The phosphoinositide phosphatase Sjl2 is recruited to cortical actin patches in the control of vesicle formation and fission during endocytosis

The Saccharomyces cerevisiae synaptojanin-like proteins (Sjl1, Sjl2, and Sjl3) are phosphoinositide (PI) phosphatases that regulate PI metabolism in the control of actin organization and membrane trafficking. However, the primary sites of action for each of the yeast synaptojanin-like proteins remain unclear. In this study, we show that Sjl2 is localized to cortical actin patches, sites of endocytosis. Cortical recruitment of Sjl2 requires the actin patch component Abp1. Consistent with this, the SH3 domain-containing protein Abp1 physically associates with Sjl2 through its proline-rich domain. Furthermore, abp1Delta mutations confer defects resembling loss of SJL2; sjl1Delta abp1Delta double-mutant cells exhibit invaginated plasma membranes and impaired endocytosis, findings similar to those for sjl1Delta sjl2Delta mutant cells. Thus, Abp1 acts as an adaptor protein in the localization or concentration of Sjl2 during late stages of endocytic vesicle formation. Overexpression of the Hip1-related protein Sla2 delayed the formation of extended plasma membrane invaginations in sjl2ts cells, indicating that Sla2 may become limiting or misregulated in cells with impaired PI phosphatase activity. Consistent with this, the cortical actin patch protein Sla2 is mislocalized in sjl1Delta sjl2Delta mutant cells. Together, our studies suggest that PI metabolism by the synaptojanin-like proteins coordinately directs actin dynamics and membrane invagination, in part by regulation of Sla2.     

Interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 is important for actin-dependent endocytic internalization

A variety of studies have implicated the lipid PtdIns(4,5)P2 in endocytic internalization, but how this lipid mediates its effects is not known. The AP180 N-terminal homology (ANTH) domain is a PtdIns(4,5)P2-binding module found in several proteins that participate in receptor-mediated endocytosis. One such protein is yeast Sla2p, a highly conserved actin-binding protein essential for actin organization and endocytic internalization. To better understand how PtdIns(4,5)P2 binding regulates actin-dependent endocytosis, we investigated the functions of Sla2p's ANTH domain. A liposome-binding assay revealed that Sla2p binds to PtdIns(4,5)P2 specifically through its ANTH domain and identified specific lysine residues required for this interaction. Mutants of Sla2p deficient in PtdIns(4,5)P2 binding showed significant defects in cell growth, actin organization, and endocytic internalization. These defects could be rescued by increasing PtdIns(4,5)P2 levels in vivo. Strikingly, mutant Sla2p defective in PtdIns(4,5)P2 binding localized with the endocytic machinery at the cell cortex, establishing that the ANTH-PtdIns(4,5)P2 interaction is not necessary for this association. In contrast, multicolor real-time fluorescence microscopy and particle-tracking analysis demonstrated that PtdIns(4,5)P2 binding is required during endocytic internalization. These results demonstrate that the interaction of Sla2p's ANTH domain with PtdIns(4,5)P2 plays a key role in regulation of the dynamics of actin-dependent endocytic internalization.     

Yeast Arf3p modulates plasma membrane PtdIns(4,5)P2 levels to facilitate endocytosis

Phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2] is a key regulator of endocytosis. PtdIns(4,5)P2 generation at the plasma membrane in yeast is mediated by the kinase Mss4p, but the mechanism underlying the temporal and spatial activation of Mss4p to increase formation of PtdIns(4,5)P2 at appropriate sites is not known. Here, we show that ADP ribosylation factor (Arf)3p, the yeast homologue of mammalian Arf6, is necessary for wild-type levels of PtdIns(4,5)P2 at the plasma membrane. Arf3p localizes to dynamic spots at the membrane, and the behaviour of these is consistent with it functioning in concert with endocytic machinery. Localization of Arf3p is disrupted by deletion of genes encoding an ArfGAP homology protein Gts1p and a guanine nucleotide exchange factor Yel1p. Significantly, deletion of arf3 causes a reduction in PtdIns(4,5)P2 at the plasma membrane, while increased levels of active Arf3p, caused by deletion of the GTPase-activating protein Gts1, increase PtdIns(4,5)P2 levels. Furthermore, elevated Arf3p correlates with an increase in the number of endocytic sites. Our data provide evidence for a mechanism in yeast to positively regulate plasma membrane production of PtdIns(4,5)P2 levels and that these changes impact on endocytosis.     

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.     

Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2

Clathrin-mediated endocytosis (CME) is facilitated by a precisely regulated burst of actin assembly. PtdIns(4,5)P2 is an important signaling lipid with conserved roles in CME and actin assembly regulation. Rhomboid family multipass transmembrane proteins regulate diverse cellular processes; however, rhomboid-mediated CME regulation has not been described. We report that yeast lacking the rhomboid protein Rbd2 exhibit accelerated endocytic-site dynamics and premature actin assembly during CME through a PtdIns(4,5)P2-dependent mechanism. Combined genetic and biochemical studies showed that the cytoplasmic tail of Rbd2 binds directly to PtdIns(4,5)P2 and is sufficient for Rbd2''s role in actin regulation. Analysis of an Rbd2 mutant with diminished PtdIns(4,5)P2-binding capacity indicates that this interaction is necessary for the temporal regulation of actin assembly during CME. The cytoplasmic tail of Rbd2 appears to modulate PtdIns(4,5)P2 distribution on the cell cortex. The syndapin-like F-BAR protein Bzz1 functions in a pathway with Rbd2 to control the timing of type 1 myosin recruitment and actin polymerization onset during CME. This work reveals that the previously unstudied rhomboid protein Rbd2 functions in vivo at the nexus of three highly conserved processes: lipid regulation, endocytic regulation, and cytoskeletal function.     

Regulation of Focal Adhesion Dynamics and Cell Migration by PLC/PI3K-Mediated Metabolism of PtdIns (4,5) P2 in a Breast Cancer Cell Line

Background:Focal adhesions (FAs) are highly dynamic complex structures that assembled and disassembled on an ongoing basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion to cell migration. Assembly and disassembly processes of FAs are regulated by a variety of cellular signaling proteins and adaptors. We previously demonstrated that local levels of Phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2) in MDA-MB-231 cells increases during FA assembly and declines during disassembly. In this study we aimed to investigate whether PtdIns(4,5)P2 regulates FA turnover.Methods:MDA-MB-231 cells were co-transfected with a labeling vinculin (or zyxin) and the PLC𝛅1-PH biosensor to visualize FA localization and PtdIns(4,5)P2 in the cell membrane. We also used pharmacological inhibitors to determine the mechanism underlying the changes of PtdIns(4,5)P2 level during FA turnover and cell migration. Immunostaining, immunoprecipitation, and Western blotting were used to examine the localization and interaction between phospholipase C (PLC)/phosphatidylinositol 3-kinase (PI3K) FA proteins.Results:We showed that inhibition of PLC, PI3K significantly reduced the decline of PtdIns(4,5)P2 levels within FA disassembly and the slowdown rate of FA turnover and cell migration. We also showed that the inhibition of enzymes implicated in the downstream pathway of PtdIns(4,5)P2, such as diacylglycerol kinase (DAGK) and protein kinase C (PKC) significantly reduced FA turnover time and the speed of cell migration. Additionally, we demonstrated that PLC but not PI3K interact with FAs. In conclusion,DiscussionThis study suggests that dynamical changes of PtdIns(4,5)P2 might regulate FA turnover and facilitate cell migration.Key Words: Cell Migration, Focal adhesion turnover, MDA-MB-231 Breast Cancer Cell Line, Ptdins(4,5)P2, PLC, PI3K     

Phosphatidylinositol-4,5-bisphosphate is required for endocytic coated vesicle formation

Receptor-mediated endocytosis via clathrin-coated vesicles has been extensively studied and, while many of the protein players have been identified, much remains unknown about the regulation of coat assembly and the mechanisms that drive vesicle formation [1]. Some components of the endocytic machinery interact with inositol polyphosphates and inositol lipids in vitro, implying a role for phosphatidylinositols in vivo [2] and [3]. Specifically, the adaptor protein complex AP2 binds phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P₂), PtdIns(3)P, PtdIns(3,4,5)P₃ and inositol phosphates. Phosphatidylinositol binding regulates AP2 self-assembly and the interactions of AP2 complexes with clathrin and with peptides containing endocytic motifs [4] and [5]. The GTPase dynamin contains a pleckstrin homology (PH) domain that binds PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ to regulate GTPase activity in vitro [6] and [7]. However, no direct evidence for the involvement of phosphatidylinositols in clathrin-mediated endocytosis exists to date. Using well-characterized PH domains as high affinity and high specificity probes in combination with a perforated cell assay that reconstitutes coated vesicle formation, we provide the first direct evidence that PtdIns(4,5)P₂ is required for both early and late events in endocytic coated vesicle formation.     

Interaction of Pik1p and Sjl proteins in membrane trafficking

Phosphatidylinositol (PtdIns) phosphates are involved in signal transduction, cytoskeletal organization, and membrane traffic. PtdIns 4-phosphate [PtdIns(4)P], produced in yeast by PtdIns 4-kinase (Pik1p), appears to regulate Golgi secretory function. PtdIns(4)P is also produced by dephosphorylation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], catalyzed by one of the three yeast Sjl proteins, homologs of the mammalian synaptic vesicle-associated PtdIns(4,5)P2 5-phosphatase, synaptojanin. To determine whether Pik1p and Sjl proteins operate in the same pathway or regulate the same process, we used a genetic approach. Mutation in the PIK1 gene displays synthetic genetic interactions with deletions of individual SJL genes. Deletion of SJL3 gene is synthetically lethal with pik1ts, and deletions of SJL1 or SJL2 genes in pik1ts cells exacerbate the temperature sensitivity, neomycin sensitivity, and defect in invertase secretion. A diminished level of PtdIns(4)P and increased level of PtdIns(4,5)P2 in pik1(ts)sjl1delta and pik1(ts)sjl2delta cells, compared with pik1ts cells, indicate that PtdIns(4)P is specifically required for secretion. Collectively, our results suggest that Pik1p and the Sjl proteins coordinately function to regulate the dynamic phosphorylation-dephosphorylation of the polar heads of phosphoinositides, and this process appears to be important for membrane trafficking pathways.     

Phosphatidylinositol-4,5-bisphosphate-rich plasma membrane patches organize active zones of endocytosis and ruffling in cultured adipocytes

A major regulator of endocytosis and cortical F-actin is thought to be phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] present in plasma membranes. Here we report that in 3T3-L1 adipocytes, clathrin-coated membrane retrieval and dense concentrations of polymerized actin occur in restricted zones of high endocytic activity. Ultrafast-acquisition and superresolution deconvolution microscopy of cultured adipocytes expressing an enhanced green fluorescent protein- or enhanced cyan fluorescent protein (ECFP)-tagged phospholipase Cdelta1 (PLCdelta1) pleckstrin homology (PH) domain reveals that these zones spatially coincide with large-scale PtdIns(4,5)P2-rich plasma membrane patches (PRMPs). PRMPs exhibit lateral dimensions exceeding several micrometers, are relatively stationary, and display extensive local membrane folding that concentrates PtdIns(4,5)P2 in three-dimensional space. In addition, a higher concentration of PtdIns(4,5)P2 in the membranes of PRMPs than in other regions of the plasma membrane can be detected by quantitative fluorescence microscopy. Vesicular structures containing both clathrin heavy chains and PtdIns(4,5)P2 are revealed immediately beneath PRMPs, as is dense F actin. Blockade of PtdIns(4,5)P2 function in PRMPs by high expression of the ECFP-tagged PLCdelta1 PH domain inhibits transferrin endocytosis and reduces the abundance of cortical F-actin. Membrane ruffles induced by the expression of unconventional myosin 1c were also found to localize at PRMPs. These results are consistent with the hypothesis that PRMPs organize active PtdIns(4,5)P2 signaling zones in the adipocyte plasma membrane that in turn control regulators of endocytosis, actin dynamics, and membrane ruffling.     

Regulation of CK2 activity by phosphatidylinositol phosphates

The process of clathrin-coated vesicle (CCV) formation/disassembly involves numerous proteins that act cooperatively. Phosphorylation is an important regulatory mechanism governing protein interactions in CCVs, and many of the core and accessory proteins of the CCV machinery are reversibly phosphorylated in vivo. CK2 is highly enriched in CCVs and is capable of phosphorylating a number of peripheral membrane proteins involved in the process of clathrin-mediated endocytosis. At least some of these phosphorylation events have been shown to be inhibitory for CCV assembly, and CK2 has been shown to be inactive when associated with intact CCVs. Here we show that CCV membranes inhibit CK2 activity even after incubation in trypsin, indicating that a component of the lipid bilayer may be the inhibitory factor. Consistent with this, we showed that liposomes containing phosphatidylinositol phosphates inhibit the activity of CK2 and that CK2 binds to those liposomes. Notably, liposomes containing phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), a component of CCVs, bind CK2 and inhibit its activity. Furthermore, we showed that the binding of CK2 to PtdIns(4,5)P(2)-containing liposomes is via the active site of CK2, thus providing a molecular explanation for the inhibition of CK2 activity when it is bound to PtdIns(4,5)P(2)-containing liposomes. Thus CK2 is inactive in CCVs because of the fact that it is bound to the CCV membrane via an interaction between PtdIns(4,5)P(2) in the CCV membrane and the active site in CK2.     

The dual PH domain protein Opy1 functions as a sensor and modulator of PtdIns(4,5)P₂ synthesis

Phosphatidylinositol-4,5-bisphosphate, PtdIns(4,5)P(2), is an essential signalling lipid that regulates key processes such as endocytosis, exocytosis, actin cytoskeletal organization and calcium signalling. Maintaining proper levels of PtdIns(4,5)P(2) at the plasma membrane (PM) is crucial for cell survival and growth. We show that the conserved PtdIns(4)P 5-kinase, Mss4, forms dynamic, oligomeric structures at the PM that we term PIK patches. The dynamic assembly and disassembly of Mss4 PIK patches may provide a mechanism to precisely modulate Mss4 kinase activity, as needed, for localized regulation of PtdIns(4,5)P(2) synthesis. Furthermore, we identify a tandem PH domain-containing protein, Opy1, as a novel Mss4-interacting protein that partially colocalizes with PIK patches. Based upon genetic, cell biological, and biochemical data, we propose that Opy1 functions as a coincidence detector of the Mss4 PtdIns(4)P 5-kinase and PtdIns(4,5)P(2) and serves as a negative regulator of PtdIns(4,5)P(2) synthesis at the PM. Our results also suggest that additional conserved tandem PH domain-containing proteins may play important roles in regulating phosphoinositide signalling.     

Movin' on up: the role of PtdIns(4,5)P(2) in cell migration

Cell migration requires the coordination of many biochemical events, including cell-matrix contact turnover and cytoskeletal restructuring. Recent advances further implicate phosphatidylinositol(4,5)-bisphosphate [PtdIns(4,5)P(2)] in the control of these events. Many proteins that are crucial to the assembly of the migration machinery are regulated by PtdIns(4,5)P(2). Coordinated synthesis of PtdIns(4,5)P(2) at these sites is dependent on the precise targeting of the type I phosphatidylinositol phosphate kinases (PIPKs). Two PIPKI isoforms target to, and generate, PtdIns(4,5)P(2) at membrane ruffles and focal adhesions during cell migration. Here, we discuss our current understanding of PtdIns(4,5)P(2) in the regulation of cell responses to migratory stimuli and how the migrating cell controls PtdIns(4,5)P(2) availability.     

Analysis of yeast endocytic site formation and maturation through a regulatory transition point

The earliest stages of endocytic site formation and the regulation of endocytic site maturation are not well understood. Here we analyzed the order in which the earliest proteins are detectable at endocytic sites in budding yeast and found that an uncharacterized protein, Pal1p/Ydr348cp, is also present at the initial stages of endocytosis. Because Ede1p (homologue of Eps15) and clathrin are the early-arriving proteins most important for cargo uptake, their roles during the early stages of endocytosis were examined more comprehensively. Ede1p is necessary for efficient recruitment of most early-arriving proteins, but not for the recruitment of the adaptor protein Yap1802p, to endocytic sites. The early-arriving proteins, as well as the later-arriving proteins Sla2p and Ent1/2p (homologues of Hip1R and epsins), were found to have longer lifetimes in CLC1-knockout yeast, which indicates that clathrin light chain facilitates the transition from the intermediate to late coat stages. Cargo also arrives during the early stages of endocytosis, and therefore its effect on endocytic machinery dynamics was investigated. Our results are consistent with a role for cargo in regulating the transition of endocytic sites from the early stages of formation to the late stages during which vesicle formation occurs.     

Regulatory mechanisms of dynamin-dependent endocytosis

Extensive studies on endocytosis in the last decade have resulted in identification of several key molecules that function in clathrin- and dynamin-dependent endocytosis. Most endocytic molecules contain multiple binding motifs that mediate protein-protein or protein-lipid interactions, which must be modulated spatially and temporally during endocytosis. Regulation of these interactions is the molecular basis of regulatory mechanisms involved in endocytosis. This review first describes current models of the mechanism of dynamin-dependent fission, then introduces several mechanisms that modulate dynamin GTPase activity and dynamin-dependent vesicle formation. Such mechanisms include regulation by inositol phospholipids, especially phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)], and their metabolism. It concludes by describing the regulation of dynamin 1 by its binding partner, amphiphysin 1, and regulation by cyclin-dependent kinase 5 (Cdk5)-dependent phosphorylation of dynamin 1 and amphiphysin 1. These mechanisms help endocytic molecules to function properly, and cooperatively regulate dynamin-dependent endocytosis.     

Essential roles of phosphatidylinositol 4-phosphate phosphatases Sac1p and Sjl3p in yeast autophagosome formation

Autophagy is regulated by phosphoinositides. We have previously shown that phosphatidylinositol 4-phosphate (PtdIns(4)P) is localized in the autophagosomal membrane. Additionally, in yeast cells, phosphatidylinositol 4-kinases Pik1p and Stt4p play important roles in the formation of the autophagosome and its fusion with the vacuole, respectively. In this study, we analyzed the primary role of PtdIns(4)P phosphatases in yeast autophagy. The PtdIns(4)P labeling densities in the membranes of the vacuoles, mitochondria, nucleus, endoplasmic reticulum, and plasma membrane dramatically increased in the phosphatase deletion mutants sac1? and sjl3?, and the temperature-sensitive mutant sac1^ts/sjl3? at the restrictive temperature. GFP-Atg8 processing assay indicated defective autophagy in the sac1? and sac1^ts/sjl3? mutants. In contrast to the localization of PtdIns(4)P in the luminal leaflet of autophagosomal membranes in the wild-type yeast, PtdIns(4)P was localized in both the luminal and cytoplasmic leaflets of the autophagosomal membranes in the sac1? strain. In addition, the number of autophagic bodies in the vacuole significantly decreased in the sac1? strain, although autophagosomes were present in the cytoplasm. In the sac1^ts/sjl3? strain, the number of autophagosomes in the cytoplasm dramatically decreased at the restrictive temperature. Considering that the numbers of autophagosomes and autophagic bodies in the sjl3? strain were comparable to those in the wild-type yeast, we found that the autophagosome could not be formed when PtdIns(4)P phosphatase activities of both Sac1p and Sjl3p were diminished. Together, these results indicate that the turnover of PtdIns(4)P by phosphatases is essential for autophagosome biogenesis.     

Elimination of host cell PtdIns(4,5)P(2) by bacterial SigD promotes membrane fission during invasion by Salmonella

Salmonella invades mammalian cells by inducing membrane ruffling and macropinocytosis through actin remodelling. Because phosphoinositides are central to actin assembly, we have studied the dynamics of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P(2)) in HeLa cells during invasion by Salmonella typhimurium. Here we show that the outermost parts of the ruffles induced by invasion show a modest enrichment in PtdIns(4,5)P(2), but that PtdIns(4,5)P(2) is virtually absent from the invaginating regions. Rapid disappearance of PtdIns(4,5)P(2) requires the expression of the Salmonella phosphatase SigD (also known as SopB). Deletion of SigD markedly delays fission of the invaginating membranes, indicating that elimination of PtdIns(4,5)P(2) may be required for rapid formation of Salmonella-containing vacuoles. Heterologous expression of SigD is sufficient to promote the disappearance of PtdIns(4,5)P(2), to reduce the rigidity of the membrane skeleton, and to induce plasmalemmal invagination and fission. Hydrolysis of PtdIns(4,5)P(2) may be a common and essential feature of membrane fission during several internalization processes including invasion, phagocytosis and possibly endocytosis.     

The yeast synaptojanin-like proteins control the cellular distribution of phosphatidylinositol (4,5)-bisphosphate

Phosphoinositides (PI) are synthesized and turned over by specific kinases, phosphatases, and lipases that ensure the proper localization of discrete PI isoforms at distinct membranes. We analyzed the role of the yeast synaptojanin-like proteins using a strain that expressed only a temperature-conditional allele of SJL2. Our analysis demonstrated that inactivation of the yeast synaptojanins leads to increased cellular levels of phosphatidylinositol (3,5)-bisphosphate and phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)), accompanied by defects in actin organization, endocytosis, and clathrin-mediated sorting between the Golgi and endosomes. The phenotypes observed in synaptojanin-deficient cells correlated with accumulation of PtdIns(4,5)P(2), because these effects were rescued by mutations in MSS4 or a mutant form of Sjl2p that harbors only PI 5-phosphatase activity. We utilized green fluorescent protein-pleckstrin homology domain chimeras (termed FLAREs for fluorescent lipid-associated reporters) with distinct PI-binding specificities to visualize pools of PtdIns(4,5)P(2) and phosphatidylinositol 4-phosphate in yeast. PtdIns(4,5)P(2) localized to the plasma membrane in a manner dependent on Mss4p activity. On inactivation of the yeast synaptojanins, PtdIns(4,5)P(2) accumulated in intracellular compartments, as well as the cell surface. In contrast, phosphatidylinositol 4-phosphate generated by Pik1p localized in intracellular compartments. Taken together, our results demonstrate that the yeast synaptojanins control the localization of PtdIns(4,5)P(2) in vivo and provide further evidence for the compartmentalization of different PI species.     

Regulation of Fab1 phosphatidylinositol 3-phosphate 5-kinase pathway by Vac7 protein and Fig4, a polyphosphoinositide phosphatase family member

The Saccharomyces cerevisiae FAB1 gene encodes the sole phosphatidylinositol 3-phosphate [PtdIns(3)P] 5-kinase responsible for synthesis of the polyphosphoinositide PtdIns(3,5)P(2). VAC7 encodes a 128-kDa transmembrane protein that localizes to vacuolar membranes. Both vac7 and fab1 null mutants have dramatically enlarged vacuoles and cannot grow at elevated temperatures. Additionally, vac7Delta mutants have nearly undetectable levels of PtdIns(3,5)P(2), suggesting that Vac7 functions to regulate Fab1 kinase activity. To test this hypothesis, we isolated a fab1 mutant allele that bypasses the requirement for Vac7 in PtdIns(3,5)P(2) production. Expression of this fab1 allele in vac7Delta mutant cells suppresses the temperature sensitivity, vacuolar morphology, and PtdIns(3,5)P(2) defects normally exhibited by vac7Delta mutants. We also identified a mutant allele of FIG4, whose gene product contains a Sac1 polyphosphoinositide phosphatase domain, which suppresses vac7Delta mutant phenotypes. Deletion of FIG4 in vac7Delta mutant cells suppresses the temperature sensitivity and vacuolar morphology defects, and dramatically restores PtdIns(3,5)P(2) levels. These results suggest that generation of PtdIns(3,5)P(2) by the Fab1 lipid kinase is regulated by Vac7, whereas turnover of PtdIns(3,5)P(2) is mediated in part by the Sac1 polyphosphoinositide phosphatase family member Fig4.     

Kinetic Changes of Ptdins (3,4,5) P3 within Fast and Slow Turnover Rates of Focal Adhesion

Background:The assembly and disassembly of the focal adhesions (FA) components occurs throughout life cycle of adhesion, with conservation of balance between removal and recruitment rate during temporal stages. Previous studies have demonstrated that phosphotidyilinositols play a role in regulating FA turnover. However, a little attention has been given to quantify the dynamics changes of Phosphatidylinositol 3,4,5-trisphosphate (PtdIns (3,4,5) P3) within and during fast and slow turnover rates of FA.Methods:In this study, we developed a protein purification MDA-MB-231 breast cancer cell line was used as a model in this study due to high metastatic and motile. These cells were co-transfected with GFP- paxillin/vinculin, as FA marker, and the GFP/mCherry-Btk-PH, as a biosensor to visualize PtdIns (3,4,5) P3. Confocal time-lapse images were used to monitor changes or differences in the local generation of PtdIns (3,4,5) P3 within and during assembly and disassembly of FA. Following transfection, immunostaining was used to examine the spatial co-localization between FA and PtdIns (3,4,5) P3.Results:Our data demonstrated that PtdIns (3,4,5) P3 co-localized with FAs and increase during assembly and decline during disassembly of FA which exhibits slow turnover rates and was in a constant level during assembly and disassembly of FA that displays fast turnover rates.DiscussionOur result suggested that the dynamic changes of PtdIns (3,4,5) P3, it may depend on components undergo turnover, such that early, nascent FA displays fast turnover rates and mature FA exhibits slow turnover rates. Thus, the local enrichment of PtdIns (3,4,5) P3 enhances FA assembly and disassembly activation.Key Words: Cancer cell migration, Focal adhesion turnover, MDA-MB-231 cell line, PtdIns (3,4,5) P3     

Signalling through phospholipase C interferes with clathrin-mediated endocytosis

We investigated if phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) hydrolysis by phospholipase C activation through cell surface receptors would interfere with clathrin-mediated endocytosis as recruitment of clathrin assembly proteins is PtdIns(4,5)P2-dependent. In the WKPT renal epithelial cell line, endocytosed insulin and beta2-glycoprotein I (beta2gpI) were observed in separate compartments, although endocytosis of both ligands was clathrin-dependent as demonstrated by expression of the clathrin-binding C-terminal domain of AP180 (AP180-C). The two uptake mechanisms were different as only insulin uptake was reduced when the mu2-subunit of the adaptor complex AP-2 was silenced by RNA interference. ATP receptors are expressed at the apical surface of renal cells and, thus, we examined the effect of extracellular ATP on insulin and beta2gpI uptake. ATP stimulated phospholipase C activity, and also suppressed uptake of insulin, but not beta2gpI. This effect was reversed by the PLC inhibitor U-73122. In polarized cell cultures, insulin uptake was apical, whereas beta2gpI uptake was through the basolateral membrane, thus providing an explanation for selective inhibition of insulin endocytosis by ATP. Taken together, these results demonstrate that stimulation of apical G-protein-coupled P2Y receptors, which are coupled to phospholipase C activation diminishes clathrin-mediated endocytosis without interfering with basolateral endocytic mechanisms.