Reverse Fountain Flow of Phosphatidylinositol-3,4-Bisphosphate Polarizes Migrating Cells
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ABSTRACT: The ability of cells to polarize and move toward external stimuli plays a crucial role in development, as well as normal and pathological physiology. Migrating cells maintain dynamic complementary distributions of Ras activity and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). Here we show lagging edge component PI(3,4)P2 also localizes to retracting leading edge protrusions and nascent macropinosomes, even in the absence of phosphatidylinositol 3,4,5-trisphosphate (PIP3). Once internalized, the macropinosomes break up into smaller PI(3,4)P2-enriched vesicles, which fuse to the plasma membrane at the cell rear. The phosphoinositide then diffuses towards the front, where it is degraded. A computational model confirmed that this cycle brings about a stable back-to-front gradient. These results uncover a surprising "reverse fountain flow" of PI(3,4)P2 that regulates polarity.
Project description:The ability of cells to polarize and move toward external stimuli plays a crucial role in development, as well as in normal and pathological physiology. Migrating cells maintain dynamic complementary distributions of Ras activity and of the phospholipid phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). Here, we show that lagging-edge component PI(3,4)P2 also localizes to retracting leading-edge protrusions and nascent macropinosomes, even in the absence of phosphatidylinositol 3,4,5-trisphosphate (PIP3). Once internalized, macropinosomes break up into smaller PI(3,4)P2-enriched vesicles, which fuse with the plasma membrane at the rear of the cell. Subsequently, the phosphoinositide diffuses toward the front of the cell, where it is degraded. Computational modeling confirms that this cycle gives rise to stable back-to-front gradient. These results uncover a surprising "reverse-fountain flow" of PI(3,4)P2 that regulates polarity.
Project description:Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5' position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] upon voltage depolarization. However, it is unclear whether VSPs also have 3' phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5' phosphatase activity of VSP toward PI(3,4,5)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) is then dephosphorylated at the 3' position. These results suggest that substrate specificity of the VSP changes with membrane potential.
Project description:Focal adhesions are multifunctional organelles that couple cell-matrix adhesion to cytoskeletal force transmission and signaling and to steer cell migration and collective cell behavior. Whereas proteomic changes at focal adhesions are well understood, little is known about signaling lipids in focal adhesion dynamics. Through the characterization of cells from mice with a kinase-inactivating point mutation in the class II PI3K-C2β, we find that generation of the phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) membrane lipid promotes focal adhesion disassembly in response to changing environmental conditions. We show that reduced growth factor signaling sensed by protein kinase N, an mTORC2 target and effector of RhoA, synergizes with the adhesion disassembly factor DEPDC1B to induce local synthesis of PtdIns(3,4)P2 by PI3K-C2β. PtdIns(3,4)P2 then promotes turnover of RhoA-dependent stress fibers by recruiting the PtdIns(3,4)P2-dependent RhoA-GTPase-activating protein ARAP3. Our findings uncover a pathway by which cessation of growth factor signaling facilitates cell-matrix adhesion disassembly via a phosphoinositide lipid switch.
Project description:Neurite initiation is critical for neuronal morphogenesis and early neural circuit development. Recent studies showed that local actin aggregation underneath the cell membrane determined the site of neurite initiation. An immediately arising question is what signaling mechanism initiated actin aggregation. Here we demonstrate that local clustering of phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), a phospholipid with relatively few known signaling functions, is necessary and sufficient for aggregating actin and promoting neuritogenesis. In contrast, the related and more extensively studied phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol (3,4,5)-trisphosphate (PIP3) molecules did not have such functions. Specifically, we showed that beads coated with PI(3,4)P2 promoted actin aggregation and neurite initiation, while pharmacological interference with PI(3,4)P2 synthesis inhibited both processes. PI(3,4)P2 clustering occurred even when actin aggregation was pharmacologically blocked, demonstrating that PI(3,4)P2 functioned as the upstream signaling molecule. Two enzymes critical for PI(3,4)P2 generation, namely, SH2 domain-containing inositol 5-phosphatase and class II phosphoinositide 3-kinase α, were complementarily and non-redundantly required for actin aggregation and neuritogenesis, as well as for subsequent dendritogenesis. Finally, we demonstrate that neural Wiskott-Aldrich syndrome protein and the Arp2/3 complex functioned downstream of PI(3,4)P2 to mediate neuritogenesis and dendritogenesis. Together, our results identify PI(3,4)P2 as an important signaling molecule during early development and demonstrate its critical role in regulating actin aggregation and neuritogenesis.
Project description:Phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2] is a phosphoinositide that plays important roles in signal transduction, endocytosis, and cell migration among others. The intracellular distribution of PtdIns(3,4)P2 has mainly been studied by observing the distribution of GFP-tagged PtdIns(3,4)P2-binding protein domains in live cells and by labeling with anti-PtdIns(3,4)P2 antibody in fixed cell samples, but these methods only offer low spatial resolution results and may have pitfalls. In the present study, we developed an electron microscopic method to observe the PtdIns(3,4)P2 distribution using the SDS-treated freeze-fracture replica labeling method. The recombinant GST-tagged pleckstrin homology (PH) domain of TAPP1 was used as the binding probe, and its binding to PtdIns(3,4)P2 in the freeze-fracture replica was confirmed by using liposomes containing different phosphoinositides and by the lack of labeling by a mutant probe, in which one amino acid in the PH domain was substituted. The method was applied to NIH3T3 cell samples and showed that the increase of PtdIns(3,4)P2 in cells treated with hydrogen peroxide occurs in the cytoplasmic leaflet of the plasma membrane, except in the caveolar membrane. The present method can define the distribution of PtdIns(3,4)P2 at a high spatial resolution and will facilitate our understanding of the physiological function of this less studied phosphoinositide.
Project description:Phosphoinositides play crucial roles in intracellular membrane dynamics and cell signaling, with phosphatidylinositol (PI) 3-phosphates being the predominant phosphoinositide lipids at endosomes and lysosomes, whereas PI 4-phosphates, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), are enriched at the cell surface including sites of endocytosis. How PI 4-phosphates and PI 3-phosphates are dynamically interconverted within the endocytic pathway and how this is controlled in space and time remains poorly understood. Here, combining live imaging, genome engineering, and acute chemical and genetic manipulations, we found that local synthesis of PI(3,4)P2 by phosphatidylinositol 3-kinase C2α at plasma membrane clathrin-coated pits is spatially segregated from its hydrolysis by the PI(3,4)P2-specific inositol polyphosphate 4-phosphatase 4A (INPP4A). We observed that INPP4A is dispensable for clathrin-mediated endocytosis and is undetectable in endocytic clathrin-coated pits. Instead, we found that INPP4A partially localizes to endosomes and that loss of INPP4A in HAP1 cancer cells perturbs signaling via AKT kinase and mTOR complex 1. These results reveal a function for INPP4-mediated PI(3,4)P2 hydrolysis in local regulation of growth factor and nutrient signals at endosomes in cancer cells. They further suggest a model whereby synthesis and turnover of PI(3,4)P2 are spatially segregated within the endocytic pathway to couple endocytic membrane traffic to growth factor and nutrient signaling.
Project description:Molecular interactions that direct trafficking of secreted proteins are not well-described in salivary glands. Here, we report that the soluble cargo protein Parotid Secretory Protein (PSP) is bound to the membranes of secretory granules isolated from rat parotids. This is apparently due to specific interaction with phosphatidylinositol phosphates (PtdInsP). PSP binds PtdIns(3,4)P(2), 10-fold greater than PtdIns(3,5)P(2) or PtdIns(4)P, and does not bind PtdIns(3)P or PtdIns(5)P. Human PSP synthesized in vitro also binds PtdIns(3,4)P(2). Bacterially expressed rat PSP binds PtdIns(3,4)P(2) with a K(d) of 2.4 x 10(-11) M. Other major secretory proteins (amylase, proline-rich protein) are not bound to isolated granule membranes and do not bind phosphatidylinositol phosphates. Immunofluorescence shows PtdIns(3,4)P(2) at the secretory granules, and fluorescent PtdIns(3,4)P(2) can flip from the outer leaflet to the inner leaflet of the membrane. Binding of PSP to PtdInsPs may contribute to sorting during the formation of the secretory granules, or sorting by retention during maturation of the granules.
Project description:The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] regulates the activity of many actin-binding proteins and as such is an important modulator of cytoskeleton organization during cell migration, for example. In migrating cells actin remodeling is tightly regulated and localized; therefore, how the PI(4,5)P2 level is spatially and temporally regulated is crucial to understanding how it controls cell migration. Here we show that the LIM protein Ajuba contributes to the cellular regulation of PI(4,5)P2 levels by interacting with and activating the enzymatic activity of the PI(4)P 5-kinase (PIPKIalpha), the predominant enzyme in the synthesis of PI(4,5)P2, in a migration stimulus-regulated manner. In migrating primary mouse embryonic fibroblasts (MEFs) from Ajuba(-/-) mice the level of PI(4,5)P2 was decreased with a corresponding increase in the level of the substrate PI(4)P. Reintroduction of Ajuba into these cells normalized PI(4,5)P2 levels. Localization of PI(4,5)P2 synthesis and PIPKIalpha in the leading lamellipodia and membrane ruffles, respectively, of migrating Ajuba(-/-) MEFs was impaired. In vitro, Ajuba dramatically activated the enzymatic activity of PIPKIalpha while inhibiting the activity of PIPKIIbeta. Thus, in addition to its effects upon Rac activity Ajuba can also influence cell migration through regulation of PI(4,5)P2 synthesis through direct activation of PIPKIalpha enzyme activity.
Project description:The Phox homology (PX) domain is a functional module that targets membranes through specific interactions with phosphoinositides. The p47(phox) PX domain preferably binds phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) and plays a pivotal role in the assembly of phagocyte NADPH oxidase. We describe the PI(3,4)P(2) binding mode of the p47(phox) PX domain as identified by a transferred cross-saturation experiment. The identified PI(3,4)P(2)-binding site, which includes the residues of helices α1 and α1' and the following loop up to the distorted left-handed PP(II) helix, is located at a unique position, as compared with the phosphoinositide-binding sites of all other PX domains characterized thus far. Mutational analyses corroborated the results of the transferred cross-saturation experiments. Moreover, experiments with intact cells demonstrated the importance of this unique binding site for the function of the NADPH oxidase. The low affinity and selectivity of the atypical phosphoinositide-binding site on the p47(phox) PX domain suggest that different types of phosphoinositides sequentially bind to the p47(phox) PX domain, allowing the regulation of the multiple events that characterize the assembly and activation of phagocyte NADPH oxidase.
Project description:Phosphatidylinositol (3,5)-bisphosphate (PtdIns(3,5)P2) is a quantitatively minor phospholipid in eukaryotic cells that plays a fundamental role in regulating endocytic membrane traffic. Despite its clear importance for cellular function and organism physiology, mechanistic details of its biology have so far not been fully elucidated. In part, this is due to a lack of experimental tools that specifically probe for PtdIns(3,5)P2 in cells to unambiguously identify its dynamics and site(s) of action. In this study, we have evaluated a recently reported PtdIns(3,5)P2 biosensor, GFP-ML1Nx2, for its veracity as such a probe. We report that, in live cells, the localization of this biosensor to sub-cellular compartments is largely independent of PtdIns(3,5)P2, as assessed after pharmacological, chemical genetic or genomic interventions that block the lipid's synthesis. We therefore conclude that it is unwise to interpret the localization of ML1Nx2 as a true and unbiased biosensor for PtdIns(3,5)P2.