Great Expectations for PIP: Phosphoinositides as Regulators of Signaling During Development and Disease

Developmental Cell

Review Great Expectations for PIP: Phosphoinositides as Regulators of Signaling During Development and Disease Lara C. Skwarek1,2 and Gabrielle L. Boulianne1,2,* 1The

Hospital for Sick Children, Program in Developmental and Stem Cell Biology, Toronto, ON M5G 1X8, Canada of Molecular Genetics, University of Toronto, Toronto, ON M5S 1A8, Canada *Correspondence: [email protected] DOI 10.1016/j.devcel.2008.12.006 2Department

Phosphoinositides function as signaling precursors as well as regulators and scaffolds of signaling molecules required for important cellular processes such as membrane trafficking. Although a picture of the biochemical and cell biological functions of phosphoinositides is emerging, less is known about how these functions impact signaling on a broader scale during development. This review summarizes recent work on the role of phosphoinositides in developmental signaling and in a number of diseases and developmental disorders. The development of a multicellular organism requires the integration of multiple cellular processes including differentiation, migration, proliferation, and growth. Each of these events must be precisely controlled and coordinated from the cellular to the tissue level. A surprisingly short list of signaling pathways is responsible for development, and the pathways are used repeatedly to create such distinct features as the limbs and brain. Signaling must therefore be subject to precise temporal and spatial regulation. During the last decade, it has become clear that basic cellular processes play important roles in the regulation of signaling, including cytokinesis, cell polarity, translation, and membrane trafficking. Moreover, this work has shown that lipids such as the polyphosphatidylinositols (PIPs or phosphoinositides) are essential for the precise regulation of signaling. PIPs can function as signaling precursors themselves or as regulators and scaffolds of signaling molecules. While the biochemical and cell biological functions of PIPs within specific cell types is a growing field of study, less emphasis has been placed on how PIPs impact signaling on a broader scale during development. This review will summarize what is known about the role of PIPs during the development of a multicellular organism and describe how perturbations in PIP function can lead to human disease. Introduction to Phosphoinositides Phosphoinositides comprise a family of phosphorylated derivatives of the membrane lipid phosphatidylinositol (PI). They are glycerophospholipids that contain a hydrophobic diacylglycerol (DAG) backbone esterified to a polar inositol headgroup (Figure 1). Three of the five hydroxyl residues on the inositol ring can be phosphorylated individually or in combination to give seven different phosphorylated phosphatidylinositols, or PIPs. Phosphorylation and dephosphorylation by lipid kinases and phosphatases, respectively, can rapidly interconvert PIP species, contributing to the dynamic production of specific PIP lipids within different cellular compartments (Figure 1). Phosphoinositides make up a very small proportion of the lipids present within cellular membranes. PI makes up 4% of 12 Developmental Cell 16, January 20, 2009 ª2009 Elsevier Inc.

cellular membrane phospholipids, and the other phosphorylated PIs together comprise 1% (Lemmon, 2008; Mulgrew-Nesbitt et al., 2006). Classical PIP signaling results from the hydrolysis of PI(4,5)P2 by phospholipase C isoforms, resulting in the production of diacylglycerol (DAG) and inositol-3,4,5-trisphosphate (IP3), which act as second messengers. Phospholipase C (PLC) activity is stimulated by signaling molecules such as G protein coupled receptors (GPCR), receptor tyrosine kinases, Ras-like GTPases, and calcium, thus linking the hydrolysis of PI(4,5)P2 to a diverse set of cellular signals (Oude Weernink et al., 2007). A second, very well-studied phosphoinositide signaling pathway results from the activation of PI 3-kinases (PI3Ks). Downstream of stimulation by growth factors, hormones, or other cellular signals, PI3Ks phosphorylate PI(4,5)P2 and PI(4)P to produce PI(3,4,5)P3 and PI(3,4)P2, respectively. These lipids then activate the downstream protein kinase B (PKB)/Akt signaling pathway. PKB/Akt signaling is important for cell growth, survival, proliferation, and motility and also provides crosstalk between signaling pathways, including those activated by growth factors, Insulin, Notch, BMP, and Shh (Cantley, 2002; Riobo et al., 2006; Taniguchi et al., 2006; Tian et al., 2005). PIPs also play a role in the specification and maturation of various intracellular compartments and can act as scaffolds for the recruitment of proteins with specific PIP binding domains (Figure 2). Many different globular PIP binding domains have been identified, including PH, C2, PX, FYVE, ENTH/ANTH, FERM, PTB, and PDZ domains (reviewed in Cho and Stahelin, 2005; Lemmon, 2008). The mechanics of membrane recruitment through PIP binding can take on various forms and includes domains or motifs that bind through a combination of mechanisms, including nonspecific electrostatic interactions, membrane insertion, oligomerization, and calcium binding. Several recent reviews have provided excellent in-depth analyses of the mechanisms of PIP binding by different domains (Cho and Stahelin, 2005; Lemmon, 2008; McLaughlin and Murray, 2005; Mulgrew-Nesbitt et al., 2006). PIP binding is important for recruitment of proteins to specific membranes or domains of

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membranes, such as the localization of FYVE domain containing trafficking proteins to endosomes. However, it is important to emphasize that PIP binding can also play a direct role in regulating protein-protein interactions and catalytic activities (see for example Corgan et al., 2004; Takei et al., 2005; Wang et al., 2007). Therefore, understanding the role of PIP binding in protein function will require an in-depth analysis of how PIPs specifically affect each unique binding partner. Cellular Effects of Phosphoinositides during Development Phosphoinositides have been extensively studied at the cellular level. They have been implicated in regulation of membrane trafficking, cell polarity, motility, chemotaxis, and transcription. One can imagine how all of these processes would be important to the survival of a single-celled organism. Importantly, however, PIPs also regulate adhesion, proliferation, apoptosis, and the transmission of signaling in response to growth factors, hormones, and morphogens. Each of these classes of factors is important for development, and signaling in response to each must be precisely coordinated. It is clear, therefore, that the impact of PIPs on any one of these processes will impact the development of a whole organism. One prime example is the role of PI(3,4,5)P3 (PIP3) in cell polarity and migration. This has been largely studied in Dictyostelium and mammalian neutrophils and is reviewed elsewhere (Janetopoulos and Firtel, 2008; Kolsch et al., 2008). Given the emphasis on single-cell chemotaxis in the literature, it remained

Figure 1. Metabolism of Phosphatidylinositol (A) Structure of phosphatidylinositol (PI). Positions 3, 4, and 5 on the inositol ring can be phosphorylated to produce seven different phosphoinositide species: PI(3)P, PI(4)P, PI(5)P, PI(3,4)P2, PI(4,5)P2, PI(3,5)P2 and PI(3,4,5)P3. (B) Metabolic pathways interconverting the different phosphoinositides are shown. Lipid kinases phosphorylate the inositol ring at specific positions to produce more highly phosphorylated forms, while lipid phosphatases dephosphorylate the inositol ring. Question marks indicate speculative pathways for which no enzymes have been identified.

unclear how related mechanisms might influence the behaviors of larger fields of cells. Recently, however, a role for PIP3 during Danio rerio (zebrafish) gastrulation was identified (Montero et al., 2003) and a clear connection between the role of PIP3 in individual cells and the regulation of cell movements during morphogenesis became apparent. Gastrulation and Tissue Rearrangements Gastrulation requires internalization of mesendodermal cells from the surface of the embryo. These cells migrate inside the ectoderm and organize to form the two internal germ layers: mesoderm and endoderm. In zebrafish, chicken, and mice, mesendodermal cells undergo an epithelial-to-mesenchymal transition to facilitate migration into the interior of the embryo. These cells then migrate as a group toward the animal pole and give rise to the prechordal plate and notochord. Ingression is triggered in response to Nodal signaling, while migration is controlled by a noncanonical Wnt planar-cell polarity pathway required for proper direction of migration (Carmany-Rampey and Schier, 2001; Ulrich et al., 2003). PDGF signaling is also required for Xenopus gastrulation, suggesting a possible role for PI3K activation in this process, since PDGF signaling stimulates PIP3 production (Ataliotis et al., 1995; Auger et al., 1989). Treatment of zebrafish embryos with the PI3K inhibitor LY294002 and interference with type I PI3K activity through the injection of dominant-negative PI3K RNA resulted in loss of PIP3 production and a shorter and broader body axis that is indicative of defects in gastrulation, including reduced convergence and extension movements (Montero et al., 2003). On a cellular level, ingressing cells were found to be less elongated, have fewer cellular processes, and reduced velocity of cell migration. Interestingly, the direction of cell migration was not overly affected, suggesting that alternate pathways are involved in establishing the direction of movement. Expression of a fluorescently tagged PH domain from Akt/PKB demonstrated accumulation of PIP3 at the leading edge of these cells, correlating with a concentration of GFP-actin in the same location. This suggests that PI3K acts to generate a localized increase of PIP3 at the leading edge, which has immediate effects on the Developmental Cell 16, January 20, 2009 ª2009 Elsevier Inc. 13

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Figure 2. Simplified Schematic of the Contribution of Different Phosphoinositides to the Trafficking of a Hypothetical Receptor A receptor transits through the Golgi to the cell surface. PI(4)P plays an important role in Golgi-mediated trafficking (1). PI(4,5)P2, PI(4)P and PI(3)P are found at the plasma membrane. PI(3,4)P2 and PI(3,4,5)P3 are generated at the plasma membrane through stimulation of various signaling pathways. Endocytosis of the receptor depends upon PI(4,5)P2 and possibly PI(3)P (2). The receptor is trafficked to the endosome, which depends upon PI(3)P for proper structure and function (3). The receptor can be trafficked from the endosome to recycling endosomes and from there back to the plasma membrane. PIPs involved specifically in recycling have yet to be identified (4). Alternatively, the receptor can traffic from the endosome to the multi-vesicular body (MVB). MVB morphology and function depends on PI(3,5)P2 and PI(5)P (5). The receptor can traffic from the MVB to the lysosome for degradation or back to the endosome or cell surface (data not shown). PI(3,5)P2 and PI(5)P are important for lysosomal biogenesis (6).

actin cytoskeleton. This was blocked by administration of a PDGF inhibitor, suggesting that PI3K is indeed responding to PDGF in these cells (Montero et al., 2003). These results demonstrate that the effects of PIP3 on single-cell polarization and migration also influence larger scale morphogenesis and that the activation of PI3K downstream of developmental signaling pathways can have tissue-specific effects during development. Axial Development The Wnt signaling pathway is essential for embryonic axial development and also contributes to bone formation and axon guidance in vertebrates through the activation of PI3K/Akt downstream of calcium signaling (Tu et al., 2007; Wolf et al., 2008). Wnt signaling involves a network of canonical and noncanonical pathways. The canonical pathway acts via LipoproteinReceptor-Related Protein 6 (LRP6) and Dishevelled to promote accumulation of cytoplasmic b-catenin, which translocates to the nucleus where it acts together with TCF/LEF transcription factors to regulate expression of Wnt effector genes. The noncanonical Wnt signaling pathway has been shown to activate phosphatidylinositol signaling and calcium release through the activation of G protein-coupled receptors and may directly link Wnt signaling to cytoskeletal dynamics (Slusarski et al., 1997). Pan 14 Developmental Cell 16, January 20, 2009 ª2009 Elsevier Inc.

et al. (2008) recently identified the lipid kinases PI4KIIa and PIP5KIb as being important for the canonical pathway. Through an elegant series of biochemical and cell biological studies, they demonstrated that PIP5KIb interacts with, and is activated by, Dishevelled, which results in an increase in PI(4,5)P2 (PIP2) production downstream of Wnt. They then went on to show that PIP2 promotes LRP6 activation and that PI4KIIa and PIP5KIb control Wnt signaling in Xenopus embryonic axial development. Together, these results indicate a role for regulated PIP metabolism in the feedback loop between Dishevelled and LRP6 that is required for Wnt signaling (see also Bilic et al., 2007). Blastoderm Survival PI3K signaling and PIP3 are also known to play important roles in cell growth and survival (reviewed in Cantley, 2002; Hawkins et al., 2006). A recent study by Halet et al. (2008) has revealed a previously uncharacterized role for PIP3 in the development and survival of early mammalian embryos. Mammalian preimplantation embryos can develop and survive in vitro in the absence of exogenous growth factors, suggesting that an intrinsic factor can act to maintain proliferation and prevent cell death. Through the use of an isolated GFP-tagged PH domain from the PIP3 binding protein GRP1, PIP3 was found in cells at all preimplantation stages, accumulating at sites of blastomere apposition during all cleavage stages, and in an apical domain up to the 8 cell stage (Halet et al., 2008). PIP3 accumulation was inhibited by loss of E-cadherin, indicating that production of PIP3 at cell-cell contacts depends on this adhesion molecule. Interference with PIP3 production or availability through use of the PI3K inhibitor LY294002, or sequestration with high levels of the PIP3 binding PH domain, interfered with development beyond the 2 cell stage, while later disruption of PIP3 synthesis induced apoptosis in 16–32 cell morulae (Halet et al., 2008). Mouse embryos lacking the type I PI3K p110b subunit die before implantation (Bi et al., 2002), suggesting that this enzyme may be responsible for production of PIP3 during early embryogenesis. Further understanding of the role of PIP3 during early embryo development will require determining how E-cadherin can activate PIP3 production in the absence of growth factors and the downstream effects of PIP3 in the 2 cell embryo at the maternal-zygotic transition. Cell and Tissue Polarity The development of multiple different cell types and tissues requires the establishment and maintenance of intrinsic cell polarity. This is especially important in epithelial cells, neurons, and dividing zygotes. While the role of conserved polarity complexes has long been established in this process, a growing body of work demonstrates an essential role for phosphoinositides in the regulation of these polarity-determining complexes and in specification of membrane-domain identity. Recent work has revealed that Par3 binds to phosphoinositides through its PDZ2 domain and that PIP binding regulates membrane localization of Par3 and is required for epithelial cell polarization (Wu et al., 2007). While Par3 shows promiscuous PIP binding in vitro, a chimeric Par3 protein containing the PLC-d PIP2 binding PH domain in place of PDZ2 can properly localize to tight junctions and establish apical-basal polarity, suggesting that Par3 binds PIP2 in vivo (Wu et al., 2007). Interestingly, both vertebrate Par3 and the Drosophila Par3 homolog Bazooka interact directly with the PIP3 lipid phosphatase

Developmental Cell

Review PTEN (von Stein et al., 2005; Wu et al., 2007). In Drosophila epithelial cells and neuroblasts, Bazooka is required for PTEN localization to the apical cell cortex in a region colocalizing with enriched PIP2 (Pinal et al., 2006; von Stein et al., 2005). PTEN may therefore locally alter the balance between PIP2 and PIP3 in the plasma membrane, possibly contributing to differences in the lipid composition of apical versus basolateral membrane domains. In support of this, the PIP3 sensor GFPPH-Akt localizes specifically to the basolateral membrane of MDCK cells, and PIP3 can regulate the formation of the basolateral plasma membrane in cultured epithelial cells (GassamaDiagne et al., 2006). Indeed, another Par3 complex component, atypical protein kinase C (aPKC), is also required for the development of basolateral-specific PIP3 expression, though not for the maintenance of this asymmetry (Takahama et al., 2008). Conversely, PIP2 is localized to the apical membrane in MDCK cells and Drosophila epithelia and is required for the localization of PIP2 binding proteins such as Annexin2 and Bitesize to the apical surface, where they recruit actin binding proteins like Cdc42 and Moesin to control actin dynamics during cell-cell junction and lumen formation (Martin-Belmonte et al., 2007; Pilot et al., 2006). Taken together, these data suggest that the polarity determining complexes may function in part by integrating lipid metabolism and actin dynamics during development of epithelia, and additional work suggests that this may hold true for generation of neuronal polarity as well (Shi et al., 2003). Proper establishment and maintenance of cell polarity and cellular junctions is essential for signaling and morphogenetic movements such as gastrulation, and loss of these connections can contribute to epithelial-mesenchymal transitions and metastasis in tumors. It therefore remains an important goal to understand the role of PIPs in both the development and maintenance of epithelial cell polarity (Lee and Vasioukhin, 2008). Asymmetric Cell Division The cell polarity complexes are also involved in regulating the localization of cell-fate determinants and in spindle alignment during asymmetric cell divisions (McCarthy and Goldstein, 2006). A recent study has demonstrated a role for the C. elegans sole PI(4)P 5-kinase PPK-1 in spindle positioning during asymmetric cell division of the 1 cell embryo. Specifically, asymmetric localization of PPK-1 downstream of the PAR proteins is required for posterior localization of LIN-5 and GPR-1/2, two conserved proteins required for asymmetric spindle positioning and dynein-mediated pulling forces (Panbianco et al., 2008). Furthermore, as asymmetric localization of LIN-5 and GPR-1/2 homologs is required for Drosophila neuroblast development and spindle orientation in the mammalian brain (McCarthy and Goldstein, 2006; Sanada and Tsai, 2005), it is likely that asymmetries in PIP2 production may also play a role during these processes. Phosphoinositides and Intracellular Trafficking Regulate Developmental Signaling All of the examples discussed thus far focus on short-range or intracellular signaling events. The following section describes how the interplay between phosphoinositides, intracellular trafficking, and hormone function can amplify single-cell polarity into directional signals that control multicellular patterning.

Hormonal Control of Plant Development The phytohormone auxin is essential for cell-type specification, cell elongation, and cell proliferation in multiple tissues within the developing plant, and it is becoming clear that phosphoinositides are crucial regulators of both auxin transport and auxin signaling itself. Auxin is highly mobile and moves throughout the plant using a process known as polar auxin transport (PAT). The local cellular concentrations of auxin are directly translated into a transcriptional response by the interactions between auxin and E3 ubiquitin ligase complexes that target auxin-dependent transcriptional effectors, resulting in immediate changes in gene expression. These local cellular concentrations of auxin result from regulation of PAT by auxin influx and efflux carriers such as AtAUX and AtPIN1/2, thereby linking auxin transport directly to auxin signaling output (Hardtke et al., 2007). Endocytic and phosphoinositide-based regulation of PAT is complex, involving contributions from pathways that include PIP2 hydrolysis and calcium signaling (Hardtke et al., 2007), PI(3)P-dependent recycling by Sorting nexin 1-positive endosomes (Jaillais et al., 2006), and sterol-based endocytic maintenance of cell polarity (reviewed in Men et al., 2008). Interestingly, some of these same factors are also regulated by auxin and act as downstream effectors of auxin signaling (Ettlinger and Lehle, 1988). Multiple feedback loops therefore exist between auxin and phosphoinositide signaling, controlling both signal strength and directionality. Endocytic Regulation of Receptor Tyrosine Kinases The vertebrate class II PI3K PI3K-C2-b is recruited to growth factor receptors upon ligand stimulation and preferentially stimulates the production of PI(3)P from PI (Arcaro et al., 2000). PI(3)P plays an important role in membrane trafficking and early endosome biogenesis (Cho and Stahelin, 2005; Lemmon, 2008), and there are many examples in the literature supporting a role for endocytic regulation in determining the strength and duration of EGFR signaling (reviewed in Hoeller et al., 2005; Peschard and Park, 2003) (Chen and De Camilli, 2005; Sigismund et al., 2008). Interestingly, type II PI3Ks, including PI3K-C2-b, bind clathrin, and clathrin binding increases PI3K activity (Wheeler and Domin, 2006). These interactions may therefore bridge activated growth factor receptors and clathrin and promote trafficking through the localized production of PI(3)P. Indeed, there is in vivo evidence that the sole Drosophila class II PI3K downregulates EGFR signaling in several developmental contexts and may also promote Notch signaling (MacDougall et al., 2004). Interestingly, the only Drosophila type I PI3K, which produces PI(3,4,5)P3 and regulates downstream Akt/PKB activation, does not result in patterning defects, displaying defects only in growth. This indicates that the class I and II PI3Ks target distinct pathways in Drosophila and that these functions can be clearly distinguished in vivo. In support of a role for increased PIP production in the promotion of endocytosis, disruption of Drosophila phosphocholine cytidylyltransferase 1 (CCT1) was shown to decrease cellular phosphatidylcholine levels with a subsequent increase in phosphatidylinositol (Weber et al., 2003). This disruption in cellular lipid balance promotes higher levels of endocytosis and disrupts EGFR and Notch signaling due to a reduction of receptors at the cell surface. Intriguingly, expression of CCT1 is developmentally Developmental Cell 16, January 20, 2009 ª2009 Elsevier Inc. 15

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Review regulated and affected by changes in both TGFb and EGFR signaling. This suggests that levels of phosphatidylinositol within a particular cell are subject to developmental regulation and participate in a feedback mechanism involved in regulation of signaling through the control of lipid precursors required for membrane trafficking. As a result of the unique role that membrane trafficking plays in individual signaling pathways, it is becoming clear that PIPs and PIP binding proteins also play unique roles that are specific to each signaling context. Phosphoinositides and the Regulation of Notch Signaling Notch signaling is emerging as a model pathway for the study of phosphoinositides during developmental signaling. Notably, regulated membrane trafficking is essential for Notch signaling. Endocytosis of Notch ligands is required in the signaling cell, and endocytosis and trafficking of Notch is important in the responding cell (reviewed in Bray, 2006; Le Borgne, 2006) (Seugnet et al., 1997). While it is unclear how endocytosis of Notch ligands results in signal transduction, it has become evident that signaling requires one of two E3 ubiquitin ligases, Neuralized or Mind bomb, as well as the endocytic adaptor protein Epsin (Bray, 2006; Le Borgne, 2006). Ubiquitination targets transmembrane proteins for endocytosis and also regulates their further trafficking within the endosomal sorting pathway. We previously identified an interaction between Neuralized and PIPs in vitro and demonstrated that this interaction is required for Neuralizedmediated Delta endocytosis in vivo, but not for the ability of Neuralized to interact with Delta nor for its ubiquitin ligase activity (Skwarek et al., 2007). This work suggests that interactions between Neuralized and PI(4,5)P2 regulate unanticipated aspects of endocytosis downstream of ubiquitination. It will be interesting to see if type II PI3K activity (see above, related to MacDougall et al., 2004) is required in the signal sending cell for ligand endocytosis and downstream Notch signaling. After ligand binding and ectodomain shedding, the remaining portion of the Notch receptor becomes a constitutive substrate for g-secretase cleavage (Figure 3). There is a growing body of evidence to suggest that the lipid microenvironment regulates g-secretase activity and possibly substrate specificity (Hur et al., 2008; Osenkowski et al., 2008; Vetrivel et al., 2004; Wrigley et al., 2005). Landman et al. (2006) observed a potential role for PIP2 in g-secretase function, as the levels of a cleavage product derived from a g-secretase target were inversely proportional to the levels of cellular PIP2. This is further supported by recent evidence suggesting that PIP2, along with other phosphoinositides, inhibits g-secretase activity in vitro (Osawa et al., 2008). Indeed, the addition of PIP2 to g-secretase containing microsomal fractions obtained from CHO cells reduced production of a FLAG-tagged Notch cleavage product in a concentrationdependent manner, while addition of phosphatidylcholine stimulated cleavage. PIPs may therefore contribute to signaling by regulating the cellular domain in which g-secretase is most active, either within a specific lipid microdomain or a specialized endosomal compartment. Whether g-secretase cleaves its targets at the plasma membrane or within endosomes is controversial, and there is mounting evidence that endocytosis may be required for Notch cleavage (Gupta-Rossi et al., 2004; Vaccari et al., 2008). Perhaps an inhibitory effect of PIP2 and other PIPs on g-secretase prevents cleavage at the plasma 16 Developmental Cell 16, January 20, 2009 ª2009 Elsevier Inc.

Figure 3. Schematic of the Regulation of Notch Signaling by Phosphoinositides Signaling initiates when a ligand on one cell (DSL) interacts with the Notch receptor on a neighboring cell (N). Upon ligand binding, an extracellular metalloprotease of the TACE/ADAM family releases the Notch extracellular domain from the membrane inserted intracellular domain. Upon ectodomain shedding, the ligand and receptor ectodomain are endocytosed into the signal sending cell. The remaining membrane inserted Notch intracellular domain acts as a constitutive cleavage substrate for the Presenilin containing g-secretase complex. Intramembrane cleavage of Notch by g-secretase releases the Notch intracellular signaling (NIC) fragment, which translocates to the nucleus. In the nucleus, NIC interacts with the CSL family of transcription factors, relieving CSL-mediated transcriptional repression and activating transcription of downstream effectors. Phosphoinositides have been implicated in regulation of Notch signaling at multiple points during signaling and receptor trafficking. The PIP binding proteins Neuralized and Epsin are important for endocytosis of ligand and transduction of the signal (1). PIPs regulate activity of the g-secretase complex (2). PIPs regulate the rate of Notch receptor endocytosis and its availability at the cell surface (3). The PIP binding Nedd4 family of E3 ubiquitin ligases regulate trafficking of Notch through endosomes (Wilkin et al., 2004) (4). The PIP binding protein Lgd regulates trafficking of Notch from a late endosomal compartment (in which the g-secretase complex may constitutively cleave Notch) to the lysosome for degradation, therefore playing an important role in signal attenuation (5).

membrane, thereby requiring that both protease and substrate first traffic to a compartment lacking inhibitory lipids. The presence of compartments that promote g-secretase activity is supported by analysis of Lethal giant discs (Lgd) mutants in Drosophila. Lgd contains a C2 domain that binds to monophosphorylated PIPs in vitro (Gallagher and Knoblich, 2006) and is required for trafficking of the Notch receptor to the lysosome. In lgd / mutant tissue, full-length Notch that has not undergone ligand-mediated cleavage becomes trapped

Developmental Cell

Review in a late endocytic compartment from which it can signal, resulting in autonomous activation of Notch signaling within mutant cells (Childress et al., 2006; Gallagher and Knoblich, 2006; Jaekel and Klein, 2006). The lgd / phenotype can be rescued by trapping Notch in an earlier compartment, suggesting that Lgd may normally be required to prevent Notch accumulation in late endosomal compartments where g-secretase activity is constitutive (Wilkin et al., 2008). Along similar lines, it is worth noting that loss of Lgd appears to increase the acidity of endosomes, and there is evidence that the catalytic activity of g-secretase may be enhanced in lower pH environments (Kanwar and Fortini, 2008; Pasternak et al., 2003). This diversity of regulatory mechanisms in Notch signaling highlights the importance of understanding the intimate connections between receptor activation, lipid production, trafficking, and signaling as a basis for the study of lipid-mediated trafficking in other developmental signaling pathways. Phosphoinositides in Disease The previous sections highlight just a few examples of what we are beginning to learn about the diverse roles of phosphoinositides during development. The list of developmental processes that may depend upon regulated phosphatidylinositol metabolism can be expanded to include neurite extension and neuronal development, angiogenesis (Im and Kazlauskas, 2006), cytokinesis (Echard, 2008; Field et al., 2005; Wong et al., 2005), cell cycle regulation (Ho et al., 2008), and flagellar biogenesis (Wei et al., 2008) to name a few. Given their integral roles during development, it is not surprising that the misregulation of PIPs and PIP binding proteins has been implicated in a growing number of diseases and developmental disorders. Mutations in genes that code for proteins that regulate the metabolism of phosphoinositides or act downstream of PIP signaling have been linked to a number of human disorders. Indeed, altered phosphoinositide metabolism and signaling is often causative in cancer (Wymann and Schneiter, 2008). For example, aberrant PI3K signaling occurs in multiple cancers, and PTEN is a known tumor suppressor, while activated Akt/ PKB is an oncogene (Cully et al., 2006). As mentioned earlier, however, a role for PIPs in maintenance of epithelial cell polarity and tight junctions raises questions about how they may contribute to metastatic processes such as epithelial-mesenchymal transition and cell migration (Wodarz and Nathke, 2007). Given the important role of phosphoinositides in the regulation of membrane trafficking, it is not surprising that a disease associated with perturbations in lysosomal enzyme function is linked to mutations that disrupt a PIP2 phosphatase (Ungewickell and Majerus, 1999). Lowe syndrome (or oculocerebrorenal syndrome of Lowe) is an X-linked condition that results in severe mental retardation, growth defects, renal Fancomi syndrome, and eye defects such as lens cataracts, glaucoma, and ultimately blindness (Halstead et al., 2005). Lowe syndrome is caused by mutations in the inositol-5-phosphatase OCRL, and renal cells lacking OCRL show elevated levels of PIP2, suggesting that defects in PIP2 metabolism may be responsible for the disease phenotypes (Zhang et al., 1998). Interestingly, OCRL has been shown to bind clathrin and localizes to endosomes and the Golgi, where it may regulate receptor-mediated endocytosis and trafficking between the trans-Golgi network (TGN) and

endosomes (Choudhury et al., 2005; Erdmann et al., 2007). The cellular causes of the varied disease phenotypes are not yet understood; however, analysis of the function of OCRL in trafficking and lipid metabolism is beginning to shed some light on possible mechanisms. Charcot-Marie-Tooth (CMT) disease is another hereditary disorder that is caused, at least in part, by defects in phosphoinositide metabolism. CMT comprises a heterogeneous group of disorders that affect peripheral nerves, leading to muscular atrophy and weakness in the distal limbs. It is one of the most common inherited neurological disorders, and several different subtypes of CMT are due to mutations in lipid phosphatases involved in dephosphorylation of PI(3,5)P2 at either the D-3 or -5 position (Chow et al., 2007; Robinson et al., 2008; Suter, 2007). One of the hallmarks of this disease is axon loss, which may or may not occur together with demyelination of peripheral nerves. When myotubularins, the PI 3-phosphatase family implicated in CMT, are mutated in mice, aberrant myelin membranes are observed that mimic those seen in CMT patients, suggesting that defects in membrane addition and/or remodeling may underlie some of the disease phenotypes (Robinson et al., 2008; Suter, 2007). FIG4 is another phosphatase implicated in CMT that is responsible for dephosphorylating PI(3,5)P2 at the D-5 position (Chow et al., 2007). While the cellular mechanisms driving development of CMT are not understood, it is clear that alterations in the cellular pool of PI(3,5)P2 contribute to the disease phenotype. In addition, several of the other proteins linked to CMT contain PIP binding domains, including Frabin/ FGD4 and Dynamin2, further supporting a role for phosphoinositides in disease etiology (Suter, 2007). The creation of mouse models harboring mutations in the myotubularins and FIG4 will hopefully lead to a greater understanding of the cellular mechanisms behind CMT pathology and will also contribute to an understanding of the role of PI(3,5)P2 during normal cellular function (Buj-Bello et al., 2002; Chow et al., 2007; Robinson et al., 2008). As was touched on earlier, the activity of Presenilin and the g-secretase complex has been shown to both regulate and be regulated by lipid metabolism. Presenilin and the g-secretase complex target many substrates in addition to Notch, including the Amyloid Precursor Protein (APP) (Selkoe and Wolfe, 2007). Mutations in either Presenilin1, Presenilin2, or APP are associated with most cases of Familial Alzheimer’s Disease (FAD), and misregulation of APP cleavage is likely associated with sporadic cases of the disease as well (Blennow et al., 2006; Koo and Kopan, 2004). Studies investigating the cellular consequences of Presenilin FAD mutations have revealed defects in calcium homeostasis and there is mounting evidence to suggest that calcium regulation may be central to the Alzheimer’s neurodegenerative phenotype. Interestingly, Presenilin, but not g-secretase function, is required to maintain PIP2 levels in cell culture, and a reduction of PIP2 observed in Presenilin FAD mutant cells is connected to defects in TRPM7-associated Ca2+ homeostasis (Landman et al., 2006). PIP2 is required for the activation of TRP channels (Hardie, 2003), and the calcium defect in Presenilin FAD cells can be rescued by restoration of PIP2 levels. Recent work from the Di Paolo lab also suggests that disruptions in PIP2 metabolism may underlie some of the phenotypes of Alzheimer’s Developmental Cell 16, January 20, 2009 ª2009 Elsevier Inc. 17

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Review disease, including dysregulation of calcium homeostasis and synaptic dysfunction (Berman et al., 2008). Treatment of cells with an oligomerized form of the amyloidogenic g-secretase cleavage product of APP, Ab-42, decreased PIP2 levels, apparently via changes in calcium/PLC signaling. Strikingly, removal of one copy of the PIP2 phosphatase Synaptojanin rescues the loss of PIP2 in cells treated with Ab-42 and also rescues Ab-42induced impairment of long-term potentiation, suggesting that some of the synaptic defects observed in Alzheimer’s pathology may result from reductions in PIP2 (Berman et al., 2008). In support of this hypothesis, impaired PIP2 synthesis in nerve terminals leads to synaptic defects in mice, due to effects on synaptic vesicle endocytosis and recycling (Di Paolo et al., 2004). In keeping with a role for PIPs in synaptic function, two PI 4-kinases, one PI3K, and the 5-phosphatase Synaptojanin1 all map to chromosomal regions linked to bipolar disorder and schizophrenia, and the concentration of PIP2 in platelet membranes of patients with bipolar disorder is significantly higher than controls (Halstead et al., 2005; Soares et al., 2001). Understanding the role of PIPs and PIP signaling in neural development and synaptic function will likely provide insight into the etiology of diseases such as Alzheimer’s and neuropsychiatric disorders and may provide valuable targets for the development of treatment options.

Ataliotis, P., Symes, K., Chou, M.M., Ho, L., and Mercola, M. (1995). PDGF signalling is required for gastrulation of Xenopus laevis. Development 121, 3099–3110.

Conclusion and Future Perspectives Phosphoinositides play crucial roles in the regulation of diverse pathways and developmental processes. To understand how they impact development, it is important to evaluate data obtained from biochemistry and cell culture in the context of in vivo signaling. Future analyses should focus on trying to understand how these ubiquitous lipids play defined roles in specific pathways. This will be a complex undertaking, for the precise regulation of PIP metabolism downstream of developmental signals must be coordinated with the role of PIPs in the promotion or attenuation of these same signals. It will therefore be essential to understand the interactions between PIPs and individual proteins during specific stages of signaling and to understand the temporal and spatial control of PIP metabolism. These studies will contribute to an understanding of how dynamic feedback systems function to ensure the proper coordination between cells during development and adult homeostasis.

Carmany-Rampey, A., and Schier, A.F. (2001). Single-cell internalization during zebrafish gastrulation. Curr. Biol. 11, 1261–1265.

ACKNOWLEDGMENTS

Corgan, A.M., Singleton, C., Santoso, C.B., and Greenwood, J.A. (2004). Phosphoinositides differentially regulate alpha-actinin flexibility and function. Biochem. J. 378, 1067–1072.

We thank W.S. Trimble, H. McNeill, M.K. Garroni, S.E. Egan, and J.A. Brill for helpful advice and for their critical review of the manuscript. This work was supported by grants to G.L.B. from the Natural Sciences and Engineering Research Council of Canada and the Canadian Institutes of Health Research (FRN #14143). L.C.S. was supported by the Natural Sciences and Engineering Research Council of Canada as well as by a Hospital for Sick Children Restracomp award. G.L.B. is the recipient of a Tier 1 Canada Research Chair in Molecular and Developmental Neurobiology. We apologize to the many researchers whose papers could not be cited due to space limitations.

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