Type B Phosphatidylinositol-4-Phosphate 5-Kinases Mediate Arabidopsis and Nicotiana tabacum Pollen Tube Growth by Regulating Apical Pectin Secretion

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P₂ production at that location are uncharacterized, functions of PtdIns(4,5)P₂ in pollen tube tip growth are unresolved. Two candidate genes encoding pollen-expressed Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinases (PI4P 5-kinases) of Arabidopsis subfamily B were identified (PIP5K4 and PIP5K5), and their recombinant proteins were characterized as being PI4P 5-kinases. Pollen of T-DNA insertion lines deficient in both PIP5K4 and PIP5K5 exhibited reduced pollen germination and defects in pollen tube elongation. Fluorescence-tagged PIP5K4 and PIP5K5 localized to an apical plasma membrane microdomain in Arabidopsis and tobacco (Nicotiana tabacum) pollen tubes, and overexpression of either PIP5K4 or PIP5K5 triggered multiple tip branching events. Further studies using the tobacco system revealed that overexpression caused massive apical pectin deposition accompanied by plasma membrane invaginations. By contrast, callose deposition and cytoskeletal structures were unaltered in the overexpressors. Morphological effects depended on PtdIns(4,5)P₂ production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P₂ production by type B PI4P 5-kinases disturbs the balance of membrane trafficking and apical pectin deposition. Polar tip growth of pollen tubes may thus be modulated by PtdIns(4,5)P₂ via regulatory effects on membrane trafficking and/or apical pectin deposition.     

Courier service for phosphatidylinositol: PITPs deliver on demand

Phosphatidylinositol is the parent lipid for the synthesis of seven phosphorylated inositol lipids and each of them play specific roles in numerous processes including receptor-mediated signalling, actin cytoskeleton dynamics and membrane trafficking. PI synthesis is localised to the endoplasmic reticulum (ER) whilst its phosphorylated derivatives are found in other organelles where the lipid kinases also reside. Phosphorylation of PI to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P₂) at the plasma membrane and to phosphatidylinositol 4-phosphate (PI4P) at the Golgi are key events in lipid signalling and Golgi function respectively. Here we review a family of proteins, phosphatidylinositol transfer proteins (PITPs), that can mobilise PI from the ER to provide the substrate to the resident kinases for phosphorylation. Recent studies identify specific and overlapping functions for the three soluble PITPs (PITPα, PITPβ and PITPNC1) in phospholipase C signalling, neuronal function, membrane trafficking, viral replication and in cancer metastases.     

Maintenance of hormone-sensitive phosphoinositide pools in the plasma membrane requires phosphatidylinositol 4-kinase IIIalpha

Type III phosphatidylinositol (PtdIns) 4-kinases (PI4Ks) have been previously shown to support plasma membrane phosphoinositide synthesis during phospholipase C activation and Ca²⁺ signaling. Here, we use biochemical and imaging tools to monitor phosphoinositide changes in the plasma membrane in combination with pharmacological and genetic approaches to determine which of the type III PI4Ks (α or β) is responsible for supplying phosphoinositides during agonist-induced Ca²⁺ signaling. Using inhibitors that discriminate between the α- and β-isoforms of type III PI4Ks, PI4KIIIα was found indispensable for the production of phosphatidylinositol 4-phosphate (PtdIns4P), phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂], and Ca²⁺ signaling in angiotensin II (AngII)-stimulated cells. Down-regulation of either the type II or type III PI4K enzymes by small interfering RNA (siRNA) had small but significant effects on basal PtdIns4P and PtdIns(4,5)P₂ levels in ³²P-labeled cells, but only PI4KIIIα down-regulation caused a slight impairment of PtdIns4P and PtdIns(4,5)P₂ resynthesis in AngII-stimulated cells. None of the PI4K siRNA treatments had a measurable effect on AngII-induced Ca²⁺ signaling. These results indicate that a small fraction of the cellular PI4K activity is sufficient to maintain plasma membrane phosphoinositide pools, and they demonstrate the value of the pharmacological approach in revealing the pivotal role of PI4KIIIα enzyme in maintaining plasma membrane phosphoinositides.     

A homogeneous and nonisotopic assay for phosphatidylinositol 4-kinases

Phosphatidylinositol 4-kinases (PI 4-kinases) catalyze the conversion of phosphatidylinositol to phosphatidylinositol 4-phosphate (PtdIns4P). The four known mammalian PI 4-kinases, PI4KA, PI4KB, PI4K2A, and PI4K2B have roles in intracellular lipid and protein trafficking. PI4KA and PI4KB also assist in the replication of several positive-sense RNA viruses. The identification of selective inhibitors of these kinases would be facilitated by assays suitable for high-throughput screening. We describe a homogeneous and nonisotopic assay for PI 4-kinase activity based on the bioluminescent detection of the ADP produced by kinase reactions. We have evaluated this assay with known nonselective inhibitors of PI 4-kinases and show that it performs similar to radiometric assay formats previously described in the literature. In addition, this assay generates Z-factor values of >0.7 for PI4KA in a 384-well format, demonstrating its suitability for high-throughput screening applications.     

Overexpression of murine phosphatidylinositol 4-phosphate 5-kinase type Ibeta disrupts a phosphatidylinositol 4,5 bisphosphate regulated endosomal pathway

The type I phosphatidylinositol 4-phosphate 5-kinases (PI4P5K) phosphorylate phosphatidylinositol 4-phosphate [PI(4)P] to produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 has been implicated in signal transduction, receptor mediated endocytosis, vesicle trafficking, cytoskeletal structure, and membrane ruffling. However, the specific type I enzymes associated with the production of PI(4,5)P2 for the specific cellular processes have not been rigorously defined. Murine PI4P5K type Ibeta (mPIP5K-Ibeta) was implicated in receptor mediated endocytosis through the isolation of a truncated and inactive form of the enzyme that blocked the ligand-dependent downregulation of the colony-stimulating factor-1 receptor. The present study shows that enforced expression of mPIP5K-Ibeta in 293T cells resulted in the accumulation of large vesicles that were linked to an endosomal pathway. Similar results were obtained after the expression of the PI(4,5)P2-binding pleckstrin homology (PH) domain of phospholipase-Cdelta (PLC-delta). Analysis of the conserved domains of mPIP5K-Ibeta led to the identification of dimerization domains in the N- and C-terminal regions. Enforced expression of the individual dimerization domains interfered with the proper subcellular localization of mPIP5K-Ibeta and the PLC-delta-PH domain and blocked the accumulation of the endocytic vesicles induced by these proteins. In addition to regulating early steps in endocytosis, these results suggest that mPIP5K-Ibeta acts through PI(4,5)P2 to regulate endosomal trafficking and/or fusion.     

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P₂ and other acidic phospholipids in a Ca²⁺-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P₂, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P₂ clustering are also capable of forming interconnections between PI(4,5)P₂-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P₂ and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P₂-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.     

Plasma membrane nano-organization specifies phosphoinositide effects on Rho-GTPases and actin dynamics in tobacco pollen tubes

Pollen tube growth requires coordination of cytoskeletal dynamics and apical secretion. The regulatory phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) is enriched in the subapical plasma membrane of pollen tubes of Arabidopsis thaliana and tobacco (Nicotiana tabacum) and can influence both actin dynamics and secretion. How alternative PtdIns(4,5)P₂ effects are specified is unclear. In tobacco pollen tubes, spinning disc microscopy (SD) reveals dual distribution of a fluorescent PtdIns(4,5)P₂-reporter in dynamic plasma membrane nanodomains vs. apparent diffuse membrane labeling, consistent with spatially distinct coexisting pools of PtdIns(4,5)P₂. Several PI4P 5-kinases (PIP5Ks) can generate PtdIns(4,5)P₂ in pollen tubes. Despite localizing to one membrane region, the PIP5Ks AtPIP5K2-EYFP and NtPIP5K6-EYFP display distinctive overexpression effects on cell morphologies, respectively related to altered actin dynamics or membrane trafficking. When analyzed by SD, AtPIP5K2-EYFP associated with nanodomains, whereas NtPIP5K6-EYFP localized diffusely. Chimeric AtPIP5K2-EYFP and NtPIP5K6-EYFP variants with reciprocally swapped membrane-associating domains evoked reciprocally shifted effects on cell morphology upon overexpression. Overall, active PI4P 5-kinase variants stabilized actin when targeted to nanodomains, suggesting a role of nanodomain-associated PtdIns(4,5)P₂ in actin regulation. This notion is further supported by interaction and proximity of nanodomain-associated AtPIP5K2 with the Rho-GTPase NtRac5, and by its functional interplay with elements of Rho of plants signaling. Plasma membrane nano-organization may thus aid the specification of PtdIns(4,5)P₂ functions to coordinate cytoskeletal dynamics and secretion.

The apical plasma membrane of pollen tubes contains nanodomains where the regulatory phospholipid PtdIns(4,5)P₂ exerts a stabilizing effect on the actin cytoskeleton.     

PtdIns4P synthesis by PI4KIIIα at the plasma membrane and its impact on plasma membrane identity

Plasma membrane phosphatidylinositol (PI) 4-phosphate (PtdIns4P) has critical functions via both direct interactions and metabolic conversion to PI 4,5-bisphosphate (PtdIns(4,5)P₂) and other downstream metabolites. However, mechanisms that control this PtdIns4P pool in cells of higher eukaryotes remain elusive. PI4KIIIα, the enzyme thought to synthesize this PtdIns4P pool, is reported to localize in the ER, contrary to the plasma membrane localization of its yeast homologue, Stt4. In this paper, we show that PI4KIIIα was targeted to the plasma membrane as part of an evolutionarily conserved complex containing Efr3/rolling blackout, which we found was a palmitoylated peripheral membrane protein. PI4KIIIα knockout cells exhibited a profound reduction of plasma membrane PtdIns4P but surprisingly only a modest reduction of PtdIns(4,5)P₂ because of robust up-regulation of PtdIns4P 5-kinases. In these cells, however, much of the PtdIns(4,5)P₂ was localized intracellularly, rather than at the plasma membrane as in control cells, along with proteins typically restricted to this membrane, revealing a major contribution of PI4KIIIα to the definition of plasma membrane identity.     

The Essential Phosphoinositide Kinase MSS-4 Is Required for Polar Hyphal Morphogenesis,Localizing to Sites of Growth and Cell Fusion in Neurospora crassa

Fungal hyphae and plant pollen tubes are among the most highly polarized cells known and pose extraordinary requirements on their cell polarity machinery. Cellular morphogenesis is driven through the phospholipid-dependent organization at the apical plasma membrane. We characterized the contribution of phosphoinositides (PIs) in hyphal growth of the filamentous ascomycete Neurospora crassa. MSS-4 is an essential gene and its deletion resulted in spherically growing cells that ultimately lyse. Two conditional mss-4-mutants exhibited altered hyphal morphology and aberrant branching at restrictive conditions that were complemented by expression of wild type MSS-4. Recombinant MSS-4 was characterized as a phosphatidylinositolmonophosphate-kinase phosphorylating phosphatidylinositol 4-phosphate (PtdIns4P) to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). PtdIns3P was also used as a substrate. Sequencing of two conditional mss-4 alleles identified a single substitution of a highly conserved Y750 to N. The biochemical characterization of recombinant protein variants revealed Y750 as critical for PI4P 5-kinase activity of MSS-4 and of plant PI4P 5-kinases. The conditional growth defects of mss-4 mutants were caused by severely reduced activity of MSS-4(Y750N), enabling the formation of only trace amounts of PtdIns(4,5)P₂. In N. crassa hyphae, PtdIns(4,5)P₂ localized predominantly in the plasma membrane of hyphae and along septa. Fluorescence-tagged MSS-4 formed a subapical collar at hyphal tips, localized to constricting septa and accumulated at contact points of fusing N. crassa germlings, indicating MSS-4 is responsible for the formation of relevant pools of PtdIns(4,5)P₂ that control polar and directional growth and septation. N. crassa MSS-4 differs from yeast, plant and mammalian PI4P 5-kinases by containing additional protein domains. The N-terminal domain of N. crassa MSS-4 was required for correct membrane association. The data presented for N. crassa MSS-4 and its roles in hyphal growth are discussed with a comparative perspective on PI-control of polar tip growth in different organismic kingdoms.     

Differential effects of the phosphatidylinositol 4-kinases, PI4KII�� and PI4KIII��, on Akt activation and apoptosis

In this study, we investigated the role of PI4P synthesis by the phosphatidylinositol 4-kinases, PI4KIIα and PI4KIIIβ, in epidermal growth factor (EGF)-stimulated phosphoinositide signaling and cell survival. In COS-7 cells, knockdown of either isozyme by RNA interference reduced basal levels of PI4P and PI(4,5)P₂, without affecting receptor activation. Only knockdown of PI4KIIα inhibited EGF-stimulated Akt phosphorylation, indicating that decreased PI(4,5)P₂ synthesis observed by loss of either isoform could not account for this PI4KIIα-specific effect. Phospholipase Cγ activation was also differentially affected by knockdown of either PI4K isozyme. Overexpression of kinase-inactive PI4KIIα, which induces defective endosomal trafficking without reducing PI(4,5)P₂ levels, also reduced Akt activation. Furthermore, PI4KIIα knockdown profoundly inhibited cell proliferation and induced apoptosis as evidenced by the cleavage of caspase-3 and its substrate poly(ADP-ribose) polymerase. However, in MDA-MB-231 breast cancer cells, apoptosis was observed subsequent to knockdown of either PI4KIIα or PI4KIIIβ and this correlated with enhanced proapoptotic Akt phosphorylation. The differential effects of phosphatidylinositol 4-kinase knockdown in the two cell lines lead to the conclusion that phosphoinositide turnover is inhibited through PI4P substrate depletion, whereas impaired antiapoptotic Akt signaling is an indirect consequence of dysfunctional endosomal trafficking.     

GABARAP-mediated targeting of PI4K2A/PI4KIIα to autophagosomes regulates PtdIns4P-dependent autophagosome-lysosome fusion

For decades, phosphatidylinositol 4-phosphate (PtdIns4P) was considered primarily as a precursor in the synthesis of phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P₂). More recently, specific functions for PtdIns4P itself have been identified, particularly in the regulation of intracellular membrane trafficking. PI4K2A/PI4KIIα (phosphatidylinositol 4-kinase type 2 α), one of the 4 enzymes that catalyze PtdIns4P production in mammalian cells, promotes vesicle formation from the trans-Golgi network (TGN) and endosomes. We recently identified a novel function for PI4K2A-derived PtdIns4P, as a facilitator of autophagosome-lysosome (A-L) fusion. We further showed that that this function requires the presence of the autophagic adaptor protein GABARAP (GABA[A] receptor-associated protein), which binds to PI4K2A and recruits it to autophagosomes. The mechanism whereby GABARAP-PI4K2A-PtdIns4P promotes A-L fusion remains to be defined. Based on other examples of phosphoinositide involvement in membrane trafficking, we speculate that it acts by recruiting elements of the membrane docking and fusion machinery.     

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.     

α-Synuclein facilitates endocytosis by elevating the steady-state levels of phosphatidylinositol 4,5-bisphosphate

α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson''s disease (PD). It is an intrinsically disordered protein that binds acidic phospholipids. Growing evidence supports a role for α-Syn in membrane trafficking, including, mechanisms of endocytosis and exocytosis, although the exact role of α-Syn in these mechanisms is currently unclear. Here we investigate the associations of α-Syn with the acidic phosphoinositides (PIPs), phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P₂). Our results show that α-Syn colocalizes with PIP₂ and the phosphorylated active form of the clathrin adaptor protein 2 (AP2) at clathrin-coated pits. Using endocytosis of transferrin as an indicator for clathrin-mediated endocytosis (CME), we find that α-Syn involvement in endocytosis is specifically mediated through PI(4,5)P₂ levels on the plasma membrane. In accord with their effects on PI(4,5)P₂ levels, the PD associated A30P, E46K, and A53T mutations in α-Syn further enhance CME in neuronal and nonneuronal cells. However, lysine to glutamic acid substitutions at the KTKEGV repeat domain of α-Syn, which interfere with phospholipid binding, are ineffective in enhancing CME. We further show that the rate of synaptic vesicle (SV) endocytosis is differentially affected by the α-Syn mutations and associates with their effects on PI(4,5)P₂ levels, however, with the exception of the A30P mutation. This study provides evidence for a critical involvement of PIPs in α-Syn–mediated membrane trafficking.     

Regulation of type IIα phosphatidylinositol phosphate kinase localisation by the protein kinase CK2

Inositol lipid synthesis is regulated by several distinct families of enzymes [1]. Members of one of these families, the type II phosphatidylinositol phosphate kinases (PIP kinases), are 4-kinases and are thought to catalyse a minor route of synthesis of the multifunctional phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) from the inositide PI(5)P [2]. Here, we demonstrate the partial purification of a protein kinase that phosphorylates the type IIα PIP kinase at a single site unique to that isoform – Ser304. This kinase was identified as protein kinase CK2 (formerly casein kinase 2). Mutation of Ser304 to aspartate to mimic its phosphorylation had no effect on PIP kinase activity, but promoted both redistribution of the green fluorescent protein (GFP)-tagged enzyme in HeLa cells from the cytosol to the plasma membrane, and membrane ruffling. This effect was mimicked by mutation of Ser304 to alanine, although not to threonine, suggesting a mechanism involving the unmasking of a latent membrane localisation sequence in response to phosphorylation.     

Interaction of neuronal calcium sensor-1 (NCS-1) with phosphatidylinositol 4-kinase beta stimulates lipid kinase activity and affects membrane trafficking in COS-7 cells

Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated PI4Kbeta in PI4Kbeta-immunoprecipitates. When expressed in COS-7 cells, PI4Kbeta and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either proteins, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Expressed NCS-1-YFP showed co-localization with endogenous PI4Kbeta primarily in the Golgi, but it was also present in the walls of numerous large perinuclear vesicles. Co-expression of a catalytically inactive PI4Kbeta inhibited the development of this vesicular phenotype. Transfection of PI4Kbeta and NCS-1 had no effect on basal PIP synthesis in permeabilized COS-7 cells, but it increased the wortmannin-sensitive [(32)P]phosphate incorporation into phosphatidylinositol 4-phosphate during Ca(2+)-induced phospholipase C activation. These results together indicate that NCS-1 is able to interact with PI4Kbeta also in mammalian cells and may play a role in the regulation of this enzyme in specific cellular compartments affecting vesicular trafficking.     

Phosphatidylinositol 4,5-bisphosphate regulates adipocyte actin dynamics and GLUT4 vesicle recycling

To investigate the potential role of phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) in the regulation of actin polymerization and GLUT4 translocation, the type I phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) were expressed in 3T3L1 adipocytes. In preadipocytes (fibroblasts) PIP5K expression promoted actin polymerization on membrane-bound vesicles to form motile actin comets. In contrast, expression of PIP5K in differentiated 3T3L1 adipocytes resulted in the formation of enlarged vacuole-like structures coated with F-actin, cortactin, dynamin, and N-WASP. Treatment with either latrunculin B (an inhibitor for actin polymerization) or Clostridium difficile toxin B (a general Rho family inhibitor) resulted in a relatively slower disappearance of coated F-actin from these vacuoles, but the vacuoles themselves remained unaffected. Functionally, the increased PI(4,5)P2 levels resulted in an inhibition of transferrin receptor and GLUT4 endocytosis and a slow accumulation of these proteins in the PI(4,5)P2-enriched vacuoles along with the non-clathrin-derived endosome marker (caveolin) and the AP-2 adaptor complex. However, these structures were devoid of early endosome markers (EEA1, clathrin) and the biosynthetic membrane secretory machinery markers p115 (Golgi) and syntaxin 6 (trans-Golgi Network). Taken together, these data demonstrate that PI(4,5)P2 has distinct morphologic and functional properties depending upon specific cell context. In adipocytes, altered PI(4,5)P2 metabolism has marked effects on GLUT4 endocytosis and intracellular vesicle trafficking due to the derangement of actin dynamics.     

Complexin-1 and synaptotagmin-1 compete for binding sites on membranes containing PtdInsP2

Complexin-1 is an essential protein for neuronal exocytosis that acts to depress spontaneous fusion events while enhancing evoked neurotransmitter release. In addition to binding soluble N-ethylmaleimide-sensitive factor attachment protein receptors, it is well established that complexin associates with membranes in a manner that depends upon membrane curvature. In the present work, we examine the membrane binding of complexin using electron paramagnetic resonance spectroscopy, fluorescence anisotropy, and total internal reflection fluorescence microscopy. The apparent membrane affinity of complexin is found to strongly depend upon the concentration of protein used in the binding assay, and this is a result of a limited number of binding sites for complexin on the membrane interface. Although both the N- and C-terminal regions of complexin associate with the membrane interface, membrane affinity is driven by its C-terminus. Complexin prefers to bind liquid-disordered membrane phases and shows an enhanced affinity toward membranes containing phosphatidylinositol 4-5-bisphosphate (PI(4,5)P₂). In the presence of PI(4,5)P₂, complexin is displaced from the membrane surface by proteins that bind to or sequester PI(4,5)P₂. In particular, the neuronal calcium sensor synaptotagmin-1 displaces complexin from the membrane but only when PI(4,5)P₂ is present. Complexin and synaptotagmin compete on the membrane interface in the presence of PI(4,5)P₂, and this interaction may play a role in calcium-triggered exocytosis by displacing complexin from its fusion-inhibiting state.     

Differential Regulation of Proton-Sensitive Ion Channels by Phospholipids: A Comparative Study between ASICs and TRPV1

Protons are released in pain-generating pathological conditions such as inflammation, ischemic stroke, infection, and cancer. During normal synaptic activities, protons are thought to play a role in neurotransmission processes. Acid-sensing ion channels (ASICs) are typical proton sensors in the central nervous system (CNS) and the peripheral nervous system (PNS). In addition to ASICs, capsaicin- and heat-activated transient receptor potential vanilloid 1 (TRPV1) channels can also mediate proton-mediated pain signaling. In spite of their importance in perception of pH fluctuations, the regulatory mechanisms of these proton-sensitive ion channels still need to be further investigated. Here, we compared regulation of ASICs and TRPV1 by membrane phosphoinositides, which are general cofactors of many receptors and ion channels. We observed that ASICs do not require membrane phosphatidylinositol 4-phosphate (PI(4)P) or phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) for their function. However, TRPV1 currents were inhibited by simultaneous breakdown of PI(4)P and PI(4,5)P₂. By using a novel chimeric protein, CF-PTEN, that can specifically dephosphorylate at the D3 position of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P₃), we also observed that neither ASICs nor TRPV1 activities were altered by depletion of PI(3,4,5)P₃ in intact cells. Finally, we compared the effects of arachidonic acid (AA) on two proton-sensitive ion channels. We observed that AA potentiates the currents of both ASICs and TRPV1, but that they have different recovery aspects. In conclusion, ASICs and TRPV1 have different sensitivities toward membrane phospholipids, such as PI(4)P, PI(4,5)P₂, and AA, although they have common roles as proton sensors. Further investigation about the complementary roles and respective contributions of ASICs and TRPV1 in proton-mediated signaling is necessary.     

OCRL1 Modulates Cilia Length in Renal Epithelial Cells

Lowe syndrome is an X-linked disorder characterized by cataracts at birth, mental retardation and progressive renal malfunction that results from loss of function of the OCRL1 (oculocerebrorenal syndrome of Lowe) protein. OCRL1 is a lipid phosphatase that converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 4-phosphate. The renal pathogenesis of Lowe syndrome patients has been suggested to result from alterations in membrane trafficking, but this cannot fully explain the disease progression. We found that knockdown of OCRL1 in zebrafish caused developmental defects consistent with disruption of ciliary function, including body axis curvature, pericardial edema, hydrocephaly and impaired renal clearance. In addition, cilia in the proximal tubule of the zebrafish pronephric kidney were longer in ocrl morphant embryos. We also found that knockdown of OCRL1 in polarized renal epithelial cells caused elongation of the primary cilium and disrupted formation of cysts in three-dimensional cultures. Calcium release in response to ATP was blunted in OCRL1 knockdown cells, suggesting changes in signaling that could lead to altered cell function. Our results suggest a new role for OCRL1 in renal epithelial cell function that could contribute to the pathogenesis of Lowe syndrome.     

Phosphatidylinositol 3-Phosphate,an Essential Lipid in Plasmodium,Localizes to the Food Vacuole Membrane and the Apicoplast

Phosphoinositides are important regulators of diverse cellular functions, and phosphatidylinositol 3-monophosphate (PI3P) is a key element in vesicular trafficking processes. During its intraerythrocytic development, the malaria parasite Plasmodium falciparum establishes a sophisticated but poorly characterized protein and lipid trafficking system. Here we established the detailed phosphoinositide profile of P. falciparum-infected erythrocytes and found abundant amounts of PI3P, while phosphatidylinositol 3,5-bisphosphate was not detected. PI3P production was parasite dependent, sensitive to a phosphatidylinositol-3-kinase (PI3-kinase) inhibitor, and predominant in late parasite stages. The Plasmodium genome encodes a class III PI3-kinase of unusual size, containing large insertions and several repetitive sequence motifs. The gene could not be deleted in Plasmodium berghei, and in vitro growth of P. falciparum was sensitive to a PI3-kinase inhibitor, indicating that PI3-kinase is essential in Plasmodium blood stages. For intraparasitic PI3P localization, transgenic P. falciparum that expressed a PI3P-specific fluorescent probe was generated. Fluorescence was associated mainly with the membrane of the food vacuole and with the apicoplast, a four-membrane bounded plastid-like organelle derived from an ancestral secondary endosymbiosis event. Electron microscopy analysis confirmed these findings and revealed, in addition, the presence of PI3P-positive single-membrane vesicles. We hypothesize that these vesicles might be involved in transport processes, likely of proteins and lipids, toward the essential and peculiar parasite compartment, which is the apicoplast. The fact that PI3P metabolism and function in Plasmodium appear to be substantially different from those in its human host could offer new possibilities for antimalarial chemotherapy.Phosphatidylinositol is a crucial phospholipid in eukaryotic cells. It is a structural membrane lipid, and phosphorylation of the hydroxyl groups of its inositol head group by specific lipid kinases leads to the production of seven different phosphoinositide species, which have been found to be enriched in distinct cellular compartments. They play key roles in a multitude of cellular processes, such as membrane traffic, cell motility, cytoskeletal reorganization, DNA synthesis, the cell cycle, adhesion, and signal transduction (9). Approximately 1% of total lipids in mammalian cells are phosphoinositides, mainly phosphatidylinositol 4-monophosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] (45). Derivatives phosphorylated at the 3 position are considerably less abundant in mammalian cells. Phosphatidylinositol 3-monophosphate (PI3P) is a ubiquitous lipid in eukaryotic cells and is present in small amounts in mammalian cells (classically <15% of PI4P), while PI3P is as abundant as PI4P in the yeast Saccharomyces cerevisiae (2). It has been suggested that one of the functions of these lipids is to establish membrane identity (46); PI4P predominates at the Golgi apparatus (33), PI(4,5)P₂ at the plasma membrane (62), PI3P on early endosomes (20), and phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] on late endocytic organelles (48). Certain phosphoinositides have been shown to play important roles in constitutive membrane traffic. PI3P is involved in endocytosis and vesicular trafficking toward the lysosome in yeast and mammalian cells (23, 39). PI3P has also been shown to be implicated in the processes of retrograde trafficking from the endosome to the Golgi apparatus and in autophagy (7, 42). PI(3,5)P₂ is found in yeast, mammalian, and plant cells (11) and is essential for protein sorting in multivesicular bodies (38), which are an intermediate compartment for the degradation of cell surface receptors within lysosomes.PI3P is the product of PI3-kinase class III (16), also termed Vps34 (vacuolar protein sorting), and PI(3,5)P₂ is synthesized by the phosphorylation of PI3P by PIKfyve (phosphoinositide kinase, FYVE finger containing; Fab1 in yeast) (38). Both enzymes are conserved across eukaryotic evolution from yeast to mammals. Vps34-type enzymes are the only PI3-kinases in unicellular organisms. In contrast, metazoan cells possess three classes of PI3-kinases (I, II, and III), which differ from each other by their activation mode and their substrate specificity (56), leading to the synthesis of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], phosphatidylinositol 3,4-bisphosphate [PI(3,4)P₂], and PI3P, respectively. The synthesis of PI(3,4,5)P₃ and PI(3,4)P₂ is controlled by agonist stimulation of cells, resulting in strong fluctuations in their cellular levels. These phosphoinositides are generally not observed in unicellular organisms.Plasmodium falciparum is the causal agent of the most severe form of human malaria. During the symptomatic phase of the disease, the parasite resides within a vacuole in mature erythrocytes, cells that are devoid of protein and lipid biosynthesis and intracellular compartments. In addition to the classically observed organelles of eukaryotic cells, Plasmodium contains certain particular compartments. These include the apicoplast, a four-membrane bounded plastid-like organelle derived from an ancestral secondary endosymbiosis event (61), and specialized secretory organelles, rhoptries and micronemes, located at the apical pole and involved in host cell invasion. Plasmodium blood stages internalize host cell hemoglobin that is degraded in a specialized compartment, the food vacuole (17). Some characteristics of the food vacuole, such as low pH and the presence of proteolytic enzymes, could qualify it as a lysosome-like organelle (21). However, this very peculiar compartment is present only during Plasmodium blood stages, is absent from mosquito and liver stages, and is not found in other apicomplexan parasites. Targeting proteins and lipids to these numerous cellular compartments requires sophisticated vesicular trafficking. Certain classical actors in vesicular trafficking in eukaryotic cells, such as Rab GTPases and SNARE-like proteins, are present in Plasmodium (3, 44).Here we addressed the question of whether the second group of classical actors in vesicle transport, i.e., the phosphoinositides PI3P and PI(3,5)P₂, are synthesized by the parasite. For this purpose, we established, for the first time, a detailed phosphoinositide profile of P. falciparum-infected red blood cells and detected important amounts of PI3P, which we found located at the food vacuole membrane and the apicoplast. In contrast, PI(3,5)P₂ could not be detected, indicating possible differences in protein sorting between Plasmodium and other eukaryotes. PI3-kinase activity is essential for Plasmodium, suggesting that PI3P-dependent functions might be an interesting target for future drug development.