Arf1 directly recruits the Pik1-Frq1 PI4K complex to regulate the final stages of Golgi maturation.

Proper Golgi complex function depends on the activity of Arf1, a GTPase whose effectors assemble and transport outgoing vesicles. Phosphatidylinositol 4-phosphate (PI4P) generated at the Golgi by the conserved PI 4-kinase Pik1 (PI4KIIIß) is also essential for Golgi function, although its precise roles in vesicle formation are less clear. Arf1 has been reported to regulate PI4P production, but whether Pik1 is a direct Arf1 effector is not established. Using a combination of live-cell time-lapse imaging analyses, acute PI4P depletion experiments, and in vitro protein-protein interaction assays on Golgi-mimetic membranes, we present evidence for a model in which Arf1 initiates the final stages of Golgi maturation by tightly controlling PI4P production through direct recruitment of the Pik1-Frq1 PI4-kinase complex. This PI4P serves as a critical signal for AP-1 and secretory vesicle formation, the final events at maturing Golgi compartments. This work therefore establishes the regulatory and temporal context surrounding Golgi PI4P production and its precise roles in Golgi maturation.

Functions of Nuclear Polyphosphoinositides.

Despite interest in phosphoinositide (PtdIns) kinases, such as PtdIns 3 kinases (PI3K), as targets for controlling plasma membrane PtdIns levels in disease, the PtdIns have another less well-known site of action in the cell nucleus.Recent studies show that PtdIns use a variety of strategies to alter DNA responses. Here, we provide an overview of these newly identified forms of gene expression control, which should be considered when studying the therapeutic use of PtdIns-directed compounds. As PI3K is one of the most important clinical targets in recent years, we will focus on two polyphosphoinositides, the PI3K substrate PtdIns(4,5)di-phosphate (PI4,5P2) and its product PtdIns(3,4,5)tri-phosphate (PI3,4,5P3).

Two isotypes of phosphatidylinositol 3-phosphate-binding sorting nexins play distinct roles in trogocytosis in Entamoeba histolytica.

Phosphatidylinositol phosphates (PIPs) function as important second messengers in many cellular events. In the human intestinal protist Entamoeba histolytica, where phagocytosis/trogocytosis plays an indispensable role in proliferation and pathophysiology during infection, various PIPs are involved in multiple steps of phago/trogocytosis. PI3-phosphate (PI3P) plays a pivotal role in the biogenesis of phagosome/trogosomes via recruitment of PI3P effectors. Because no known PI3P downstream effectors are conserved in E. histolytica, we exploited a unique method to identify the proteins PI3P dependently recruited to phagosomes. We rationalised that overexpression of PI3P-binding GFP-HrsFYVE competes for PI3P on phagosomal membranes and results in dissociation of PI3P effectors from phagosomes. EhVps26 and EhVps35, but not sorting nexins (SNXs), of the retromer complex were detected from phagosomes only without GFP-HrsFYVE overexpression. Two potential SNXs, EhSNX1 and EhSNX2, identified in the genome, possess only phox homology domain and specifically bound to PI3P, but retromer components, EhVps26 and EhVps35, did not bind to PI3P. Live and immunofluorescence imaging showed that EhSNX1 was recruited to the trogocytic cup and tunnel-like structures, and subsequently, EhSNX2 was recruited to trogosomes. Furthermore, EhSNX1, but not EhSNX2, specifically bound to Arp2/3 and EhVps26, which were localised to the tunnel-like structures and the trogosomes, respectively. EhSNX2 gene silencing increased trogocytosis, suggesting that EhSNX2 plays an inhibitory role in trogocytosis.

WDFY2 restrains matrix metalloproteinase secretion and cell invasion by controlling VAMP3-dependent recycling.

Cancer cells secrete matrix metalloproteinases to remodel the extracellular matrix, which enables them to overcome tissue barriers and form metastases. The membrane-bound matrix metalloproteinase MT1-MMP (MMP14) is internalized by endocytosis and recycled in endosomal compartments. It is largely unknown how endosomal sorting and recycling of MT1-MMP are controlled. Here, we show that the endosomal protein WDFY2 controls the recycling of MT1-MMP. WDFY2 localizes to endosomal tubules by binding to membranes enriched in phosphatidylinositol 3-phosphate (PtdIns3P). We identify the v-SNARE VAMP3 as an interaction partner of WDFY2. WDFY2 knockout causes a strong redistribution of VAMP3 into small vesicles near the plasma membrane. This is accompanied by increased, VAMP3-dependent secretion of MT1-MMP, enhanced degradation of extracellular matrix, and increased cell invasion. WDFY2 is frequently lost in metastatic cancers, most predominantly in ovarian and prostate cancer. We propose that WDFY2 acts as a tumor suppressor by serving as a gatekeeper for VAMP3 recycling.

PtdIns(3,5)P2 mediates root hair shank hardening in Arabidopsis.

Root hairs elongate by tip growth and simultaneously harden the shank by constructing the inner secondary cell wall layer. While much is known about the process of tip growth1, almost nothing is known about the mechanism by which root hairs harden the shank. Here we show that phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2), the enzymatic product of FORMATION OF APLOID AND BINUCLEATE CELLS 1 (FAB1), is involved in the hardening of the shank in root hairs in Arabidopsis. FAB1 and PtdIns(3,5)P2 localize to the plasma membrane along the shank of growing root hairs. By contrast, phosphatidylinositol 4-phosphate 5-kinase 3 (PIP5K3) and PtdIns(4,5)P2 localize to the apex of the root hair where they are required for tip growth. Reduction of FAB1 function results in the formation of wavy root hairs while those of the wild type are straight. The localization of FAB1 in the plasma membrane of the root hair shank requires the activity of Rho-related GTPases from plants 10 (ROP10) and localization of ROP10 requires FAB1 activity. Computational modelling of root hair morphogenesis successfully reproduces the wavy root hair phenotype. Taken together, these data demonstrate that root hair shank hardening requires PtdIns(3,5)P2/ROP10 signalling.

Quantitative Lipid Imaging Reveals a New Signaling Function of Phosphatidylinositol-3,4-Bisphophate: Isoform- and Site-Specific Activation of Akt.

Activation of class I phosphatidylinositol 3-kinase (PI3K) leads to formation of phosphatidylinositol-3,4,5-trisphophate (PIP3) and phosphatidylinositol-3,4-bisphophate (PI34P2), which spatiotemporally coordinate and regulate a myriad of cellular processes. By simultaneous quantitative imaging of PIP3 and PI34P2 in live cells, we here show that they have a distinctively different spatiotemporal distribution and history in response to growth factor stimulation, which allows them to selectively induce the membrane recruitment and activation of Akt isoforms. PI34P2 selectively activates Akt2 at both the plasma membrane and early endosomes, whereas PIP3 selectively stimulates Akt1 and Akt3 exclusively at the plasma membrane. These spatiotemporally distinct activation patterns of Akt isoforms provide a mechanism for their differential regulation of downstream signaling molecules. Collectively, our studies show that different spatiotemporal dynamics of PIP3 and PI34P2 and their ability to selectively activate key signaling proteins allow them to mediate class I PI3K signaling pathways in a spatiotemporally specific manner.

Atg9 establishes Atg2-dependent contact sites between the endoplasmic reticulum and phagophores.

The autophagy-related (Atg) proteins play a key role in the formation of autophagosomes, the hallmark of autophagy. The function of the cluster composed by Atg2, Atg18, and transmembrane Atg9 is completely unknown despite their importance in autophagy. In this study, we provide insights into the molecular role of these proteins by identifying and characterizing Atg2 point mutants impaired in Atg9 binding. We show that Atg2 associates to autophagosomal membranes through lipid binding and independently from Atg9. Its interaction with Atg9, however, is key for Atg2 confinement to the growing phagophore extremities and subsequent association of Atg18. Assembly of the Atg9-Atg2-Atg18 complex is important to establish phagophore-endoplasmic reticulum (ER) contact sites. In turn, disruption of the Atg2-Atg9 interaction leads to an aberrant topological distribution of both Atg2 and ER contact sites on forming phagophores, which severely impairs autophagy. Altogether, our data shed light in the interrelationship between Atg9, Atg2, and Atg18 and highlight the possible functional relevance of the phagophore-ER contact sites in phagophore expansion.

Adhesion force and attachment lifetime of the KIF16B-PX domain interaction with lipid membranes.

KIF16B is a highly processive kinesin-3 family member that participates in the trafficking and tubulation of early endosomes along microtubules. KIF16B attaches to lipid cargoes via a PX motif at its C-terminus, which has nanomolar affinity for bilayers containing phosphatidylinositol-3-phosphate (PI[3]P). As the PX domain has been proposed to be a primary mechanical anchor for the KIF16B-cargo attachment, we measured the adhesion forces and detachment kinetics of the PX domain as it interacts with membranes containing 2% PI(3)P and 98% phosphatidylcholine. Using optical tweezers, we found that the adhesion strength of a single PX domain ranged between 19 and 54 pN at loading rates between 80 and 1500 pN/s. These forces are substantially larger than the interaction of the adhesion of a pleckstrin homology domain with phosphatidylinositol 4,5-bisphosphate. This increased adhesion is the result of the membrane insertion of hydrophobic residues adjacent to the PI(3)P binding site, in addition to electrostatic interactions with PI(3)P. Attachment lifetimes under load decrease monotonically with force, indicating slip-bond behavior. However, the lifetime of membrane attachment under load appears to be well matched to the duration of processive motility of the KIF16B motor, indicating the PX domain is a suitable mechanical anchor for intracellular transport.

An intramolecular interaction within the lipid kinase Fab1 regulates cellular phosphatidylinositol 3,5-bisphosphate lipid levels.

Phosphorylated phosphoinositide lipids (PPIs) are low-abundance signaling molecules that control signal transduction pathways and are necessary for cellular homeostasis. The PPI phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P2) is essential in multiple organ systems. PI(3,5)P2 is generated from PI3P by the conserved lipid kinase Fab1/PIKfyve. Defects in the dynamic regulation of PI(3,5)P2 are linked to human diseases. However, few mechanisms that regulate PI(3,5)P2 have been identified. Here we report an intramolecular interaction between the yeast Fab1 kinase region and an upstream conserved cysteine-rich (CCR) domain. We identify mutations in the kinase domain that lead to elevated levels of PI(3,5)P2 and impair the interaction between the kinase and CCR domain. We also identify mutations in the CCR domain that lead to elevated levels of PI(3,5)P2 Together these findings reveal a regulatory mechanism that involves the CCR domain of Fab1 and contributes to dynamic control of cellular PI(3,5)P2 synthesis.

Multiphasic dynamics of phosphatidylinositol 4-phosphate during phagocytosis.

We analyzed the distribution, fate, and functional role of phosphatidylinositol 4-phosphate (PtdIns4P) during phagosome formation and maturation. To this end, we used genetically encoded probes consisting of the PtdIns4P-binding domain of the bacterial effector SidM. PtdIns4P was found to undergo complex, multiphasic changes during phagocytosis. The phosphoinositide, which is present in the plasmalemma before engagement of the target particle, is transiently enriched in the phagosomal cup. Soon after the phagosome seals, PtdIns4P levels drop precipitously due to the hydrolytic activity of Sac2 and phospholipase C, becoming undetectable for ∼10 min. PtdIns4P disappearance coincides with the emergence of phagosomal PtdIns3P. Conversely, the disappearance of PtdIns3P that signals the transition from early to late phagosomes is accompanied by resurgence of PtdIns4P, which is associated with the recruitment of phosphatidylinositol 4-kinase 2A. The reacquisition of PtdIns4P can be prevented by silencing expression of the kinase and can be counteracted by recruitment of a 4-phosphatase with a heterodimerization system. Using these approaches, we found that the secondary accumulation of PtdIns4P is required for proper phagosomal acidification. Defective acidification may be caused by impaired recruitment of Rab7 effectors, including RILP, which were shown earlier to displace phagosomes toward perinuclear lysosomes. Our results show multimodal dynamics of PtdIns4P during phagocytosis and suggest that the phosphoinositide plays important roles during the maturation of the phagosome.

Two-pore channels (TPCs): Novel voltage-gated ion channels with pleiotropic functions.

Two-pore channels (TPC1-3) comprise a subfamily of the eukaryotic voltage-gated ion channels (VGICs) superfamily that are mainly expressed in acidic stores in plants and animals. TPCS are widespread across the animal kingdom, with primates, mice and rats lacking TPC3, and mainly act as Ca+ and Na+ channels, although it was also suggested that they could be permeable to other ions. Nowadays, TPCs have been related to the development of different diseases, including Parkinson´s disease, obesity or myocardial ischemia. Due to this, their study has raised the interest of the scientific community to try to understand their mechanism of action in order to be able to develop an efficient drug that could regulate TPCs activity. In this review, we will provide an updated view regarding TPCs structure, function and activation, as well as their role in different pathophysiological processes.

[Phosphoinositides: lipidic essential actors in the intracellular traffic]. / Les phosphoinositides, des lipides acteurs essentiels du trafic intracellulaire.

Phosphoinositides (PPIn) are lipids involved in the vesicular transport of proteins between the different intracellular compartments. They act by recruiting and/or activating effector proteins and are thus involved in crucial cellular functions including vesicle budding, fusion and dynamics of membranes and regulation of the cytoskeleton. Although they are present in low concentrations in membranes, their activity is essential for cell survival and needs to be tightly controlled. Therefore, phosphatases and kinases specific of the various cellular membranes can phosphorylate/dephosphorylate their inositol ring on the positions D3, D4 and/or D5. The differential phosphorylation determines the intracellular localisation and the activity of the PPIn. Indeed, non-phosphorylated phosphatidylinositol (PtdIns) is the basic component of the PPIn and can be found in all eukaryotic cells at the cytoplasmic face of the ER, the Golgi, mitochondria and microsomes. It can get phosphorylated on position D4 to obtain PtdIns4P, a PPIn enriched in the Golgi compartment and involved in the maintenance of this organelle as well as anterograde and retrograde transport to and from the Golgi. PtdIns phosphorylation on position D3 results in PtdIns3P that is required for endosomal transport and multivesicular body (MVB) formation and sorting. These monophosphorylated PtdIns can be further phosphorylated to produce bisphophorylated PtdIns. Thus, PtdIns(4,5)P2, mainly produced by PtdIns4P phosphorylation, is enriched in the plasma membrane and involved in the regulation of actin cytoskeleton and endocytosis. PtdIns(3,5)P2, mainly produced by PtdIns3P phosphorylation, is enriched in late endosomes, MVBs and the lysosome/vacuole and plays a role in endosome to vacuole transport. PtdIns(3,4)P2 is absent in yeast, cells and mainly produced by PtdIns4P phosphorylation in human cells; PtdIns(3,4)P2 is localised in the plasma membrane and plays an important role as a second messenger by recruiting specific protein kinases (Akt and PDK1). Finally the triple phosphorylated PPIn, PtdIns(3,4,5)P3 also absent in yeast, is produced by the phosphorylation of PtdIns(3,4)P2 and localized at the plasma membrane of human cells where it binds proteins via their PH domain. Interaction partners include members of the Arf (ADP-ribosylation factors) family, PDK1 (Phosphoinositide Dependent Kinase 1) and Akt. Therefore this last PPIn is essential for the control of cell proliferation and its deregulation leads to the development of numerous cancers. In conclusion, the regulation of PPIn phosphorylation/dephosphorylation is complex and needs to be very precisely regulated. Indeed phosphatases and kinases allow the maintenance of the equilibrium between the different forms. PPIn play a crucial role in numerous cellular functions and a loss in their synthesis or regulation results in severe genetic diseases.

PI(5)P regulates autophagosome biogenesis.

Phosphatidylinositol 3-phosphate (PI(3)P), the product of class III PI3K VPS34, recruits specific autophagic effectors, like WIPI2, during the initial steps of autophagosome biogenesis and thereby regulates canonical autophagy. However, mammalian cells can produce autophagosomes through enigmatic noncanonical VPS34-independent pathways. Here we show that PI(5)P can regulate autophagy via PI(3)P effectors and thereby identify a mechanistic explanation for forms of noncanonical autophagy. PI(5)P synthesis by the phosphatidylinositol 5-kinase PIKfyve was required for autophagosome biogenesis, and it increased levels of PI(5)P, stimulated autophagy, and reduced the levels of autophagic substrates. Inactivation of VPS34 impaired recruitment of WIPI2 and DFCP1 to autophagic precursors, reduced ATG5-ATG12 conjugation, and compromised autophagosome formation. However, these phenotypes were rescued by PI(5)P in VPS34-inactivated cells. These findings provide a mechanistic framework for alternative VPS34-independent autophagy-initiating pathways, like glucose starvation, and unravel a cytoplasmic function for PI(5)P, which previously has been linked predominantly to nuclear roles.

Actin dynamics is rapidly regulated by the PTEN and PIP2 signaling pathways leading to myocyte hypertrophy.

Mature cardiac myocytes are terminally differentiated, and the heart has limited capacity to replace lost myocytes. Thus adaptation of myocyte size plays an important role in the determination of cardiac function. The hypothesis tested is that regulation of the dynamic exchange of actin leads to cardiac hypertrophy. ANG II was used as a hypertrophic stimulant in mouse heart and neonatal rat ventricular myocytes (NRVMs) in culture for assessment of a mechanism for regulation of actin dynamics by phosphatidylinositol 4,5-bisphosphate (PIP2). Actin dynamics in NRVMs rapidly increased in a PIP2-dependent manner, measured by imaging and fluorescence recovery after photobleaching (FRAP). A significant increase in PIP2 levels was found by immunoblotting in both adult mouse heart tissue and cultured NRVMs. Inhibition of phosphatase and tensin homolog (PTEN) in NRVMs markedly blunted ANG II-induced increases in actin dynamics, the PIP2 level, and cell size. Furthermore, PTEN activity was dramatically upregulated in ANG II-treated NRVMs but downregulated when PTEN inhibitors were used. The time course of the rise in the PIP2 level was inversely related to the fall in the PIP3 level, which was significant by 30 min in ANG II-treated NRVMs. However, significant translocation of PTEN to the plasma membrane occurred by 10 min, suggesting a crucial initial step for PTEN for the cellular responses to ANG II. In conclusion, PTEN and PIP2 signaling may play an important role in myocyte hypertrophy by the regulation of actin filament dynamics, which is induced by ANG II stimulation.

Selective phosphorylation modulates the PIP2 sensitivity of the CaM-SK channel complex.

Phosphatidylinositol bisphosphate (PIP2) regulates the activities of many membrane proteins, including ion channels, through direct interactions. However, the affinity of PIP2 is so high for some channel proteins that its physiological role as a modulator has been questioned. Here we show that PIP2 is a key cofactor for activation of small conductance Ca2+-activated potassium channels (SKs) by Ca(2+)-bound calmodulin (CaM). Removal of the endogenous PIP2 inhibits SKs. The PIP2-binding site resides at the interface of CaM and the SK C terminus. We further demonstrate that the affinity of PIP2 for its target proteins can be regulated by cellular signaling. Phosphorylation of CaM T79, located adjacent to the PIP2-binding site, by casein kinase 2 reduces the affinity of PIP2 for the CaM-SK channel complex by altering the dynamic interactions among amino acid residues surrounding the PIP2-binding site. This effect of CaM phosphorylation promotes greater channel inhibition by G protein-mediated hydrolysis of PIP2.

A cascade of ER exit site assembly that is regulated by p125A and lipid signals.

The inner and outer layers of COPII mediate cargo sorting and vesicle biogenesis. Sec16A and p125A (officially known as SEC23IP) proteins interact with both layers to control coat activity, yet the steps directing functional assembly at ER exit sites (ERES) remain undefined. By using temperature blocks, we find that Sec16A is spatially segregated from p125A-COPII-coated ERES prior to ER exit at a step that required p125A. p125A used lipid signals to control ERES assembly. Within p125A, we defined a C-terminal DDHD domain found in phospholipases and PI transfer proteins that recognized PA and phosphatidylinositol phosphates in vitro and was targeted to PI4P-rich membranes in cells. A conserved central SAM domain promoted self-assembly and selective lipid recognition by the DDHD domain. A basic cluster and a hydrophobic interface in the DDHD and SAM domains, respectively, were required for p125A-mediated functional ERES assembly. Lipid recognition by the SAM-DDHD module was used to stabilize membrane association and regulate the spatial segregation of COPII from Sec16A, nucleating the coat at ERES for ER exit.

PIP3 induces the recycling of receptor tyrosine kinases.

Down-regulation of receptor tyrosine kinases such as the epidermal growth factor receptor (EGFR) is achieved by endocytosis of the receptor followed by degradation or recycling. We demonstrated that in the absence of ligand, increased phosphatidylinositol 3,4,5-trisphosphate (PIP3) concentrations induced clathrin- and dynamin-mediated endocytosis of EGFR but not that of transferrin or G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. Endocytosis of the receptor in response to binding of EGF resulted in a decrease in the abundance of the EGFR, but PIP3-induced internalization decreased receptor ubiquitination and phosphorylation and resulted in recycling of the receptor to the plasma membrane. An RNA interference (RNAi) screen directed against lipid-binding domain-containing proteins identified polarity complex proteins, including PARD3 (partitioning defective 3), as essential for PIP3-induced receptor tyrosine kinase recycling. Thus, PIP3 and polarity complex proteins regulate receptor tyrosine kinase trafficking, which may enhance cellular responsiveness to growth factors.

Analysis, regulation, and roles of endosomal phosphoinositides.

Phosphoinositides (PIs) are minor lipid components of cellular membranes that play critical roles in membrane dynamics, trafficking, and cellular signaling. Among the seven naturally occurring PIs, the monophosphate phosphatidylinositol 3-phosphate (PtdIns3P) and the bisphosphate phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P2] have been mainly associated with endosomes and endosomal functions. Metabolic labeling and HPLC analysis revealed that a bulk of PtdIns3P is constitutively present in cells, making it the only detectable product of the enzymes phosphoinositide 3-kinases in unstimulated, normal cells. The use of specific tagged-PtdIns3P-binding domains later demonstrated that this constitutive PtdIns3P accumulates in endosomes where it critically regulates trafficking and membrane dynamics.

PIP3 regulates spinule formation in dendritic spines during structural long-term potentiation.

Dendritic spines are small, highly motile structures on dendritic shafts that provide flexibility to neuronal networks. Spinules are small protrusions that project from spines. The number and the length of spinules increase in response to activity including theta burst stimulation and glutamate application. However, what function spinules exert and how their formation is regulated still remains unclear. Phosphatidylinositol-3,4,5-trisphosphate (PIP3) plays important roles in cell motility such as filopodia and lamellipodia by recruiting downstream proteins such as Akt and WAVE to the membrane, respectively. Here we reveal that PIP3 regulates spinule formation during structural long-term potentiation (sLTP) of single spines in CA1 pyramidal neurons of hippocampal slices from rats. Since the local distribution of PIP3 is important to exert its functions, the subcellular distribution of PIP3 was investigated using a fluorescence lifetime-based PIP3 probe. PIP3 accumulates to a greater extent in spines than in dendritic shafts, which is regulated by the subcellular activity pattern of proteins that produce and degrade PIP3. Subspine imaging revealed that when sLTP was induced in a single spine, PIP3 accumulates in the spinule whereas PIP3 concentration in the spine decreased.

Role of phosphatidylinositol 3,4,5-trisphosphate in cell signaling.

Many lipids present in cellular membranes are phosphorylated as part of signaling cascades and participate in the recruitment, localization, and activation of downstream protein effectors. Phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) is one of the most important second messengers and is capable of interacting with a variety of proteins through specific PtdIns(3,4,5)P3 binding domains. Localization and activation of these effector proteins controls a myriad of cellular functions including cell survival, proliferation, cytoskeletal rearrangement, and gene expression. Aberrations in the production and metabolism of PtdIns(3,4,5)P3 have been implicated in many human diseases including cancer, diabetes, inflammation, and heart disease. This chapter provides an overview of the role of PtdIns(3,4,5)P3 in cellular regulation and the implications of PtdIns(3,4,5)P3 dysregulation in human diseases. Additionally, recent attempts at targeting PtdIns(3,4,5)P3 signaling via small molecule inhibitors are summarized.