Receptor tyrosine kinases (RTKs) induce Ca 2+ 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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-2xP4M SidM , PH PLCδ1 -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)P 2 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)P 2 is localized at the base of cilia. Knocking down or knocking out INPP5E alters this distribution, resulting in the distribution of PI(4,5)P 2 along the ciliary membrane and the disappearance of PI4P from the cilia. Meanwhile, PI(4,5)P 2 is located in the ciliary membrane labeled by SMO-tRFP., Conclusions: INPP5E regulates the distribution of phosphoinositide on cilia. PI(4,5)P 2 localizes at the ciliary membrane labeled with SMO-tRFP, indicating that ciliary pocket membrane contains PI(4,5)P 2 , and phosphoinositide composition in early membrane structures may differ from that in mature ciliary membrane., (© 2024 The Authors. Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

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)P 2 . PI(4,5)P 2 is a substrate for phospholipase C and during cell signaling, PI(4,5)P 2 levels are reduced. Here I discuss a family of proteins, phosphatidylinositol transfer proteins (PITPs) that can restore PI(4,5)P 2 levels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

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 β2 adrenergic receptor (β2AR) from the PM to early endosomes. In this study, we tested the effect of hormone-induced PM PIP2 depletion on β2AR internalization using type-1 angiotensin receptor (AT1R) or M3 muscarinic acetylcholine receptor (M3R). We followed the endocytic route of β2ARs 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, β2AR 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 β2AR 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 β2AR internalization upon either AT1R or M3R activation, indicating the importance of a locally synthetized phosphoinositide pool in the regulation of this process., (© 2024. The Author(s).)

Phosphatidylinositol (PI)-4-phosphate (PI4P) is a lipid found at the plasma membrane (PM) and Golgi in cells from yeast to humans. PI4P is generated from PI by PI4-kinases and can be converted into PI-4,5-bisphosphate [PI(4,5)P2]. Schizosaccharomyces pombe have two essential PI4-kinases - Stt4 and Pik1. Stt4 localizes to the PM, and its loss from the PM results in a decrease of PM PI4P and PI(4,5)P2. As a result, cells divide non-medially due to disrupted cytokinetic ring-PM anchoring. However, the localization and function of S. pombe Pik1 has not been thoroughly examined. Here, we found that Pik1 localizes exclusively to the trans-Golgi and is required for Golgi PI4P production. We determined that Ncs1 regulates Pik1, but unlike in other organisms, it is not required for Pik1 Golgi localization. When Pik1 function was disrupted, PM PI4P but not PI(4,5)P2 levels were reduced, a major difference compared with Stt4. We conclude that Stt4 is the chief enzyme responsible for producing the PI4P that generates PI(4,5)P2. Also, that cells with disrupted Pik1 do not divide asymmetrically highlights the specific importance of PM PI(4,5)P2 for cytokinetic ring-PM anchoring., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Anomalous aggregation of α-synuclein (α-Syn) is a pathological hallmark of many degenerative synucleinopathies including Lewy body dementia (LBD) and Parkinson's disease (PD). Despite its strong link to disease, the precise molecular mechanisms that link α-Syn aggregation to neurodegeneration have yet to be elucidated. Here, we find that elevated α-Syn leads to an increase in the plasma membrane (PM) phosphoinositide PI(4,5)P 2 , which precipitates α-Syn aggregation and drives toxic increases in mitochondrial Ca 2+ and reactive oxygen species leading to neuronal death. Upstream of this toxic signaling pathway is PIP5K1γ, whose abundance and localization is enhanced at the PM by α-Syn-dependent increases in ARF6. Selective inhibition of PIP5K1γ or knockout of ARF6 in neurons rescues α-Syn aggregation and cellular phenotypes of toxicity. Collectively, our data suggest that modulation of phosphoinositide metabolism may be a therapeutic target to slow neurodegeneration for PD and other related neurodegenerative disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

α-Synuclein and family members β- and γ-synuclein are presynaptic proteins that sense and generate membrane curvature, properties important for synaptic vesicle (SV) cycling. αβγ-synuclein triple knockout neurons exhibit SV endocytosis deficits. Here, we investigated if α-synuclein affects clathrin assembly in vitro. Visualizing clathrin assembly on membranes using a lipid monolayer system revealed that α-synuclein increases clathrin lattices size and curvature. On cell membranes, we observe that α-synuclein is colocalized with clathrin and its adapter AP180 in a concentric ring pattern. Clathrin puncta that contain both α-synuclein and AP180 were significantly larger than clathrin puncta containing either protein alone. We determined that this effect occurs in part through colocalization of α-synuclein with the phospholipid PI(4,5)P2 in the membrane. Immuno-electron microscopy (EM) of synaptosomes uncovered that α-synuclein relocalizes from SVs to the presynaptic membrane upon stimulation, positioning α-synuclein to function on presynaptic membranes during or after stimulation. Additionally, we show that deletion of synucleins impacts brain-derived clathrin-coated vesicle size. Thus, α-synuclein affects the size and curvature of clathrin structures on membranes and functions as an endocytic accessory protein., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The lipid molecule phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] controls all aspects of plasma membrane (PM) function in animal cells, from its selective permeability to the attachment of the cytoskeleton. Although disruption of PI(4,5)P2 is associated with a wide range of diseases, it remains unclear how cells sense and maintain PI(4,5)P2 levels to support various cell functions. Here, we show that the PIP4K family of enzymes, which synthesize PI(4,5)P2 via a minor pathway, also function as sensors of tonic PI(4,5)P2 levels. PIP4Ks are recruited to the PM by elevated PI(4,5)P2 levels, where they inhibit the major PI(4,5)P2-synthesizing PIP5Ks. Perturbation of this simple homeostatic mechanism reveals differential sensitivity of PI(4,5)P2-dependent signaling to elevated PI(4,5)P2 levels. These findings reveal that a subset of PI(4,5)P2-driven functions might drive disease associated with disrupted PI(4,5)P2 homeostasis., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The phospholipid phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] acts as a signaling lipid at the plasma membrane (PM) with pleiotropic regulatory actions on multiple cellular processes. Signaling specificity might result from spatiotemporal compartmentalization of the lipid and from combinatorial binding of PI(4,5)P2 effector proteins to additional membrane components. Here, we analyzed the spatial distribution of tubbyCT, a paradigmatic PI(4,5)P2-binding domain, in live mammalian cells by total internal reflection fluorescence (TIRF) microscopy and molecular dynamics simulations. We found that unlike other well-characterized PI(4,5)P2 recognition domains, tubbyCT segregates into distinct domains within the PM. TubbyCT enrichment occurred at contact sites between PM and endoplasmic reticulum (ER) (i.e. at ER-PM junctions) as shown by colocalization with ER-PM markers. Localization to these sites was mediated in a combinatorial manner by binding to PI(4,5)P2 and by interaction with a cytosolic domain of extended synaptotagmin 3 (E-Syt3), but not other E-Syt isoforms. Selective localization to these structures suggests that tubbyCT is a novel selective reporter for a ER-PM junctional pool of PI(4,5)P2. Finally, we found that association with ER-PM junctions is a conserved feature of tubby-like proteins (TULPs), suggesting an as-yet-unknown function of TULPs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) plays an essential role in neuronal activities through interaction with various proteins involved in signaling at membranes. However, the distribution pattern of PI(4,5)P 2 and the association with these proteins on the neuronal cell membranes remain elusive. In this study, we established a method for visualizing PI(4,5)P 2 by SDS-digested freeze-fracture replica labeling (SDS-FRL) to investigate the quantitative nanoscale distribution of PI(4,5)P 2 in cryo-fixed brain. We demonstrate that PI(4,5)P 2 forms tiny clusters with a mean size of ∼1000 nm 2 rather than randomly distributed in cerebellar neuronal membranes in male C57BL/6J mice. These clusters show preferential accumulation in specific membrane compartments of different cell types, in particular, in Purkinje cell (PC) spines and granule cell (GC) presynaptic active zones. Furthermore, we revealed extensive association of PI(4,5)P 2 with Ca V 2.1 and GIRK3 across different membrane compartments, whereas its association with mGluR1α was compartment specific. These results suggest that our SDS-FRL method provides valuable insights into the physiological functions of PI(4,5)P 2 in neurons. SIGNIFICANCE STATEMENT In this study, we established an electron microscopic method to visualize and analyze the quantitative distribution pattern of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P 2 ) on cell membranes using cryo-fixed brain tissues and SDS-digested freeze-fracture replica labeling. PI(4,5)P 2 interacts with various ion channels and receptors to regulate membrane signaling but its nanoscale distribution and association with these proteins remain elusive. This method revealed PI(4,5)P 2 clusters preferentially accumulated in specific membrane compartments and its distinct associations with Ca V 2.1, GIRK3, and mGluR1α in the mouse cerebellum. These results demonstrate usefulness of the method for gaining insights into the physiological functions of PI(4,5)P 2 ., (Copyright © 2023 Eguchi et al.)

T-cell acute lymphoblastic leukemia (T-ALL) is one of the deadliest and most aggressive hematological malignancies, but its pathological mechanism in controlling cell survival is not fully understood. Oculocerebrorenal syndrome of Lowe is a rare X-linked recessive disorder characterized by cataracts, intellectual disability, and proteinuria. This disease has been shown to be caused by mutation of oculocerebrorenal syndrome of Lowe 1 (OCRL1; OCRL), encoding a phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] 5-phosphatase involved in regulating membrane trafficking; however, its function in cancer cells is unclear. Here, we uncovered that OCRL1 is overexpressed in T-ALL cells, and knockdown of OCRL1 results in cell death, indicating the essential role of OCRL in controlling T-ALL cell survival. We show OCRL is primarily localized in the Golgi and can translocate to plasma membrane (PM) upon ligand stimulation. We found OCRL interacts with oxysterol-binding protein-related protein 4L, which facilitates OCRL translocation from the Golgi to the PM upon cluster of differentiation 3 stimulation. Thus, OCRL represses the activity of oxysterol-binding protein-related protein 4L to prevent excessive PI(4,5)P 2 hydrolysis by phosphoinositide phospholipase C β3 and uncontrolled Ca 2+ release from the endoplasmic reticulum. We propose OCRL1 deletion leads to accumulation of PI(4,5)P 2 in the PM, disrupting the normal Ca 2+ oscillation pattern in the cytosol and leading to mitochondrial Ca 2+ overloading, ultimately causing T-ALL cell mitochondrial dysfunction and cell death. These results highlight a critical role for OCRL in maintaining moderate PI(4,5)P 2 availability in T-ALL cells. Our findings also raise the possibility of targeting OCRL1 to treat T-ALL disease., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The discovery and application of genome editing introduced a new era of plant breeding by giving researchers efficient tools for the precise engineering of crop genomes 1 . Here we demonstrate the power of genome editing for engineering broad-spectrum disease resistance in rice (Oryza sativa). We first isolated a lesion mimic mutant (LMM) from a mutagenized rice population. We then demonstrated that a 29-base-pair deletion in a gene we named RESISTANCE TO BLAST1 (RBL1) caused broad-spectrum disease resistance and showed that this mutation caused an approximately 20-fold reduction in yield. RBL1 encodes a cytidine diphosphate diacylglycerol synthase that is required for phospholipid biosynthesis 2 . Mutation of RBL1 results in reduced levels of phosphatidylinositol and its derivative phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ). In rice, PtdIns(4,5)P 2 is enriched in cellular structures that are specifically associated with effector secretion and fungal infection, suggesting that it has a role as a disease-susceptibility factor 3 . By using targeted genome editing, we obtained an allele of RBL1, named RBL1 Δ12 , which confers broad-spectrum disease resistance but does not decrease yield in a model rice variety, as assessed in small-scale field trials. Our study has demonstrated the benefits of editing an LMM gene, a strategy relevant to diverse LMM genes and crops., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is an important molecule located at the inner leaflet of cell membrane, where it serves as anchoring sites for a cohort of membrane-associated molecules and as a broad-reaching signaling intermediate. The lipid raft is thought as the major platform recruiting proteins for signal transduction and also known to mediate PIP 2 accumulation across the membrane. While the significance of this cross-membrane coupling is increasingly appreciated, it remains unclear whether and how PIP 2 senses the dynamic change of the ordered lipid domains over the packed hydrophobic core of the bilayer. Herein, by means of molecular dynamic simulation, we reveal that inner PIP 2 molecules can sense the outer lipid domain via inter-leaflet coupling, and the coupling manner is dictated by the acyl chain length of sphingomyelin (SM) partitioned to the lipid domain. Shorter SM promotes membrane domain registration, whereby PIP 2 accumulates beneath the domain across the membrane. In contrast, the anti-registration is thermodynamically preferred if the lipid domain has longer SM due to the hydrophobic mismatch between the corresponding acyl chains in SM and PIP 2 . In this case, PIP 2 is expelled by the domain with a higher diffusivity. These results provide molecular insights into the regulatory mechanism of correlation between the outer lipid domain and inner PIP 2 , both of which are critical components for cell signal transduction.

Many membrane proteins including ion channels and ion transporters are regulated by membrane phospholipids such as phosphoinositides in cell membranes and organelles. Voltage-sensing phosphatase, VSP, is a voltage-sensitive phosphoinositide phosphatase which dephosphorylates PI(4,5)P2 into PI(4)P. VSP rapidly reduces the level of PI(4,5)P2 upon membrane depolarization, thus serving as a useful tool to quantitatively study phosphoinositide-regulation of ion channels and ion transporters using a cellular electrophysiology system. In this review, we focus on the application of VSPs to Kv7 family potassium channels, which have been important research targets in biophysics, pharmacology and medicine., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

The four-point-one ezrin-radixin-moesin homology (FERM) protein domain is a multifunctional protein-lipid binding site, constituting an integral part of numerous membrane-associated proteins. Its interaction with the lipid phosphatidylinositol-4,5-bisphosphate (PIP 2 ), located at the inner leaflet of eukaryotic plasma membranes, is important for localization, anchorage, and activation of FERM-containing proteins. FERM-PIP 2 complexes structurally determined so far exclusively feature a 1:1 binding stoichiometry of protein and lipid, with a few basic FERM residues neutralizing the -4 charge of the bound PIP 2 . Whether this picture from static crystal structures also applies to the dynamic interaction of FERM domains on PIP 2 membranes is unknown. We here quantified the stoichiometry of FERM-PIP 2 binding in a lipid bilayer using atomistic molecular dynamics simulations and experiments on solid supported membranes for the FERM domains of focal adhesion kinase and ezrin. In contrast to the structural data, we find much higher average stoichiometries of FERM-PIP 2 binding, amounting to 1:3 or 1:4 ratios, respectively. In simulations, the full set of basic residues at the membrane interface, 7 and 15 residues for focal adhesion kinase and ezrin, respectively, engages in PIP 2 interactions. In addition, Na ions enter the FERM-membrane binding interface, compensating negative PIP 2 charges in case of high charge surpluses from bound PIP 2 . We propose the multivalent binding of FERM domains to PIP 2 in lipid bilayers to significantly enhance the stability of FERM-membrane binding and to render the FERM-membrane linkage highly adjustable., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Ca V 1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through G q -coupled AT1 receptors, angiotensin II triggers a decrease in PIP 2 , a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP 2 depletion suppresses Ca V 1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that Ca V 1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP 2 stabilizes Ca V 1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP 2 depletion and destabilization of Ca V 1.2 expression. We tested this hypothesis and report that Ca V 1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP 2 depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular Ca V 1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP 2 supplementation. Functional data revealed acute angiotensin II reduced Ca V 1.2 currents and Ca 2+ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP 2 are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP 2 stabilizes Ca V 1.2 membrane lifetimes, and angiotensin II-induced PIP 2 depletion destabilizes sarcolemmal Ca V 1.2, triggering their removal, and the acute reduction of Ca V 1.2 currents and contractility.

Synaptic plasticity involves proper establishment and rearrangement of structural and functional microdomains. Yet, visualization of the underlying lipid cues proved challenging. Applying a combination of rapid cryofixation, membrane freeze-fracturing, immunogold labeling and electron microscopy, we visualize and quantitatively determine the changes and the distribution of phosphatidylinositol-4,5-bisphosphate (PIP 2 ) in the plasma membrane of dendritic spines and subareas thereof at ultra-high resolution. These efforts unravel distinct phases of PIP 2 signals during induction of long-term depression (LTD). During the first minutes PIP 2 rapidly increases in a PIP5K-dependent manner forming nanoclusters. PTEN contributes to a second phase of PIP 2 accumulation. The transiently increased PIP 2 signals are restricted to upper and middle spine heads. Finally, PLC-dependent PIP 2 degradation provides timely termination of PIP 2 cues during LTD induction. Together, this work unravels the spatial and temporal cues set by PIP 2 during different phases after LTD induction and dissects the molecular mechanisms underlying the observed PIP 2 dynamics., (© 2023. The Author(s).)

Here, we present a protocol for optogenetic dephosphorylation of the phosphoinositide PI(4,5)P 2 at the plasma membrane of Xenopus laevis oocytes. We first describe the co-injection of oocytes with cRNAs encoding (1) a light-activated PI(4,5)P 2 5-phosphatase fusion protein, (2) its dimerization partner fused to the plasma membrane, and (3) the potassium channel reporter for PI(4,5)P 2 dephosphorylation. We then detail blue light illumination to induce PI(4,5)P 2 dephosphorylation, combined with simultaneous two-electrode voltage clamp electrophysiological recording to assess potassium channel current responses. For complete details on the use and execution of this protocol, please refer to Xu et al. (2022). 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Phosphatidylinositol 3-phosphate 5-kinase Type III PIKfyve is the main source for selectively generated phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ), a known regulator of membrane protein trafficking. PI(3,5)P 2 facilitates the cardiac KCNQ1/KCNE1 channel plasma membrane abundance and therewith increases the macroscopic current amplitude. Functional-physical interaction of PI(3,5)P 2 with membrane proteins and its structural impact is not sufficiently understood. This study aimed to identify molecular interaction sites and stimulatory mechanisms of the KCNQ1/KCNE1 channel via the PIKfyve-PI(3,5)P 2 axis. Mutational scanning at the intracellular membrane leaflet and nuclear magnetic resonance (NMR) spectroscopy identified two PI(3,5)P 2 binding sites, the known PIP 2 site PS1 and the newly identified N-terminal α-helix S0 as relevant for functional PIKfyve effects. Cd 2+ coordination to engineered cysteines and molecular modeling suggest that repositioning of S0 stabilizes the channel s open state, an effect strictly dependent on parallel binding of PI(3,5)P 2 to both sites., (© 2022 Walter de Gruyter GmbH, Berlin/Boston.)

Proton-activated chloride (PAC) channel is a ubiquitously expressed pH-sensing ion channel, encoded by PACC1 ( TMEM206 ). PAC regulates endosomal acidification and macropinosome shrinkage by releasing chloride from the organelle lumens. It is also found at the cell surface, where it is activated under pathological conditions related to acidosis and contributes to acid-induced cell death. However, the pharmacology of the PAC channel is poorly understood. Here, we report that phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) potently inhibits PAC channel activity. We solved the cryo-electron microscopy structure of PAC with PIP 2 at pH 4.0 and identified its putative binding site, which, surprisingly, locates on the extracellular side of the transmembrane domain (TMD). While the overall conformation resembles the previously resolved PAC structure in the desensitized state, the TMD undergoes remodeling upon PIP 2 -binding. Structural and electrophysiological analyses suggest that PIP 2 inhibits the PAC channel by stabilizing the channel in a desensitized-like conformation. Our findings identify PIP 2 as a new pharmacological tool for the PAC channel and lay the foundation for future drug discovery targeting this channel., Competing Interests: LM, ZR, JO, WL, ZQ No competing interests declared, (© 2023, Mihaljević, Mihaljević, Ruan et al.)

The molecular mechanisms underlying the establishment of the monopolar growth of fission yeast spores have been less characterized. Here, we report that the Cdc42 GTPase-activating protein (GAP) Rga6 is required for promoting monopolar growth during spore germination. The absence of Rga6 increases the number of spores that grow in a bipolar fashion. Rga6 decorates the non-growing cortical region, binds phosphatidylinositol 4,5-bisphosphate, and colocalizes with the phosphatidylinositol 4,5-bisphosphate-binding protein Opy1. Overexpression of Opy1 diminishes the cortical localization of Rga6. The characteristic localization of Rga6 on the cell cortex depends on the C-terminal PBR region of Rga6. Moreover, engineered chimera composed of the Rga6 C-terminal PBR region fused to the GAP domain of Rga3 or Rga4 are sufficient to rescue the spore growth phenotype caused by the absence of Rga6. Hence, our work establishes a paradigm in which the lipid composition of the plasma membrane directs polarized cell growth by specifying the cortical localization of a GAP protein., (© 2022 Wei et al.)

Lysosomes are central regulators of cellular growth and signaling. Once considered the acidic garbage can of the cell, their ever-expanding repertoire of functions include the regulation of cell growth, gene regulation, metabolic signaling, cell migration, and cell death. In this chapter, we detail how another of the lysosome's crucial roles, cholesterol transport, plays a vital role in the control of ion channel function and neuronal excitability through its ability to influence the abundance of the plasma membrane signaling lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). This chapter will introduce the biosynthetic pathways of cholesterol and PI(4,5)P 2 , discuss the molecular mechanisms through which each lipid distinctly regulates ion channels, and consider the interdependence of these lipids in the control of ion channel function., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)