The Oxysterol-Binding Protein Cycle: Burning Off PI(4)P to Transport Cholesterol.

To maintain an asymmetric distribution of ions across membranes, protein pumps displace ions against their concentration gradient by using chemical energy. Here, we describe a functionally analogous but topologically opposite process that applies to the lipid transfer protein (LTP) oxysterol-binding protein (OSBP). This multidomain protein exchanges cholesterol for the phosphoinositide phosphatidylinositol 4-phosphate [PI(4)P] between two apposed membranes. Because of the subsequent hydrolysis of PI(4)P, this counterexchange is irreversible and contributes to the establishment of a cholesterol gradient along organelles of the secretory pathway. The facts that some natural anti-cancer molecules block OSBP and that many viruses hijack the OSBP cycle for the formation of intracellular replication organelles highlight the importance and potency of OSBP-mediated lipid exchange. The architecture of some LTPs is similar to that of OSBP, suggesting that the principles of the OSBP cycle-burning PI(4)P for the vectorial transfer of another lipid-might be general.

PIP5 Kinases Regulate Membrane Phosphoinositide and Actin Composition for Targeted Granule Secretion by Cytotoxic Lymphocytes.

How cytotoxic T lymphocytes (CTLs) sense T cell receptor (TCR) signaling in order to specialize an area of plasma membrane for granule secretion is not understood. Here, we demonstrate that immune synapse formation led to rapid localized changes in the phosphoinositide composition of the plasma membrane, both reducing phosphoinositide-4-phosphate (PI(4)P), PI(4,5)P2, and PI(3,4,5)P3 and increasing diacylglycerol (DAG) and PI(3,4)P2 within the first 2 min of synapse formation. These changes reduced negative charge across the synapse, triggering the release of electrostatically bound PIP5 kinases that are required to replenish PI(4,5)P2. As PI(4,5)P2 decreased, actin was depleted from the membrane, allowing secretion. Forced localization of PIP5Kß across the synapse prevented actin depletion, blocking both centrosome docking and secretion. Thus, PIP5Ks act as molecular sensors of TCR activation, controlling actin recruitment across the synapse, ensuring exquisite co-ordination between TCR signaling and CTL secretion.

Osh Proteins Control Nanoscale Lipid Organization Necessary for PI(4,5)P2 Synthesis.

The plasma membrane (PM) is composed of a complex lipid mixture that forms heterogeneous membrane environments. Yet, how small-scale lipid organization controls physiological events at the PM remains largely unknown. Here, we show that ORP-related Osh lipid exchange proteins are critical for the synthesis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2], a key regulator of dynamic events at the PM. In real-time assays, we find that unsaturated phosphatidylserine (PS) and sterols, both Osh protein ligands, synergistically stimulate phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity. Biophysical FRET analyses suggest an unconventional co-distribution of unsaturated PS and phosphatidylinositol 4-phosphate (PI4P) species in sterol-containing membrane bilayers. Moreover, using in vivo imaging approaches and molecular dynamics simulations, we show that Osh protein-mediated unsaturated PI4P and PS membrane lipid organization is sensed by the PIP5K specificity loop. Thus, ORP family members create a nanoscale membrane lipid environment that drives PIP5K activity and PI(4,5)P2 synthesis that ultimately controls global PM organization and dynamics.

Reconstitution of ORP-mediated lipid exchange coupled to PI4P metabolism.

Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism.

Fission yeast Duc1 links to ER-PM contact sites and influences PM lipid composition and cytokinetic ring anchoring.

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.

Redundant function of the Arabidopsis phosphatidylinositol 4-phosphate 5-kinase genes PIP5K4-6 is essential for pollen germination.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a key enzyme producing the signaling lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2 ] in eukaryotes. Although PIP5K genes are reported to be involved in pollen tube germination and growth, the essential roles of PIP5K in these processes remain unclear. Here, we performed a comprehensive genetic analysis of the Arabidopsis thaliana PIP5K4, PIP5K5, and PIP5K6 genes and revealed that their redundant function is essential for pollen germination. Pollen with the pip5k4pip5k5pip5k6 triple mutation was sterile, while pollen germination efficiency and pollen tube growth were reduced in the pip5k6 single mutant and further reduced in the pip5k4pip5k6 and pip5k5pip5k6 double mutants. YFP-fusion proteins, PIP5K4-YFP, PIP5K5-YFP, and PIP5K6-YFP, which could rescue the sterility of the triple mutant pollen, preferentially localized to the tricolpate aperture area and the future germination site on the plasma membrane prior to germination. Triple mutant pollen grains under the germination condition, in which spatiotemporal localization of the PtdIns(4,5)P2 fluorescent marker protein 2xmCHERRY-2xPHPLC as seen in the wild type was abolished, exhibited swelling and rupture of the pollen wall, but neither the conspicuous protruding site nor site-specific deposition of cell wall materials for germination. These data indicate that PIP5K4-6 and their product PtdIns(4,5)P2 are essential for pollen germination, possibly through the establishment of the germination polarity in a pollen grain.

Identification of inositol monophosphatase as a broad-spectrum antiviral target of ivermectin.

Ivermectin has broad-spectrum antiviral activities. Despite the failure in clinical application of COVID-19, it can serve as a lead compound for the development of more effective broad-spectrum antivirals, for which a better understanding of its antiviral mechanisms is essential. We thus searched for potential novel targets of ivermectin in host cells by label-free thermal proteomic profiling using Huh-7 cells. Inositol monophosphatase (IMPase) was found among the proteins with shifted thermal stability by ivermectin. Ivermectin could inhibit IMPase activity and reduce cellular myo-inositol and phosphatidylinositol-4-phosphate levels. On the other hand, inositol could impair the antiviral activity of ivermectin and lithium, an IMPase inhibitor with known antiviral activity. As phosphatidylinositol phosphate is crucial for the replication of many RNA viruses, inhibition of cellular myo-inositol biosynthesis may be an important antiviral mechanism of ivermectin. Hence, inhibition of IMPase could serve as a potential target for broad-spectrum antiviral development.

Signal Transduction of Transient Receptor Potential TRPM8 Channels: Role of PIP5K, Gq-Proteins, and c-Jun.

Transient receptor potential melastatin-8 (TRPM8) is a cation channel that is activated by cold and "cooling agents" such as menthol and icilin, which induce a cold sensation. The stimulation of TRPM8 activates an intracellular signaling cascade that ultimately leads to a change in the gene expression pattern of the cells. Here, we investigate the TRPM8-induced signaling pathway that links TRPM8 channel activation to gene transcription. Using a pharmacological approach, we show that the inhibition of phosphatidylinositol 4-phosphate 5 kinase α (PIP5K), an enzyme essential for the biosynthesis of phosphatidylinositol 4,5-bisphosphate, attenuates TRPM8-induced gene transcription. Analyzing the link between TRPM8 and Gq proteins, we show that the pharmacological inhibition of the ßγ subunits impairs TRPM8 signaling. In addition, genetic studies show that TRPM8 requires an activated Gα subunit for signaling. In the nucleus, the TRPM8-induced signaling cascade triggers the activation of the transcription factor AP-1, a complex consisting of a dimer of basic region leucine zipper (bZIP) transcription factors. Here, we identify the bZIP protein c-Jun as an essential component of AP-1 within the TRPM8-induced signaling cascade. In summary, with PIP5K, Gq subunits, and c-Jun, we identified key molecules in TRPM8-induced signaling from the plasma membrane to the nucleus.

MiR-4649-5p acts as a tumor-suppressive microRNA in triple negative breast cancer by direct interaction with PIP5K1C, thereby potentiating growth-inhibitory effects of the AKT inhibitor capivasertib.

BACKGROUND: Triple negative breast cancer (TNBC) is a particularly aggressive and difficult-to-treat subtype of breast cancer that requires the development of novel therapeutic strategies. To pave the way for such developments it is essential to characterize new molecular players in TNBC. MicroRNAs (miRNAs) constitute interesting candidates in this regard as they are frequently deregulated in cancer and contribute to numerous aspects of carcinogenesis. METHODS AND RESULTS: Here, we discovered that miR-4649-5p, a miRNA yet uncharacterized in breast cancer, is associated with better overall survival of TNBC patients. Ectopic upregulation of the otherwise very low endogenous expression levels of miR-4646-5p significantly decreased the growth, proliferation, and migration of TNBC cells. By performing whole transcriptome analysis and physical interaction assays, we were able to identify the phosphatidylinositol phosphate kinase PIP5K1C as a direct target of miR-4649-5p. Downregulation or pharmacologic inhibition of PIP5K1C phenocopied the growth-reducing effects of miR-4649-5p. PIP5K1C is known to play an important role in migration and cell adhesion, and we could furthermore confirm its impact on downstream PI3K/AKT signaling. Combinations of miR-4649-5p upregulation and PIP5K1C or AKT inhibition, using the pharmacologic inhibitors UNC3230 and capivasertib, respectively, showed additive growth-reducing effects in TNBC cells. CONCLUSION: In summary, miR-4649-5p exerts broad tumor-suppressive effects in TNBC and shows potential for combined therapeutic approaches targeting the PIP5K1C/PI3K/AKT signaling axis.

S-nitrosylation of RABG3E positively regulates vesicle trafficking to promote salt tolerance.

Nitric oxide (NO) is a key signaling molecule affecting the response of plants to salt stress; however, the underlying molecular mechanism is poorly understood. In this study, we conducted a phenotype analysis and found that the small GTPase RABG3E (RAB7) promotes salt tolerance in Arabidopsis thaliana. NO promotes the S-nitrosylation of RAB7 at Cys-171, which in turn helps maintain the ion balance in salt-stressed plants. Furthermore, the S-nitrosylation of RAB7 at Cys-171 enhances the enzyme's GTPase activity, thereby promoting vesicle trafficking and increasing its interaction with phosphatidylinositol phosphates-especially phosphatidylinositol-4-phosphate (PI4P). Exogenously applied PI4P increases vesicle trafficking and promotes salt tolerance depending on the S-nitrosylation of RAB7 at Cys-171. These findings illustrate a unique mechanism in salt tolerance, by which NO regulates vesicle trafficking and ion homeostasis through the S-nitrosylation of RAB7 and its interaction with PI4P.

Modulation of phagosome phosphoinositide dynamics by a Legionella phosphoinositide 3-kinase.

The phagosome harboring the bacterial pathogen Legionella pneumophila is known to be enriched with phosphatidylinositol 4-phosphate (PtdIns4P), which is important for anchoring a subset of its virulence factors and potentially for signaling events implicated in the biogenesis of the Legionella-containing vacuole (LCV) that supports intracellular bacterial growth. Here we demonstrate that the effector MavQ is a phosphoinositide 3-kinase that specifically catalyzes the conversion of phosphatidylinositol (PtdIns) into PtdIns3P. The product of MavQ is subsequently phosphorylated by the effector LepB to yield PtdIns(3,4)P2, whose 3-phosphate is then removed by another effector SidF to generate PtdIns4P. We also show that MavQ is associated with the LCV and the ∆mavQ mutant displays phenotypes in the anchoring of a PtdIns4P-binding effector similar to those of ∆lepB or ∆sidF mutants. Our results establish a mechanism of de novo PtdIns4P biosynthesis by L. pneumophila via a catalysis axis comprised of MavQ, LepB, and SidF on the surface of its phagosome.

Fission Yeast PUF Proteins Puf3 and Puf4 Are Novel Regulators of PI4P5K Signaling.

Phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) is a highly conserved enzyme that generates phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) by phosphorylating phosphatidylinositol 4-phosphate (PI(4)P). Schizosaccharomyces pombe (S. pombe) its3-1 is a loss-of-function mutation in the essential its3+ gene that encodes a PI4P5K. Its3 regulates cell proliferation, cytokinesis, cell integrity, and membrane trafficking, but little is known about the regulatory mechanisms of Its3. To identify regulators of Its3, we performed a genetic screening utilizing the high-temperature sensitivity (TS) of its3-1 and identified puf3+ and puf4+, encoding Pumilio/PUF family RNA-binding proteins as multicopy suppressors of its3-1 cells. The deletions of the PUF domains in the puf3+ and puf4+ genes resulted in the reduced ability to suppress its3-1, suggesting that the suppression by Puf3 and Puf4 may involve their RNA-binding activities. The gene knockout of Puf4, but not that of Puf3, exacerbated the TS of its3-1. Interestingly, mutant Its3 expression levels both at mRNA and protein levels were lower than those of the wild-type (WT) Its3. Consistently, the overexpression of the mutant its3-1 gene suppressed the its3-1 phenotypes. Notably, Puf3 and Puf4 overexpression increased the mRNA and protein expression levels of both Its3 and Its3-1. Collectively, our genetic screening revealed a functional relationship between the Pumilio/PUF family RNA-binding proteins and PI4P5K.

Roles of Phosphatidylinositol 4-Phosphorylation in Non-vesicular Cholesterol Trafficking.

Cholesterol (Chol) is an essential component of all eukaryotic cell membranes that affects the function of numerous peripheral as well as integral membrane proteins. Chol is synthesized in the ER, but it is selectively enriched within the plasma membrane (PM) and other endomembranes, which requires Chol to cross the aqueous phase of the cytoplasm. In addition to the classical vesicular trafficking pathways that are known to facilitate the bulk transport of membrane intermediates, Chol is also transported via non-vesicular lipid transfer proteins that work primarily within specialized membrane contact sites. Some of these transport pathways work against established concentration gradients and hence require energy. Recent studies highlight the unique role of phosphoinositides (PPIns), and phosphatidylinositol 4-phosphate (PI4P) in particular, for the control of non-vesicular Chol transport. In this chapter, we will review the emerging connection between Chol, PPIns, and lipid transfer proteins that include the important family of oxysterol-binding protein related proteins, or ORPs.

Stimulator of interferon genes (STING) is an essential proviral host factor for human rhinovirus species A and C.

Human rhinoviruses (RVs) are positive-strand RNA viruses that cause respiratory tract disease in children and adults. Here we show that the innate immune signaling protein STING is required for efficient replication of members of two distinct RV species, RV-A and RV-C. The host factor activity of STING was identified in a genome-wide RNA interference (RNAi) screen and confirmed in primary human small airway epithelial cells. Replication of RV-A serotypes was strictly dependent on STING, whereas RV-B serotypes were notably less dependent. Subgenomic RV-A and RV-C RNA replicons failed to amplify in the absence of STING, revealing it to be required for a step in RNA replication. STING was expressed on phosphatidylinositol 4-phosphate (PI4P)-enriched membranes and was enriched in RV-A16 compared with RV-B14 replication organelles isolated in isopycnic gradients. The host factor activity of STING was species-specific, as murine STING (mSTING) did not rescue RV-A16 replication in STING-deficient cells. This species specificity mapped primarily to the cytoplasmic, ligand-binding domain of STING. Mouse-adaptive mutations in the RV-A16 2C protein allowed for robust replication in cells expressing mSTING, suggesting a role for 2C in recruiting STING to RV-A replication organelles. Palmitoylation of STING was not required for RV-A16 replication, nor was the C-terminal tail of STING that mediates IRF3 signaling. Despite co-opting STING to promote its replication, interferon signaling in response to STING agonists remained intact in RV-A16 infected cells. These data demonstrate a surprising requirement for a key host mediator of innate immunity to DNA viruses in the life cycle of a small pathogenic RNA virus.

Ceramide-1-phosphate transfer protein (CPTP) regulation by phosphoinositides.

Ceramide-1-phosphate transfer proteins (CPTPs) are members of the glycolipid transfer protein (GLTP) superfamily that shuttle ceramide-1-phosphate (C1P) between membranes. CPTPs regulate cellular sphingolipid homeostasis in ways that impact programmed cell death and inflammation. CPTP downregulation specifically alters C1P levels in the plasma and trans-Golgi membranes, stimulating proinflammatory eicosanoid production and autophagy-dependent inflammasome-mediated cytokine release. However, the mechanisms used by CPTP to target the trans-Golgi and plasma membrane are not well understood. Here, we monitored C1P intervesicular transfer using fluorescence energy transfer (FRET) and showed that certain phosphoinositides (phosphatidylinositol 4,5 bisphosphate (PI-(4,5)P2) and phosphatidylinositol 4-phosphate (PI-4P)) increased CPTP transfer activity, whereas others (phosphatidylinositol 3-phosphate (PI-3P) and PI) did not. PIPs that stimulated CPTP did not stimulate GLTP, another superfamily member. Short-chain PI-(4,5)P2, which is soluble and does not remain membrane-embedded, failed to activate CPTP. CPTP stimulation by physiologically relevant PI-(4,5)P2 levels surpassed that of phosphatidylserine (PS), the only known non-PIP stimulator of CPTP, despite PI-(4,5)P2 increasing membrane equilibrium binding affinity less effectively than PS. Functional mapping of mutations that led to altered FRET lipid transfer and assessment of CPTP membrane interaction by surface plasmon resonance indicated that di-arginine motifs located in the α-6 helix and the α3-α4 helix regulatory loop of the membrane-interaction region serve as PI-(4,5)P2 headgroup-specific interaction sites. Haddock modeling revealed specific interactions involving the PI-(4,5)P2 headgroup that left the acyl chains oriented favorably for membrane embedding. We propose that PI-(4,5)P2 interaction sites enhance CPTP activity by serving as preferred membrane targeting/docking sites that favorably orient the protein for function.

PIP5KIγ90-generated phosphatidylinositol-4,5-bisphosphate promotes the uptake of Staphylococcus aureus by host cells.

Staphylococcus aureus, a Gram-positive pathogen, invades cells mainly in an integrin-dependent manner. As the activity or conformation of several integrin-associated proteins can be regulated by phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2 ), we investigated the roles of PI-4,5-P2 and PI-4,5-P2 -producing enzymes in cellular invasion by S. aureus. PI-4,5-P2 accumulated upon contact of S. aureus with the host cell, and targeting of an active PI-4,5-P2 phosphatase to the plasma membrane reduced bacterial invasion. Knockdown of individual phosphatidylinositol-4-phosphate 5-kinases revealed that phosphatidylinositol-4-phosphate 5-kinase γ (PIP5KIγ) plays an important role in bacterial internalization. Specific ablation of the talin and FAK-binding motif in PIP5KIγ90 reduced bacterial invasion, which could be rescued by reexpression of an active, but not inactive PIP5KIγ90. Furthermore, PIP5KIγ90-deficient cells showed normal basal PI-4,5-P2 levels in the plasma membrane but reduced the accumulation of PI-4,5-P2 and talin at sites of S. aureus attachment and overall lower levels of FAK phosphorylation. These results highlight the importance of local synthesis of PI-4,5-P2 by a focal adhesion-associated lipid kinase for integrin-mediated internalization of S. aureus.

Enhanced fitness of hepatitis C virus increases resistance to direct-acting antivirals.

Drug resistance mutations of hepatitis C virus (HCV) negatively impact viral replicative fitness. RNA viruses are known to change their replication behaviour when subjected to suboptimal selection pressure. Here, we assess whether mutation supply in HCV is sufficiently large to allow the selection of its variants during dual or triple direct-acting antiviral (DAA) treatment associated with augmented virus fitness or impairment. We engineered randomly mutagenized full-genome libraries to create a highly diverse population of replication-competent HCV variants in cell culture. These variants exhibited escape when treated with NS5A/NS5B inhibitors (daclatasvir/sofosbuvir), and relapse on treatment with a combination of NS3/NS5A/NS5B inhibitors (simeprevir or paritaprevir/daclatasvir/sofosbuvir). Analysis of the relationship between virus fitness and drug resistance of JFH1-derived NS5A-5B variants showed a significant positive correlation (P=0.003). At the earliest time points, intracellular RNA levels remain unchanged in both the subgenomic replicon and infection assays, whereas extracellular RNA levels increased upto ten-fold compared to wild-type JFH1. Beneficial substitutions hyperstimulated phosphatidylinositol 4-phosphate during DAA treatment, and showed decreased dependence on cyclophilins during cyclosporine A treatment, indicating an interplay of virus-host molecular mechanisms in beneficial substitution selection that may necessitate infectious virus production. This comprehensive study demonstrates a possible role for HCV fitness of overcoming drug-mediated selection pressure.

Lipid kinases PIP5K7 and PIP5K9 are required for polyamine-triggered K+ efflux in Arabidopsis roots.

Polyamines, such as putrescine, spermidine and spermine (Spm), are low-molecular-weight polycationic molecules present in all living organisms. Despite their implication in plant cellular processes, little is known about their molecular mode of action. Here, we demonstrate that polyamines trigger a rapid increase in the regulatory membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2 ), and that this increase is required for polyamine effects on K+ efflux in Arabidopsis roots. Using in vivo 32 Pi -labelling of Arabidopsis seedlings, low physiological (µm) concentrations of Spm were found to promote a rapid PIP2 increase in roots that was time- and dose-dependent. Confocal imaging of a genetically encoded PIP2 biosensor revealed that this increase was triggered at the plasma membrane. Differential 32 Pi -labelling suggested that the increase in PIP2 was generated through activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity rather than inhibition of a phospholipase C or PIP2 5-phosphatase activity. Systematic analysis of transfer DNA insertion mutants identified PIP5K7 and PIP5K9 as the main candidates involved in the Spm-induced PIP2 response. Using non-invasive microelectrode ion flux estimation, we discovered that the Spm-triggered K+ efflux response was strongly reduced in pip5k7 pip5k9 seedlings. Together, our results provide biochemical and genetic evidence for a physiological role of PIP2 in polyamine-mediated signalling controlling K+ flux in plants.

Sterol transfer, PI4P consumption, and control of membrane lipid order by endogenous OSBP.

The network of proteins that orchestrate the distribution of cholesterol among cellular organelles is not fully characterized. We previously proposed that oxysterol-binding protein (OSBP) drives cholesterol/PI4P exchange at contact sites between the endoplasmic reticulum (ER) and the trans-Golgi network (TGN). Using the inhibitor OSW-1, we report here that the sole activity of endogenous OSBP makes a major contribution to cholesterol distribution, lipid order, and PI4P turnover in living cells. Blocking OSBP causes accumulation of sterols at ER/lipid droplets at the expense of TGN, thereby reducing the gradient of lipid order along the secretory pathway. OSBP consumes about half of the total cellular pool of PI4P, a consumption that depends on the amount of cholesterol to be transported. Inhibiting the spatially restricted PI4-kinase PI4KIIIß triggers large periodic traveling waves of PI4P across the TGN These waves are cadenced by long-range PI4P production by PI4KIIα and PI4P consumption by OSBP Collectively, these data indicate a massive spatiotemporal coupling between cholesterol transport and PI4P turnover via OSBP and PI4-kinases to control the lipid composition of subcellular membranes.

VAPB depletion alters neuritogenesis and phosphoinositide balance in motoneuron-like cells: relevance to VAPB-linked amyotrophic lateral sclerosis.

VAPB and VAPA are ubiquitously expressed endoplasmic reticulum membrane proteins that play key roles in lipid exchange at membrane contact sites. A mutant, aggregation-prone, form of VAPB (P56S) is linked to a dominantly inherited form of amyotrophic lateral sclerosis; however, it has been unclear whether its pathogenicity is due to toxic gain of function, to negative dominance, or simply to insufficient levels of the wild-type protein produced from a single allele (haploinsufficiency). To investigate whether reduced levels of functional VAPB, independently from the presence of the mutant form, affect the physiology of mammalian motoneuron-like cells, we generated NSC34 clones, from which VAPB was partially or nearly completely depleted. VAPA levels, determined to be over fourfold higher than those of VAPB in untransfected cells, were unaffected. Nonetheless, cells with even partially depleted VAPB showed an increase in Golgi- and acidic vesicle-localized phosphatidylinositol-4-phosphate (PI4P) and reduced neurite extension when induced to differentiate. Conversely, the PI4 kinase inhibitors PIK93 and IN-10 increased neurite elongation. Thus, for long-term survival, motoneurons might require the full dose of functional VAPB, which may have unique function(s) that VAPA cannot perform.