Plasma Membrane Phosphatidylinositol 4-phosphate Research Articles

PI4P-mediated solid-like Merlin condensates orchestrate Hippo pathway regulation.

Despite recent studies implicating liquid-like biomolecular condensates in diverse cellular processes, many biomolecular condensates exist in a solid-like state, and their function and regulation are less understood. We show that the tumor suppressor Merlin, an upstream regulator of the Hippo pathway, localizes to both cell junctions and medial apical cortex in Drosophila epithelia, with the latter forming solid-like condensates that activate Hippo signaling. Merlin condensation required phosphatidylinositol-4-phosphate (PI4P)-mediated plasma membrane targeting and was antagonistically controlled by Pez and cytoskeletal tension through plasma membrane PI4P regulation. The solid-like material properties of Merlin condensates are essential for physiological function and protect the condensates against external perturbations. Collectively, these findings uncover an essential role for solid-like condensates in normal physiology and reveal regulatory mechanisms for their formation and disassembly.

Orthogonal Targeting of SAC1 to Mitochondria Implicates ORP2 as a Major Player in PM PI4P Turnover.

Oxysterol-binding protein (OSBP)-related proteins (ORPs) 5 and 8 have been shown to deplete the lipid phosphatidylinositol 4-phosphate (PI4P) at sites of membrane contact between the endoplasmic reticulum (ER) and plasma membrane (PM). This is believed to be caused by transport of PI4P from the PM to the ER, where PI4P is degraded by an ER-localized SAC1 phosphatase. This is proposed to power the anti-port of phosphatidylserine (PS) lipids from ER to PM, up their concentration gradient. Alternatively, ORPs have been proposed to sequester PI4P, dependent on the concentration of their alternative lipid ligand. Here, we aimed to distinguish these possibilities in living cells by orthogonal targeting of PI4P transfer and degradation to PM-mitochondria contact sites. Surprisingly, we found that orthogonal targeting of SAC1 to mitochondria enhanced PM PI4P turnover independent of targeting to contact sites with the PM. This turnover could be slowed by knock-down of soluble ORP2, which also has a major impact on PM PI4P levels even without SAC1 over-expression. The data reveal a role for contact site-independent modulation of PM PI4P levels and lipid antiport.

Characterization of Pik1 function in fission yeast reveals its conserved role in lipid synthesis and not cytokinesis.

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 to PI-4,5-bisphosphate [PI(4,5)P2]. Schizosaccharomyces pombe have 2 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 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.

LIPID transfer proteins regulate store-operated calcium entry via control of plasma membrane phosphoinositides

The ER-resident proteins STIM1 together with the plasma membrane (PM)-localized Orai1 channels constitute the molecular components of the store-operated Ca2+ entry (SOCE) pathway. Prepositioning of STIM1 to the peripheral ER close to the PM ensures its efficient interaction with Orai1 upon a decrease in the ER luminal Ca2+ concentration. The C-terminal polybasic domain of STIM1 has been identified as mediating the interaction with PM phosphoinositides and hence positions the molecule to ER-PM contact sites. Here we show that STIM1 requires PM phosphatidylinositol 4-phosphate (PI4P) for efficient PM interaction. Accordingly, oxysterol binding protein related proteins (ORPs) that work at ER-PM junctions and consume PI4P gradients exert important control over the Ca2+ entry process. These studies reveal an important connection between non-vesicular lipid transport at ER-PM contact sites and regulation of ER Ca2+store refilling.

Hypoxia controls plasma membrane targeting of polarity proteins by dynamic turnover of PI4P and PI(4,5)P2.

Phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-biphosphate (PIP2) are key phosphoinositides that determine the identity of the plasma membrane (PM) and regulate numerous key biological events there. To date, mechanisms regulating the homeostasis and dynamic turnover of PM PI4P and PIP2 in response to various physiological conditions and stresses remain to be fully elucidated. Here, we report that hypoxia in Drosophila induces acute and reversible depletion of PM PI4P and PIP2 that severely disrupts the electrostatic PM targeting of multiple polybasic polarity proteins. Genetically encoded ATP sensors confirmed that hypoxia induces acute and reversible reduction of cellular ATP levels which showed a strong real-time correlation with the levels of PM PI4P and PIP2 in cultured cells. By combining genetic manipulations with quantitative imaging assays we showed that PI4KIIIα, as well as Rbo/EFR3 and TTC7 that are essential for targeting PI4KIIIα to PM, are required for maintaining the homeostasis and dynamic turnover of PM PI4P and PIP2 under normoxia and hypoxia. Our results revealed that in cells challenged by energetic stresses triggered by hypoxia, ATP inhibition and possibly ischemia, dramatic turnover of PM PI4P and PIP2 could have profound impact on many cellular processes including electrostatic PM targeting of numerous polybasic proteins.

Membrane pools of phosphatidylinositol-4-phosphate regulate KCNQ1/KCNE1 membrane expression

Plasma membrane phosphatidylinositol 4-phosphate (PI4P) is a precursor of PI(4,5)P2, an important regulator of a large number of ion channels. Although the role of the phospholipid PI(4,5)P2 in stabilizing ion channel function is well established, little is known about the role of phospholipids in channel membrane localization and specifically the role of PI4P in channel function and localization. The phosphatidylinositol 4-kinases (PI4Ks) synthesize PI4P. Our data show that inhibition of PI4K and prolonged decrease of levels of plasma membrane PI4P lead to a decrease in the KCNQ1/KCNE1 channel membrane localization and function. In addition, we show that mutations linked to Long QT syndrome that affect channel interactions with phospholipids lead to a decrease in membrane expression. We show that expression of a LQT1-associated C-terminal deletion mutant abolishes PI4Kinase-mediated decrease in membrane expression and rescues membrane expression for phospholipid-targeting mutations. Our results indicate a novel role for PI4P on ion channel regulation. Our data suggest that decreased membrane PI4P availability to the channel, either due to inhibition of PI4K or as consequence of mutations, dramatically inhibits KCNQ1/KCNE1 channel membrane localization and current. Our results may have implications to regulation of other PI4P binding channels.

Plasma Membrane PI4P Regulates Vesicle Docking in Neuronal Exocytosis

Plasma membrane (PM) phosphatidylinositol 4-phosphate (PI4P) plays a crucial role in diverse cellular functions, but its role beyond being a precursor of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in secretory pathways including neurotransmitter release and hormone secretion remains unclear. Here, using structured illumination microscopy on the PM of PC12 cells, we show that PI4P is an inner-leaflet lipid that is clustered in microdomain and involved in clustering of t-SNARE protein syntaxin 1 essential for vesicle docking. We found that PI4P interacted with the C2 domain of synaptotagmin 1 and synaptotagmin-like protein 4 in a Ca2+-independent manner, resulting in recruiting SNARE proteins to the target membrane. Interestingly, the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) was decreased by PI4P depletion in cultured hippocampal neurons, particularly it was most significantly decreased when PI(4,5)P2 depleted together. Thus, PI4P contributes to anionic PM lipid pool for SNARE-lipid interaction at vesicle docking stage of neuronal exocytosis.

Regulation of PI4P levels by PI4KIIIα during G-protein-coupled PLC signaling in Drosophila photoreceptors.

The activation of phospholipase C (PLC) is a conserved mechanism of receptor-activated cell signaling at the plasma membrane. PLC hydrolyzes the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], and continued signaling requires the resynthesis and availability of PI(4,5)P2 at the plasma membrane. PI(4,5)P2 is synthesized by the phosphorylation of phosphatidylinositol 4-phosphate (PI4P). Thus, a continuous supply of PI4P is essential to support ongoing PLC signaling. While the enzyme PI4KA has been identified as performing this function in cultured mammalian cells, its function in the context of an in vivo physiological model has not been established. In this study, we show that, in Drosophila photoreceptors, PI4KIIIα activity is required to support signaling during G-protein-coupled PLC activation. Depletion of PI4KIIIα results in impaired electrical responses to light, and reduced plasma membrane levels of PI4P and PI(4,5)P2 Depletion of the conserved proteins Efr3 and TTC7 [also known as StmA and L(2)k14710, respectively, in flies], which assemble PI4KIIIα at the plasma membrane, also results in an impaired light response and reduced plasma membrane PI4P and PI(4,5)P2 levels. Thus, PI4KIIIα activity at the plasma membrane generates PI4P and supports PI(4,5)P2 levels during receptor activated PLC signaling.

PI(4,5)P2 controls plasma membrane PI4P and PS levels via ORP5/8 recruitment to ER-PM contact sites.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critically important regulatory lipid of the plasma membrane (PM); however, little is known about how cells regulate PM PI(4,5)P2 levels. Here, we show that the phosphatidylinositol 4-phosphate (PI4P)/phosphatidylserine (PS) transfer activity of the endoplasmic reticulum (ER)-resident ORP5 and ORP8 is regulated by both PM PI4P and PI(4,5)P2 Dynamic control of ORP5/8 recruitment to the PM occurs through interactions with the N-terminal Pleckstrin homology domains and adjacent basic residues of ORP5/8 with both PI4P and PI(4,5)P2 Although ORP5 activity requires normal levels of these inositides, ORP8 is called on only when PI(4,5)P2 levels are increased. Regulation of the ORP5/8 attachment to the PM by both phosphoinositides provides a powerful means to determine the relative flux of PI4P toward the ER for PS transport and Sac1-mediated dephosphorylation and PIP 5-kinase-mediated conversion to PI(4,5)P2 Using this rheostat, cells can maintain PI(4,5)P2 levels by adjusting the availability of PI4P in the PM.

Plasma membrane phosphatidylinositol 4-phosphate and 4,5-bisphosphate determine the distribution and function of K-Ras4B but not H-Ras proteins

Plasma membrane (PM) localization of Ras proteins is crucial for transmitting signals upon mitogen stimulation. Post-translational lipid modification of Ras proteins plays an important role in their recruitment to the PM. Electrostatic interactions between negatively charged PM phospholipids and basic amino acids found in K-Ras4B (K-Ras) but not in H-Ras are important for permanent K-Ras localization to the PM. Here, we investigated how acute depletion of negatively charged PM polyphosphoinositides (PPIns) from the PM alters the intracellular distribution and activity of K- and H-Ras proteins. PPIns depletion from the PM was achieved either by agonist-induced activation of phospholipase C β or with a rapamycin-inducible system in which various phosphatidylinositol phosphatases were recruited to the PM. Redistribution of the two Ras proteins was monitored with confocal microscopy or with a recently developed bioluminescence resonance energy transfer-based approach involving fusion of the Ras C-terminal targeting sequences or the entire Ras proteins to Venus fluorescent protein. We found that PM PPIns depletion caused rapid translocation of K-Ras but not H-Ras from the PM to the Golgi. PM depletion of either phosphatidylinositol 4-phosphate (PtdIns4P) or PtdIns(4,5)P2 but not PtdIns(3,4,5)P3 was sufficient to evoke K-Ras translocation. This effect was diminished by deltarasin, an inhibitor of the Ras-phosphodiesterase interaction, or by simultaneous depletion of the Golgi PtdIns4P. The PPIns depletion decreased incorporation of [3H]leucine in K-Ras-expressing cells, suggesting that Golgi-localized K-Ras is not as signaling-competent as its PM-bound form. We conclude that PPIns in the PM are important regulators of K-Ras-mediated signals.

Rab27a controls HIV-1 assembly by regulating plasma membrane levels of phosphatidylinositol 4,5-bisphosphate.

During the late stages of the HIV-1 replication cycle, the viral polyprotein Pr55(Gag) is recruited to the plasma membrane (PM), where it binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and directs HIV-1 assembly. We show that Rab27a controls the trafficking of late endosomes carrying phosphatidylinositol 4-kinase type 2 α (PI4KIIα) toward the PM of CD4(+) T cells. Hence, Rab27a promotes high levels of PM phosphatidylinositol 4-phosphate and the localized production of PI(4,5)P2, therefore controlling Pr55(Gag) membrane association. Rab27a also controls PI(4,5)P2 levels at the virus-containing compartments of macrophages. By screening Rab27a effectors, we identified that Slp2a, Slp3, and Slac2b are required for the association of Pr55(Gag) with the PM and that Slp2a cooperates with Rab27a in the recruitment of PI4KIIα to the PM. We conclude that by directing the trafficking of PI4KIIα-positive endosomes toward the PM, Rab27a controls PI(4,5)P2 production and, consequently, HIV-1 replication.

A new role for plasma membrane phosphatidylinositol 4‐phosphate (PI4P)?

Many crucial functions of the plasma membrane (PM) rely on the minor inner leaflet lipid, phosphatidylinositol 4,5‐bisphosphate [PI(4,5)P2]. The major intermediate in PI(4,5)P2 synthesis, PI4P, seems paradoxically to be superfluous for maintenance or function of PI(4,5)P2. Inhibiting the enzyme that generates PM PI4P, PI 4‐kinase (PI4K) type III α (PI4KA), leads to depletion of PI(4,5)P2 levels only when phospholipase C (PLC) is activated, suggesting that other PI4K activities synthesize PI(4,5)P2 in resting cells. Here, we set out to test this hypothesis. Using a combination of live cell imaging with fluorescent PI4P and PI(4,5)P2 biosensors, we show that type II or type III β PI4K inhibitors lead to reductions in endosomal and Golgi‐localized PI4P pools, whereas only an inhibitor of PI4KA produces a reduction in PM PI4P. However, no combination of these agents led to substantial depletion of PM PI(4,5)P2. Use of radiolabelled tracers demonstrated that PI4KA inhibitor caused reduced absolute levels of PI4P with little change in PI(4,5)P2, despite a dramatic reduction in acute PI4P and PI(4,5)P2 synthesis. Therefore, PI(4,5)P2 levels are maintained when PI4KA is blocked not because there is an alternative route of synthesis, but because PI(4,5)P2 turnover simply stops. Consistently, in cells depleted of PM PI4P by a PI4KA inhibitor, forced degradation of PI(4,5)P2 with a 5‐phosphatase yields a pool of PI4P, which rapidly runs down due to the absence of PI4KA activity to maintain it. These results suggest a novel function for PM PI4P, which is as a metabolic “buffer” whose levels and turnover can be rapidly sensed and adjusted in order to maintain the functionally crucial PI(4,5)P2 pool. This work was supported by the intramural research program of the Eunice Kennedy Shriver National Institute for Child Health and Human Development, NIH.

Phosphatidylinositol 4,5-Bisphosphate Phospholipase C and Phosphomonoesterase in Dunaliella salina Membranes

In comparison with other cell organelles, the Dunaliella salina plasma membrane was found to be highly enriched in phospholipase C activity toward exogenous [(3)H]phosphatidylinositol 4,5-bisphosphate (PIP(2)). Based on release of [(3)H]inositol phosphates, the plasma membrane exhibited a PIP(2)-phospholipase C activity nearly tenfold higher than the nonplasmalemmal, nonchloroplast ;bottom phase' (BP) membrane fraction and 47 times higher than the chloroplast membrane fraction. The majority of phospholipase activity was clearly of a phospholipase C nature since over 80% of [(3)H]inositol phosphates released were recovered as [(3)H]inositol trisphosphate (IP(3)). These results suggest a plausible mechanism for the rapid breakdown of PIP(2) and phosphatidylinositol 4-phosphate (PIP) following hypoosmotic shock. Quantitative analysis of major [(3)H]inositol phospholipids during these assays revealed that some of the [(3)H]-PIP(2) was converted to [(3)H]phosphatidylinositol 4-monophosphate (PIP) and to [(3)H]phosphatidyl-inositol (PI) in the BP fraction of membrane remaining after removal of plasmalemma and chloroplasts. This latter fraction is enriched more than fivefold in PIP(2)/PIP phosphomonoesterase activity when compared to the plasmalemma or chloroplast membrane fractions. We have also examined some of the in vitro characteristics of the plasma membrane phospholipase C activity and have found it to be calcium sensitive, reaching maximal activity at 10 micromolar free [Ca(2+)]. We also report here that 100 micromolar GTPgammaS stimulates phosphospholipase C activity over a range of free [Ca(2+)]. Together, these results provide evidence that the plasma membrane PIP(2)-phospholipase C of D. salina may be subject to Ca(2+) and G-protein regulation.

Rapid changes in polyphosphoinositide metabolism associated with the response of Dunaliella salina to hypoosmotic shock.

The inositol phospholipids phosphatidylinositol, phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol 4,5-bisphosphate (PIP2) comprise 14.8, 1.2, and 0.3 mol %, respectively, of Dunaliella salina phospholipids. In isolated plasma membrane fractions, PIP and PIP2 are highly concentrated, together comprising 9.5 mol % of plasmalemma phospholipids. The metabolism of these inositol phospholipids and phosphatidic acid (PA) is very rapid under normal growth conditions. Within 5 min after introduction of 32Pi into the growth medium, over 75% of lipid-bound label was found in these quantitatively minor phospholipids. Within 2 min after a sudden hypoosmotic shock, the levels of PIP2 and PIP dropped to 65 and 79%, respectively, of controls. Within the same time frame, PA rose to 141% of control values. These data suggest that a rapid breakdown of the polyphosphoinositides may mediate the profound morphological and physiological changes which allow this organism to survive drastic hypoosmotic stress. In contrast to hypoosmotic shock, hyperosmotic shock induced a rise in PIP2 levels to 131% of control values, whereas the level of PA dropped to 56% of controls after 4 min. These two different types of osmotic stress affect inositol phospholipid metabolism in a fundamentally opposite manner, with only hypoosmotic shock inducing a net decrease in polyphosphoinositides.

Measurement of subcellular sites of polyphosphoinositide metabolism in isolated rat hepatocytes

Publisher Summary The products of phosphatidylinositol bisphosphate (PIP 2 ) degradation––diacylglycerol and D-myo-inositol 1, 4, 5-trisphosphate (IP 3 )––have both been identified as intracellular second messengers. Numerous cell types respond to a variety of stimuli with the enhanced hydrolysis of PIP 2 and to a lesser extent of phosphatidylinositol 4-phosphate (PIP). The plasma membrane PIP 2 phosphodiesterase does not appear to hydrolyze phosphatidylinositol (PI) in liver. One approach to the investigation of the subcellular location of polyphosphoinositide metabolic reactions is to fractionate cell homogenates by the best procedures available and to assay the respective enzymes with added substrates under V max conditions. A second approach for determining the subcellular sites of the metabolism of PIP and PIP 2 is to analyze the distribution of PIP and PIP 2 , the products of PI and PIP kinase, in a cell; however, their low levels make analysis by existing chemical means virtually impossible. The chapter explains the preparation of phosphatidylinositol [4- 32 P] phosphate and phosphatidylinositol [4,5- 32 p]bisphosphate.