PtdIns(4,5)P2 and PtdIns3P coordinate to regulate phagosomal sealing for apoptotic cell clearance

Phagocytosis requires phosphoinositides (PIs) as both signaling molecules and localization cues. How PIs coordinate to control phagosomal sealing and the accompanying switch of organelle identity is unclear. In this study, we followed dynamic changes in PIs during apoptotic cell clearance in Caenorhabditis elegans. We found that phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂) and phosphatidylinositol-3-phosphate (PtdIns3P), which accumulate transiently on unsealed and fully sealed phagosomes, respectively, are both involved in phagosome closure. We identified PtdIns3P phosphatase MTM-1 as an effector of PtdIns(4,5)P₂ to promote phagosomal sealing. MTM-1 coordinates with the class II PI3 kinase PIKI-1 to control PtdIns3P levels on unsealed phagosomes. The SNX9 family protein LST-4 is required for sealing, and its association with unsealed phagosomes is regulated by PtdIns(4,5)P₂, PIKI-1, and MTM-1. Loss of LST-4 or its retention on phagosomes disrupts sealing and suppresses PtdIns3P accumulation, indicating close coupling of the two events. Our findings support a coincidence detection mechanism by which phagosomal sealing is regulated and coupled with conversion from PtdIns(4,5)P₂ enrichment on unsealed phagosomes to PtdIns3P enrichment on fully sealed phagosomes.     

Three sorting nexins drive the degradation of apoptotic cells in response to PtdIns(3)P signaling

Apoptotic cells are swiftly engulfed by phagocytes and degraded inside phagosomes. Phagosome maturation requires phosphatidylinositol 3-phosphate [PtdIns(3)P], yet how PtdIns(3)P triggers phagosome maturation remains largely unknown. Through a genomewide PtdIns(3)P effector screen in the nematode Caenorhabditis elegans , we identified LST-4/SNX9, SNX-1, and SNX-6, three BAR domain-containing sorting nexins, that act in two parallel pathways to drive PtdIns(3)P-mediated degradation of apoptotic cells. We found that these proteins were enriched on phagosomal surfaces through association with PtdIns(3)P and through specific protein-protein interaction, and they promoted the fusion of early endosomes and lysosomes to phagosomes, events essential for phagosome maturation. Specifically, LST-4 interacts with DYN-1 (dynamin), an essential phagosome maturation initiator, to strengthen DYN-1's association to phagosomal surfaces, and facilitates the maintenance of the RAB-7 GTPase on phagosomal surfaces. Furthermore, both LST-4 and SNX-1 promote the extension of phagosomal tubules to facilitate the docking and fusion of intracellular vesicles. Our findings identify the critical and differential functions of two groups of sorting nexins in phagosome maturation and reveal a signaling cascade initiated by phagocytic receptor CED-1, mediated by PtdIns(3)P, and executed through these sorting nexins to degrade apoptotic cells.     

Requirement for Rho GTPases and PI 3-kinases during apoptotic cell phagocytosis by macrophages

In vivo, apoptotic cells are removed by surrounding phagocytes, a process thought to be essential for tissue remodeling and the resolution of inflammation [1]. Although apoptotic cells are known to be efficiently phagocytosed by macrophages, the mechanisms whereby their interaction with the phagocytes triggers their engulfment have not been described in mammals. Here, we report that primary murine bone marrow-derived macrophages (using alpha(v)beta(3) integrin for apoptotic cell uptake) extend lamellipodia to engulf apoptotic cells and form an actin cup where phosphotyrosine accumulates. Rho GTPases and PI 3-kinases have been widely implicated in the regulation of the actin cytoskeleton [2, 3]. We show that inhibition of Rho GTPases by Clostridium difficile toxin B prevents apoptotic cell phagocytosis and inhibits the accumulation of both F-actin and phosphotyrosine. Importantly, the Rho GTPases Rac1 and Cdc42 are required for apoptotic cell uptake whereas Rho inhibition enhances uptake. The PI 3-kinase inhibitor LY294002 also prevents apoptotic cell phagocytosis but has no effect on the accumulation of F actin and phosphotyrosine. These results indicate that both Rho GTPases and PI 3-kinases are involved in apoptotic cell phagocytosis but that they play distinct roles in this process.     

PtdIns(3,4,5)P3 regulates spindle orientation in adherent cells

Cultured adherent cells divide on the substratum, leading to formation of the cell monolayer. However, how the orientation of this anchorage-dependent cell division is regulated remains unknown. We have previously shown that integrin-dependent adhesion orients the spindle parallel to the substratum, which ensures this anchorage-dependent cell division. Here, we show that phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) is essential for this spindle orientation control. In metaphase, PtdIns(3,4,5)P3 is accumulated in the midcortex in an integrin-dependent manner. Inhibition of phosphatidylinositol-3-OH kinase (PI(3)K) reduces the accumulation of PtdIns(3,4,5)P3 and induces spindle misorientation. Introduction of PtdIns(3,4,5)P3 to these cells restores the midcortical accumulation of PtdIns(3,4,5)P3 and proper spindle orientation. PI(3)K inhibition causes dynein-dependent spindle rotations along the z-axis, resulting in spindle misorientation. Moreover, dynactin, a dynein-binding partner, is accumulated in the midcortex in a PtdIns(3,4,5)P3-dependent manner. We propose that PtdIns(3,4,5)P3 directs dynein/dynactin-dependent pulling forces on spindles to the midcortex, and thereby orients the spindle parallel to the substratum.     

Two waves of platelet secretion induced by thromboxane A2 receptor and a critical role for phosphoinositide 3-kinases

Thromboxane A2 (TXA2)-mediated platelet secretion and aggregation are important in thrombosis. Here, we present a novel finding that the stable TXA2 analogue, U46619, induces two waves of platelet secretion, each of which precedes a distinct wave of platelet aggregation. ADP released from platelets during the first wave of secretion played a major role in augmenting the first wave of platelet aggregation. The second wave of platelet secretion and aggregation required the first wave of both ADP secretion and aggregation and were blocked by either the integrin inhibitor RGDS or a P2Y12 receptor antagonist, indicating a requirement for both the integrin outside-in signal and ADP-activated Gi pathway. U46619 stimulated phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of Akt, which was augmented by ADP but did not require integrin outside-in signaling. Platelets from PI3Kgamma knock-out mice or PI3K inhibitor-treated platelets showed an impaired second wave of platelet secretion and aggregation. However, the second wave of platelet aggregation was restored by addition of exogenous ADP to PI3Kgamma deficient or PI3K inhibitor-treated platelets. Thus, our data indicate that PI3K, together with the integrin outside-in signaling, play a central role in inducing the second wave of platelet secretion, which leads to the second wave of irreversible platelet aggregation.     

A monoclonal antibody to visualize PtdIns(3,4,5)P(3) in cells.

Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] is a second messenger produced in response to agonist stimulation. Traditionally, visualization of phosphoinositide polyphosphates (PtdInsP(n)) in living cells is accomplished using chimeric green fluorescent protein (GFP)-pleckstrin homology (PH) domain proteins, while PtdInsP(n) quantitation is accomplished by extraction and separation of radiolabeled cellular PtdInsP(n)s. Here we describe preparation of a covalent protein-PtdIns(3,4,5)P(3) immunogen, characterization of binding selectivity of an anti-PtdIns(3,4,5)P(3) IgM, and immunodetection of PtdIns(3,4,5)P(3) in stimulated mammalian cells. This antibody has greater than three orders of magnitude selectivity for binding PtdIns(3,4,5)P(3) relative to its precursor, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), and is therefore optimal for studies of cell function. The immunodetection in platelet-derived growth factor (PDGF)-stimulated NIH 3T3 cells was benchmarked against HPLC analysis of [3H]-myo-inositol-labeled cellular PtdInsP(n)s. In addition, the changes in subcellular amounts and localizations of both PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2) in stimulated NIH 3T3 fibroblasts and human neutrophils were observed by immunofluorescence. In insulin- or PDGF-stimulated fibroblasts, PtdIns(3,4,5)P(3) levels increased in the cytoplasm, peaking at 10 min. In contrast, increases in the PtdIns(4,5)P(2) levels were detected in nuclei, corresponding to the production of new substrate following depletion by phosphoinositide (PI) 3-kinase.     

Dynamics and function of PtdIns(3)P in autophagy

Phosphorylation of phosphatidylinositol (PtdIns) by PtdIns 3-kinase is essential for autophagy. However, the distribution and function of the enzymatic product, PtdIns 3-phosphate (PtdIns(3)P), has been unknown. We monitored PtdIns(3)P distribution during autophagy by live imaging, biochemistry, and electron microscopy, and found that PtdIns(3)P is massively delivered into the vacuole via autophagy. PtdIns(3)P is highly enriched as a membrane component of the elongating isolation membranes and autophagosome membranes rather than as an enclosed cargo, implying direct involvement of PtdIns(3)P in autophagosome formation. This observation also provides important basic information on the nature of the autophagosome membrane, which is still poorly understood. Notably, PtdIns(3)P is highly enriched on the inner (concave) surfaces of the isolation membrane and autophagosome compared to the outer surfaces. PtdIns(3)P is also enriched on ambiguous structures juxtaposed to the elongating tips of isolation membranes. We also investigated the function of PtdIns(3)P in autophagy, and show that PtdIns(3)P recruits the Atg18-Atg2 complex to autophagic membranes through an Atg18-PtdIns(3)P interaction. Interestingly, PtdIns(3)P is required only for the association of the Atg18-Atg2 complex to autophagic membranes but not for any subsequent functional activity of the Atg18-Atg2 complex, suggesting that PtdIns(3)P does not act allosterically on Atg18. Based on these results we discuss the function of PtdIns(3)P in autophagy.     

Ocrl1, a PtdIns(4,5)P(2) 5-phosphatase, is localized to the trans-Golgi network of fibroblasts and epithelial cells.

PtdIns(4,5)P(2) and PtdIns(4,5)P(2) 5-phosphatases play important roles in diverse aspects of cell metabolism, including protein trafficking. However, the relative importance of the PtdIns(4,5)P(2) 5-phosphatases in regulating PtdIns(4,5)P(2) levels for specific cell processes is not well understood. Ocrl1 is a PtdIns(4,5)P(2) 5-phosphatase that is deficient in the oculocerebrorenal syndrome of Lowe, a disorder characterized by defects in kidney and lens epithelial cells and mental retardation. Ocrl1 was originally localized to the Golgi in fibroblasts, but a subsequent report suggested a lysosomal localization in a kidney epithelial cell line. In this study we defined the localization of ocrl1 in fibroblasts and in two kidney epithelial cell lines by three methods: immunofluorescence, subcellular fractionation, and a dynamic perturbation assay with brefeldin A. We found that ocrl1 was a Golgi-localized protein in all three cell types and further identified it as a protein of the trans-Golgi network (TGN). The TGN is a major sorting site and has the specialized function in epithelial cells of directing proteins to the apical or basolateral domains. The epithelial cell phenotype in Lowe syndrome and the localization of ocrl1 to the TGN imply that this PtdIns(4,5)P(2) 5-phosphatase plays a role in trafficking. (J Histochem Cytochem 48:179-189, 2000)     

Modulation of Local PtdIns3P Levels by the PI Phosphatase MTMR3 Regulates Constitutive Autophagy

Autophagy is a catabolic process that delivers cytoplasmic material to the lysosome for degradation. The mechanisms regulating autophagosome formation and size remain unclear. Here, we show that autophagosome formation was triggered by the overexpression of a dominant‐negative inactive mutant of Myotubularin‐related phosphatase 3 (MTMR3). Mutant MTMR3 partially localized to autophagosomes, and PtdIns3P and two autophagy‐related PtdIns3P‐binding proteins, GFP‐DFCP1 and GFP‐WIPI‐1α (WIPI49/Atg18), accumulated at sites of autophagosome formation. Knock‐down of MTMR3 increased autophagosome formation, and overexpression of wild‐type MTMR3 led to significantly smaller nascent autophagosomes and a net reduction in autophagic activity. These results indicate that autophagy initiation depends on the balance between PI 3‐kinase and PI 3‐phosphatase activity. Local levels of PtdIns3P at the site of autophagosome formation determine autophagy initiation and the size of the autophagosome membrane structure.     

PtdIns(4,5)P2 and PtdIns(3,4,5)P3 dynamics during focal adhesions assembly and disassembly in a cancer cell line

Focal adhesions (FAs) are large assemblies of proteins that mediate intracellular signals between the cytoskeleton and the extracellular matrix (ECM). The turnover of FA proteins plays a critical regulatory role in cancer cell migration. Plasma membrane lipids locally generated or broken down by different inositide kinases and phosphatase enzymes to activate and recruit proteins to specific regions in the plasma membrane. Presently, little attention has been given to the use of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) fluorescent biosensors in order to determine the spatiotemporal organisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 within and around or during assembly and disassembly of FAs. In this study, specific biosensors were used to detect PtdIns(4,5)P2, PtdIns(3,4,5)P3, and FAs proteins conjugated to RFP/GFP in order to monitor changes of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 levels within FAs. We demonstrated that the localisation of PtdIns(4,5)P2 and PtdIns(3,4,5)P3 were moderately correlated with that of FA proteins. Furthermore, we demonstrate that local levels of PtdIns(4,5)P2 increased within FA assembly and declined within FA disassembly. However, PtdIns(3,4,5)P3 levels remained constant within FAs assembly and disassembly. In conclusion, this study shows that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 localised in FAs may be regulated differently during FA assembly and disassembly.     

A Comparative Study to Visualize PtdIns(4,5) P2 and PtdIns(3,4,5) P3 in MDA-MB-231 Breast Cancer Cell Line

Background:Phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5) P3) and Phosphatidylinositol 4,5-trisphosphate (PtdIns(4,5) P2] form an insignificant amount of phospholipids but play important roles in controlling membrane-bound signalling. Little attention has been given to visualize and monitor changes or differences in the local generation of PtdIns(4,5) P2 and PtdIns(3,4,5) P3 in the cell membranes of MDA-MB-231 breast cancer cell lines.Methods:PLCδ1-PH-GFP and Btk-PH-GFP were used as biosensors to detected PtdIns(4,5) P2 and PtdIns(3,4,5)P3 respectively. These biosensors and antibodies were transfected, immuostained and then visualized by confocal microscopy on different cell surfaces.Results:Our results showed that PLCδ1-PH-GFP/mCherry was localized at the cell membrane, while Btk-PH-GFP/mCherry was sometimes localized at the cell membrane but there was also a large amount of fluorescence present in the cytosol and nucleus. Our results also showed that the cells that expressed low levels of Btk-PH-GFP the fluorescence was predominantly localised to the cell membrane. While the cells that expressed high levels of Btk-PH-GFP the fluorescence was localization in the cytosol and cell membrane. Our results demonstrated that both anti-PtdIns(4,5)P2 and anti-PtdIns(3,4,5)P3 antibodies were localized everywhere in cell.Conclusion:Our results suggest that PLCδ1-PH-GFP and Btk-PH-GFP/mCherry have more specificity, reliability, suitability and accuracy than antibodies in binding with and detecting PtdIns(4,5)P2 and PtdIns(3,4,5)P3 and in studying the molecular dynamics of phospholipids in live and fixed cells.Key Words: Antibodies, Biosensors, MDA-MB-231, Phosphatidylinositol     

The Phox Domain of Sorting Nexin 5 Lacks Phosphatidylinositol 3-Phosphate (PtdIns(3)P) Specificity and Preferentially Binds to Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2)

Subcellular retrograde transport of cargo receptors from endosomes to the trans-Golgi network is critically involved in a broad range of physiological and pathological processes and highly regulated by a genetically conserved heteropentameric complex, termed retromer. Among the retromer components identified in mammals, sorting nexin 5 and 1 (SNX5; SNX1) have recently been found to interact, possibly controlling the membrane binding specificity of the complex. To elucidate how the unique sequence features of the SNX5 phox domain (SNX5-PX) influence retrograde transport, we have determined the SNX5-PX structure by NMR and x-ray crystallography at 1.5 Å resolution. Although the core fold of SNX5-PX resembles that of other known PX domains, we found novel structural features exclusive to SNX5-PX. It is most noteworthy that in SNX5-PX, a long helical hairpin is added to the core formed by a new α2′-helix and a much longer α3-helix. This results in a significantly altered overall shape of the protein. In addition, the unique double PXXP motif is tightly packed against the rest of the protein, rendering this part of the structure compact, occluding parts of the putative phosphatidylinositol (PtdIns) binding pocket. The PtdIns binding and specificity of SNX5-PX was evaluated by NMR titrations with eight different PtdIns and revealed that SNX5-PX preferentially and specifically binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). The distinct structural and PtdIns binding characteristics of SNX5-PX impart specific properties on SNX5, influencing retromer-mediated regulation of retrograde trafficking of transmembrane cargo receptors.The early work on retromer revealed its role in the trafficking of cargo proteins between endosomes and the trans-Golgi network (TGN),² although recently, retromer involvement in many other physiological and developmental processes has been uncovered (1, 2). The best studied proteins associated with retromer activity are intracellular sorting receptors such as the yeast vacuolar protein-10 (Vps10) and mammalian mannose 6-phosphate receptors (3, 4). These receptors sort acid hydrolases, enzymes essential for protein degradation, out of the TGN into the yeast vacuole or the mammalian lysosome. Upon releasing their substrates, these cargos traffic back to the TGN to mediate further rounds of cargo-hydrolase transportation. Similar retrograde trafficking of cargo proteins involving signaling molecules such as Wnt and amyloid precursor protein (APP) are thought to be critical for their secretion and function (5, 6). Retrograde transportation is highly regulated by the heteropentameric retromer complex that consists of a sorting nexin (SNX) dimer (e.g. Vps5 and Vps17 in yeast) and a Vps26/29/35 trimer (7). In mammals, the binding of the SNX dimer to specific phosphatidylinositol (PtdIns) determines its subcellular membrane association and governs the recruitment of the Vps trimer to endosomal compartments. Mammalian orthologs of the trimer have been biochemically characterized, and their interaction and function in cargo protein trafficking is well established (8). More recently, crystal structures of three Vps proteins in the trimer suggested how this trimer interacts with the SNX dimer and cargo proteins as well as with curved membranes (9–12). In the SNX dimer, SNX1 and SNX2 are thought to be interchangeable Vps5 orthologs (13, 14). The NMR structure of SNX1 revealed details of PI(3)P specific binding, thereby explaining its role in endosomal trafficking (15). The identity for SNX5 as a potential functional mammalian ortholog of Vps17, however, was not revealed until recently.Although initially identified as a Fanconi anemia complementation group A (FANCA)-binding protein (16), SNX5 was later shown to play an important role in membrane trafficking (17–19). SNX5 contains a PX domain (SNX5-PX) that is the signature feature in defining the SNX family, composed of 30 members at present (20) (Fig. 1B). In addition, SNX5 possesses a C-terminal BAR (Bin/Amphiphysin/Rvs) domain that has been reported to interact with a number of other proteins involved in endosomal trafficking (17, 21–27). It functions as a dimerization module that senses and/or induces membrane curvature (28, 29). Our previous biochemical study suggested a specific interaction between SNX5 and SNX1 through which the two SNXs mutually influence each other''s effect in endosomal trafficking of epidermal growth factor receptor upon epidermal growth factor stimulation (17). In support of this observation are several recent reports that indicate a critical role of SNX5 and the closely related SNX6, beyond that of SNX1 and SNX2, on retrograde sorting of mannose 6-phosphate receptor (24, 27). Therefore, SNX5 and SNX6 may be functionally interchangeable orthologs of Vps17 in mammalian cells (7, 24). Furthermore, in contrast to some reports (18, 30), SNX5 partially localizes to late endosomes and the TGN, exhibiting very low binding affinity for PtdIns(3)P (17), the substrate for phox domain proteins associating with early endosome association. Therefore, the subcellular localization and function of the SNX dimer in SNX5 function may depend on its unique structure that is different from other known PX domains.Open in a separate windowFIGURE 1.Amino Acid sequence alignment of phox domains and domain architecture of the mammalian sorting nexin family. A, comparative sequence alignment of PX domains for residues equivalent to Gly⁴⁹–Leu¹¹⁹ of the p40-PX domain (adapted from Worby and Dixon (21)). Prolines in the Pro-X-X-Pro motif are highlighted in yellow, and residues involved in phospholipid binding in the p40-PX domain are boxed in magenta. Arg⁵⁸ and Arg¹⁰⁵ are marked with magenta triangles, and Tyr⁵⁹ and Lys⁹² are marked with black stars at the bottom of the sequences. The two conserved Arg residues and Lys⁹² of p40-PX in other PX domains are highlighted in dark blue boxes; those corresponding to Tyr⁵⁹ are boxed in green. The secondary structure elements of p40-PX are indicated by yellow arrows (β-sheets) and red ovals (α-helices). The three sequence stretches that are unique in SNX5-PX (or SNX6-PX) are enclosed in a bright blue box. B, domain architecture of SNX family members. The four classes within the SNX family are designated as PX SNXs, PX-BAR SNXs, SH3-PX-BAR, and PX-other domain SNXs. Each individual domain is depicted in a different color and/or shape. The following domains are depicted: PX (phox), BAR (Bin-Amphiphysin-Rvs), SH3 (Src homology 3), TM (transmembrane), PXA (PX domain-associated), RGS (regulator of G-protein signaling), MIT (microtubule interacting and trafficking), B41 (band 4.1 homology), TPR (tetratricopeptide repeat), PDZ (postsynaptic protein PSD-95/SAP90, the Drosophila melanogaster septate junction protein Discs-large, and the tight junction protein ZO-1), and RA (Ras association).Most PX domains of SNX family proteins preferentially bind PtdIns(3)P (30–34), with few exceptions that interact with other PtdIns (30, 32, 35). There are about a dozen structurally characterized PX domains from the SNX family or other PX domain-containing proteins currently deposited in the Protein Data Bank (PDB) data base. Their structures all share common core features, a three-stranded β-sheet that is abutted by three α-helices and an irregular strand containing the PXXP region. Analyses of the representative p47-PX and SNX3-PX domain structures suggested that PtdIns(3)P binding involves two conserved Arg residues at positions equivalent to Arg⁵⁸ and Arg¹⁰⁵ in p40-PX (36). Because equivalent Arg residues are found in the PX domains of most SNX family members, it is generally assumed that all SNX proteins interact with the PtdIns(3)P-enriched elements of the early endocytic compartments. The amino acid sequences of the PX domains of both SNX5 and SNX6, however, lack the two conserved Arg residues that are involved in PtdIns(3)P binding as well as comprising a ∼30-residue insertion immediately after the PXXP motif (Fig. 1A). In addition, the PXXP motif is extended into a double PXXP motif with the sequence PXXPXXP. These unique sequence features set SNX5/6 apart from the other SNX family members. In the p40-PX domain and yeast SNX3, the two conserved Arg residues, the loop between the PXXP motif, and the α3-helix are involved in forming the binding pocket for the phosphate groups of PtdIns(3)P (36, 37). Therefore, changes in length and sequence in this region in SNX5/6-PX are expected to have profound impact on the specific structure and conformation required for PtdIns recognition.To elucidate how its unique sequence features influence the function of SNX5 in retromer-mediated retrograde membrane trafficking, we structurally investigated the SNX5-PX domain by NMR spectroscopy and x-ray crystallography. Using direct NMR titrations, we established the PtdIns binding specificity of SNX5-PX. The high resolution (1.5 Å) crystal structure of the domain revealed its distinct features when compared with previously known family members. Our results demonstrate that the SNX5-PX domain is indeed unique, both with respect to its structure as well as with respect to ligand binding. These findings have important implications for the function of SNX5 in the subcellular membrane trafficking and retrograde sorting.     

Molecular control of PtdIns(3,4,5)P3 signaling in neutrophils

Neutrophils play critical roles in innate immunity and host defense. However, excessive neutrophil accumulation or hyper-responsiveness of neutrophils can be detrimental to the host system. Thus, the response of neutrophils to inflammatory stimuli needs to be tightly controlled. Many cellular processes in neutrophils are mediated by localized formation of an inositol phospholipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3), at the plasma membrane. The PtdIns(3,4,5)P3 signaling pathway is negatively regulated by lipid phosphatases and inositol phosphates, which consequently play a critical role in controlling neutrophil function and would be expected to act as ideal therapeutic targets for enhancing or suppressing innate immune responses. Here, we comprehensively review current understanding about the action of lipid phosphatases and inositol phosphates in the control of neutrophil function in infection and inflammation.     

Receptor-mediated regulation of PI3Ks confines PI(3,4,5)P3 to the leading edge of chemotaxing cells

Recent studies have demonstrated that PH domains specific for PI(3,4,5)P3 accumulate at the leading edge of a number of migrating cells and that PI3Ks and PTEN associate with the membrane at the front and back, respectively, of chemotaxing Dictyostelium discoideum cells. However, the dependence of chemoattractant induced changes in PI(3,4,5)P3 on PI3K and PTEN activities have not been defined. We find that bulk PI(3,4,5)P3 levels increase transiently upon chemoattractant stimulation, and the changes are greater and more prolonged in pten- cells. PI3K activation increases within 5 s of chemoattractant addition and then declines to a low level of activity identically in wild-type and pten- cells. Reconstitution of the PI3K activation profile can be achieved by mixing membranes from stimulated pi3k1-/pi3k2- cells with cytosolic PI3Ks from unstimulated cells. These studies show that significant control of chemotaxis occurs upstream of the PI3Ks and that regulation of the PI3Ks and PTEN cooperate to shape the temporal and spatial localization of PI(3,4,5)P3.     

PtdIns(3,4,5)P(3)-dependent and -independent roles for PTEN in the control of cell migration

BACKGROUND: Phosphatase and tensin homolog (PTEN) mediates many of its effects on proliferation, growth, survival, and migration through its PtdIns(3,4,5)P(3) lipid phosphatase activity, suppressing phosphoinositide 3-kinase (PI3K)-dependent signaling pathways. PTEN also possesses a protein phosphatase activity, the role of which is less well characterized. RESULTS: We have investigated the role of PTEN in the control of cell migration of mesoderm cells ingressing through the primitive streak in the chick embryo. Overexpression of PTEN strongly inhibits the epithelial-to-mesenchymal transition (EMT) of mesoderm cells ingressing through the anterior and middle primitive streak, but it does not affect EMT of cells located in the posterior streak. The inhibitory activity on EMT is completely dependent on targeting PTEN through its C-terminal PDZ binding site, but can be achieved by a PTEN mutant (PTEN G129E) with only protein phosphatase activity. Expression either of PTEN lacking the PDZ binding site or of the PTEN C2 domain, or inhibition of PI3K through specific inhibitors, does not inhibit EMT, but results in a loss of both cell polarity and directional migration of mesoderm cells. The PTEN-related protein TPTE, which normally lacks any detectable lipid and protein phosphatase activity, can be reactivated through mutation, and only this reactivated mutant leads to nondirectional migration of these cells in vivo. CONCLUSIONS: PTEN modulates cell migration of mesoderm cells in the chick embryo through at least two distinct mechanisms: controlling EMT, which involves its protein phosphatase activity; and controlling the directional motility of mesoderm cells, through its lipid phosphatase activity.     

The inositol 5-phosphatase dOCRL controls PI(4,5)P2 homeostasis and is necessary for cytokinesis

During cytokinesis, constriction of an equatorial actomyosin ring physically separates the two daughter cells. At the cleavage furrow, the phosphoinositide PI(4,5)P2 plays an?important role by recruiting and regulating essential proteins of the cytokinesis machinery [1]. Accordingly, perturbation of PI(4,5)P2 regulation leads to abortive furrowing and binucleation [2-4]. To determine how PI(4,5)P2 is regulated during cytokinesis, we individually knocked down each of the enzymes controlling the phosphoinositide (PIP) cycle in Drosophila. We show that depletion of the Drosophila ortholog of human oculocerebrorenal syndrome of Lowe 1 (OCRL1), an inositol 5-phosphatase mutated in the X-linked disorder oculocerebrorenal Lowe syndrome, triggers a high rate of cytokinesis failure. In absence of dOCRL, several essential components of the cleavage furrow were found to be incorrectly localized on giant cytoplasmic vacuoles rich in PI(4,5)P2 and in endocytic markers. We demonstrate that dOCRL is associated with endosomes and that it dephosphorylates PI(4,5)P2 on internal membranes to restrict this phosphoinositide at the plasma membrane and thereby regulates cleavage furrow formation and ingression. Identification of dOCRL as essential for cell division may be important to understand the molecular basis of the phenotypic manifestations of Lowe syndrome.     

Regulated and polarized PtdIns(3,4,5)P3 accumulation is essential for apical membrane morphogenesis in photoreceptor epithelial cells

BACKGROUND: In a specialized epithelial cell such as the Drosophila photoreceptor, a conserved set of proteins is essential for the establishment of polarity, its maintenance, or both--in Drosophila, these proteins include the apical factors Bazooka, D-atypical protein kinase C, and D-Par6 together with D-Ecadherin. However, little is known about the mechanisms by which such apical factors might regulate the differentiation of the apical membrane into functional domains such as an apical-most stack of microvilli or more lateral sub-apical membrane. RESULTS: We show that in photoreceptors Bazooka (D-Par3) recruits the tumor suppressor lipid phosphatase PTEN to developing cell-cell junctions (Zonula Adherens, za). za-localized PTEN controls the spatially restricted accumulation of optimum levels of the lipid PtdIns(3,4,5)P3 within the apical membrane domain. This in turn finely tunes activation of Akt1, a process essential for proper morphogenesis of the light-gathering organelle, consisting of a stack of F-actin rich microvilli within the apical membrane. CONCLUSIONS: Spatially localized PtdIns(3,4,5)P3 mediates directional sensing during neutrophil and Dictyostelium chemotaxis. We conclude that a conserved mechanism also operates during photoreceptor epithelial cell morphogenesis in order to achieve normal differentiation of the apical membrane.