Phosphoinositides in endocytosis.

BBAMCB-57693; No. of pages: 11; 4C: 3, 4, 6 Biochimica et Biophysica Acta xxx (2014) xxx–xxx

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Review

Phosphoinositides in endocytosis☆ York Posor 1, Marielle Eichhorn-Grünig, Volker Haucke ⁎ Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Straße 10, 13125 Berlin, Germany

a r t i c l e

i n f o

Article history: Received 5 July 2014 Received in revised form 21 August 2014 Accepted 17 September 2014 Available online xxxx Keywords: Clathrin Endocytosis Endosome Phosphatidylinositol-4,5-bisphosphate Phosphatidylinositol-3,4-bisphosphate CLIC/GEEC

a b s t r a c t The internalization and subsequent endosomal trafficking of proteins and membrane along the endocytic pathway is a fundamental cellular process. Over the last two decades, this pathway has emerged to be subject to extensive regulation by phosphoinositides (PIs), phosphorylated derivatives of the minor membrane phospholipid phosphatidylinositol. Clathrin-mediated endocytosis (CME) is the endocytic mechanism characterized in most detail. It now represents a prime example of a process spatiotemporally organized by the interplay of PI metabolizing enzymes. The most abundant PI, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], serves as a denominator of plasma membrane identity and together with cargo proteins is instrumental for the initiation of clathrin-coated pit (CCP) formation. During later stages of the process, the generation of phosphatidylinositol3,4-bisphosphate [PI(3,4)P2] and the dephosphorylation of PI(4,5)P2 regulate CCP maturation and vesicle uncoating. Here we provide an overview of the mechanisms by which PIs are made and consumed to regulate CME and other endocytic pathways and how conversion of PIs en route to endosomes may be accomplished. Mutations in PI converting enzymes are linked to multiple diseases ranging from mental retardation and neurodegeneration, to inherited muscle and kidney disorders suggesting that the tight control of PI levels along the endocytic pathway plays a critical role in cell physiology. This article is part of a Special Issue entitled Phosphoinositides. © 2014 Elsevier B.V. All rights reserved.

1. Introduction While phosphoinositides (PIs) have been known to regulate Ca2+ and growth factor signaling as well as cellular transformation since the 1980s [1,2] a landmark study by Emr and colleagues in yeast provided the first evidence for a function of PIs in membrane traffic [3]. A link between PIs and endocytosis emerged from the observation that inositol-1,4,5-trisphosphate (IP3), the second messenger that releases Ca2 + from intracellular stores, inhibits the self-association of the endocytic clathrin adaptor AP-2 in vitro [4]. The finding that AP-2 as well as the brain-specific endocytic adaptor AP180 can bind to inositol-polyphosphates [5,6] suggested that lipid signaling metabolites may directly regulate endocytic vesicle coats [7]. Subsequent work established a role for PI(4,5)P2 in clathrin-coated vesicle (CCV) formation [8] including dynamin-mediated membrane fission [9,10]. Over the last 15 years, we have gathered detailed knowledge on the mechanisms by which PIs are synthesized and turned over at endocytic sites and how their association with endocytic proteins may govern cargo selection as well as membrane deformation and internalization. PI(4,5)P2 is essential for endocytic vesicle formation by clathrin-mediated

☆ This article is part of a Special Issue entitled Phosphoinositides. ⁎ Corresponding author. E-mail address: [email protected] (V. Haucke). 1 Present address: UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley Street London WC1E 6DD, UK.

endocytosis (CME) and via macropinosomes. Both of these pathways also are regulated by the activities of phosphatidylinositol-3-kinases (PI3Ks), while PI 5-phosphatases are crucial for the final stages of vesicle formation. The endosomal system in contrast is marked by PI(3)P, which is required for homotypic endosomal fusion, endosomal sorting, and for the formation of intraluminal vesicles along the multivesicular body (MVB) pathway en route to lysosomes. Here, we provide an overview of the different roles of PIs in endocytosis with a focus on CME. We also summarize the current state of knowledge with respect to the role of PIs in clathrin-independent internalization routes such as macropinocytosis. Finally, we suggest a speculative model as to how conversion of PIs on endocytic membranes en route to endosomes may occur and how dysfunction of this conversion system could lead to disease. 1.1. PIs and membrane identity Given that the cytoplasmic leaflets of distinct cellular compartments are differentially enriched in distinct PI species, PIs have been postulated to serve as denominators of membrane identity [11,12], a feature intimately linked to their role in membrane traffic. The differential distribution of PIs is recognized by proteins harboring specific lipidbinding modules [13] thereby aiding the targeting of these proteins and their associated factors to their correct subcellular destination. The characteristic PI of the plasma membrane is PI(4,5)P2, which regulates a wide range of physiological processes including signal

http://dx.doi.org/10.1016/j.bbalip.2014.09.014 1388-1981/© 2014 Elsevier B.V. All rights reserved.

Please cite this article as: Y. Posor, et al., Phosphoinositides in endocytosis, Biochim. Biophys. Acta (2014), http://dx.doi.org/10.1016/ j.bbalip.2014.09.014

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transduction, endocytosis, actin dynamics and cell migration, as well as the function of ion channels [14,15]. PI(4,5)P2 is synthesized locally from PI(4)P by PI(4)P-5-kinases (PIP5Ks) [16], and for a long time plasma membrane PI(4)P was considered a mere precursor to PI(4,5)P2. However, depletion of plasma membrane PI(4)P or genetic disruption of plasma membrane PI(4)P synthesis was shown to interfere with the localization and function of plasma membrane proteins, in spite of the fact that total PI(4,5)P2 levels were not significantly decreased [17, 18]. These studies indicate a crucial role of PI(4)P in conferring plasma membrane identity that is independent of its role as a precursor of PI(4,5)P2. Other subcellular compartments are believed to contain comparably low levels of PI(4,5)P2 and instead are enriched in other PIs [12]. For example, PI(4)P is highly enriched at the Golgi complex in addition to the plasma membrane, whereas PI(3)P predominates in the endosomal system. Among the endosomal processes shown to depend on PI(3)P are the homotypic fusion of early endosomes, the endosomal sorting of endocytosed proteins as well as the formation of intraluminal vesicles for degradative sorting within MVBs [19]. Class III PI3K, hVps34, is believed to produce the majority of PI(3)P, yet accumulating evidence suggests that a fraction of endosomal PI(3)P may be synthesized by class II PI3Ks, either by phosphorylation of PI or indirectly via dephosphorylation of PI(3,4)P2, another major product of class II PI3Ks [20–22]. As membranes exchange between compartments conversion of their PI identity has to occur. For example, fission of endocytic vesicles from the plasma membrane and subsequent fusion with early endosomes must be accompanied by PI conversion of the internalizing membrane from PI(4,5)P2 to PI(3)P. How this is accomplished precisely is not completely understood but recent data indicate that part of this conversion may already occur at the cell surface through acquisition of PI(3,4)P2 [22], possibly followed by hydrolysis of its 4-phosphate moiety to yield PI(3)P.

1.2. Phosphoinositide regulation of clathrin-mediated endocytosis CME is a fundamental cell biological process that is of central importance to various aspects of cellular physiology ranging from nutrient uptake and signal transduction to synaptic transmission and development [23,24]. During CME small portions of the plasma membrane are internalized into small 80 nm to 140 nm sized vesicles coated with clathrin (CCVs). CCVs are formed by the assembly of a bilayered protein coat on the cytoplasmic face of the plasma membrane. At its core, this coat consists of the heterotetrameric adaptor protein AP-2, comprising α-, β2-, μ2-, and σ2-adaptin, surrounded by hexagonal and pentagonal assemblies of clathrin triskelia, composed of three tightly associated heavy and light chains [25]. AP-2, together with a host of other adaptors and accessory proteins, forms the membrane-proximal layer of the coat that directly contacts the membrane and binds to transmembrane cargo, e.g. receptors or channels. Cargo-specific adaptors enable the internalization of a large variety of distinct cargoes via CME by recognizing recurring internalization motifs. For example, AP-2 via its μ- and σsubunits recognizes tyrosine-based as well as dileucine-based motifs within the cytoplasmic domains of receptors or other types of membrane proteins. In addition, more specialized dedicated adaptors sort subsets of cargo, i.e. arrestins enable CME of G protein-coupled receptors [26]. Clathrin and AP-2 serve as central protein interaction hubs within the endocytic protein network [27,28]. The clathrin terminal domain (CTD), a globular seven-bladed β-propeller fold at the distal end of each triskelion leg, and the appendage domains of AP-2α- and β, via recognition of simple degenerate peptide motifs, recruit a large variety of endocytic proteins [25] that drive progression of the pathway (Fig. 1). These endocytic proteins include cargo adaptors, membrane deforming scaffolds, actin modulatory factors — and PI phosphatases and kinases [28].

The first PI metabolizing enzyme to be implicated in CME was the 5-phosphatase synaptojanin, an enzyme highly enriched at nerve terminals, where synaptic vesicles undergo local exo-endocytic cycling [29–32]. Genetic ablation of synaptojanin 1 leads to increased levels of PI(4,5)P2 and an accumulation of CCVs at synapses [33]. This suggests a crucial role for PI(4,5)P2 in stabilizing clathrin coats at membranes. Conversely, knockout of PIP5KIγ, the major PI(4,5)P2-synthesizing enzyme at synapses [34], causes reduced PI(4,5)P2 levels and concomitant impairments in both exocytic neurotransmitter release and in the endocytic recycling of synaptic vesicle membranes [34]. Furthermore, constitutive [35] or rapamycin-induced membrane recruitment of an inositol 5-phosphatase [36] results in the enzymatic depletion of PI(4,5)P2 and a complete loss of clathrin-coated pits (CCPs) from the plasma membrane [37], suggesting that CCPs can neither form nor persist in the absence of PI(4,5)P2. 1.2.1. PI(4,5)P2-binding proteins within the CME proteome The stringent requirement for PI(4,5)P2 in endocytosis is reflected in the abundance of PI(4,5)P2-binding proteins within the endocytic proteome. These include the early-acting clathrin adaptors AP-2 [38–41] (Figs. 1 and 2A), AP180/CALM [42,43] (Figs. 1 and 2B), and epsins 1–3, which recognize ubiquitylated cargo [42,44], the NPxY-motif adaptors disabled 2 (Dab2), autosomal recessive hypercholesterolemia (ARH), the integrin adaptor Numb [45,46], and β-arrestins, adaptors for the CME of G-protein coupled receptors (GPCRs) [47,48] (Fig. 1). In addition, PI(4,5)P2 associates with and recruits membrane remodeling proteins of the Bin/Amphiphysin/Rvs (BAR)-domain superfamily, e.g. Fer/Cip4 homology domain-only (FCHo) 1/2 [49], which in conjunction with AP-2 and clathrin acts during early stages of CCP formation. Lateacting BAR domain proteins such as endophilin and amphiphysin can associate with a variety of negatively charged lipids including PIs [50]. An exception is the PX-BAR domain scaffold sorting nexin 9 (SNX9) [51–53], which via its PX domain specifically binds to PI(4,5)P2 and to PI(3,4)P2 (as well as to PI(3)P, a lipid absent from the plasma membrane in most cell types) (Fig. 2C). Finally, PI(4,5)P2 aids the function of the fissioning GTPase dynamin [54–56], possibly by positioning the molecule towards the membrane during assembly at the neck of invaginated CCPs. Thus, in addition to clathrin and AP-2, PI(4,5)P2 serves as the third interaction hub within the endocytic network (Fig. 1). Due to its specific enrichment in the plasma membrane, PI(4,5)P2 ensures compartmental fidelity of CCP assembly. Cargo internalized by CME frequently cycles between the cell surface and internal membranes, yet assembly of endocytic coats occurs only at the cell surface. Such compartmental specificity may be achieved by a coincidence detection system that capitalizes on the ability of early-acting endocytic adaptors to associate with transmembrane cargo and with PI(4,5)P2 in the same membrane, effectively limiting the site of endocytic CCV formation to the plasma membrane. 1.2.2. PI(4,5)P2 synthesis and CCP nucleation CME depends on the general abundance of PI(4,5)P2 in the plasma membrane but further creates its own, dedicated pool of PI(4,5)P2. PI(4,5)P2 is synthesized from PI(4)P by type I PIP5K α, β, and γ [15]. All three isoforms of PIP5KI [57,58] can associate with the μ-subunit of the AP-2 adaptor complex [59], while the p90 isoform of PIP5KIγ in addition can bind to AP-2β. PIP5KIγ is present in 6 different splice variants [15]; its p90 isoform of PIP5KI γ (now referred to as PIP5KIγ-v2) carries a 28 amino acid carboxy (C)-terminal splice insert that interacts with both the μ2 subunit and the β2-appendage of AP-2 [60–62]. Binding of AP-2-cargo complexes to PIP5KIγ-v2 results in a potent stimulation of its kinase activity [59,63], suggesting a crucial role for this complex in the initial stages of CCP formation. Consistent with this scenario, enhanced PI(4,5)P2 synthesis increases the rate of CCP nucleation [64] and single molecule imaging suggests that AP-2, perhaps together with FCHo1/2 [49], is among the first endocytic proteins arriving at newly forming CCPs [65]. Newly recruited AP-2 when bound to



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Fig. 1. The PI interactome in CME. Clathrin-mediated endocytosis (CME) is organized around PI(4,5)P2, the heterotetrameric clathrin adaptor complex AP-2 and clathrin itself as major interaction hubs. AP-2 in its open conformation binds to plasma membrane PI(4,5)P2 via its α, β2, and μ2 subunits. In addition, it associates with and recruits clathrin and a number of additional endocytic proteins, many of which also bind to PI(4,5)P2 (red). These include AP180/CALM, ARH, β-arrestin, Dab2, dynamin, FCHo 1/2, epsins 1–3, and Numb. PI(4,5)P2 is synthesized from PI(4)P by PI 4-phosphate 5-kinase type Iγ (PIPKIγ) during clathrin-coated pit (CCP) nucleation. Maturation of CCPs is accompanied by the clathrin-dependent recruitment and activation of PI3K C2α, a class II PI 3-kinase that converts PI(4)P to PI(3,4)P2. Synthesis of PI(3,4)P2 is required for assembly of the PX-BAR domain protein SNX9 (green) at constricting CCPs and may occur in parallel with PI(4,5)P2 hydrolysis to PI(4)P via synaptojanin and possibly OCRL, thereby facilitating auxilin-dependent vesicle uncoating. Overall, these reactions may result in the conversion of PI(4,5)P2 to PI(3,4)P2 en route to endosomal PI(3)P.

transmembrane cargo can stimulate PIP5KIγ-v2 activity [59] and thereby generate a feed-forward loop that may drive the initial stages of CCP formation by creating a local, CME-dedicated pool of PI(4,5)P2. Displacement of PIP5KIγ-v2 from AP-2β by the assembling clathrin coat may break this feed-forward loop [61] and thereby restrict PI(4,5)P2 synthesis to the early stages of CCP nucleation. A similar role is played by β-arrestin adaptors during CME of GPCRs. β-Arrestins directly associate with PIs including PI(4,5)P2 [47] (and potentially also PI(3,4,5)P3; [66]) and bind the α isoform of PIP5KI [48] to faciliate local production of PI(4,5)P2. Acute depletion of PI(4,5)P2 [67] or mutation of the PI binding site in β-arrestin 2 [47] interferes with the internalization of GPCRs such as the β2-adrenergic receptor but does not affect the plasma membrane targeting or receptor association of arrestins [47,67]. Thus, PI(4,5)P2 acts as a gatekeeper for GPCR endocytosis via regulating the function of β-arrestins. Elegant structural studies have revealed that the function of PI(4,5) P2 during early stages of CCP nucleation likely goes beyond that of a simple glue for AP-2 and other endocytic proteins. Rather, association with PI(4,5)P2 facilitates the conformational activation of AP-2 from an initial closed to the open state [41] (Fig. 2A). In the closed, likely cytosolic form of AP-2, the binding sites for Yxx≤ϕ- (where ϕ represents a bulky hydrophobic amino acid) and [DE]xxxL[LI]-cargo peptides on the C-terminal portion of μ2 (C-μ2) and the α-σ2 interface, respectively, are occluded by contacts with AP-2β. Initial membrane association of AP-2 critically depends on PI(4,5)P2-binding sites formed by patches of basic residues within AP-2α [40,68] and AP-2β [41] (Fig. 2A). The high local concentration of PI(4,5)P2 together with transmembrane cargo and aided by phosphorylation of AP-2 μ is then believed to trigger a large scale conformational change within AP-2 that exposes two additional PI(4,5)P2-binding sites on the C-terminal domain of AP-2 μ. During this large scale intramolecular rearrangement C-μ2 is displaced from the core and β2 frees the Yxxϕ- and [DE]xxxL[LI]-motif binding sites to enable sorting of transmembrane cargo [41]. In the open conformation, all four PI(4,5)P2-binding sites on AP-2 are oriented coplanar with the

plasma membrane and with the recognition sites for Yxxϕ- and [DE] xxxL[LI]-motifs. Thus, PI(4,5)P2 enables conformational opening and cargo sorting of AP-2, while restricting its activity to the plasma membrane. 1.2.3. 5-phosphatases are present during CCP maturation Accumulating evidence suggests that PI(4,5)P2 levels are tightly controlled during CCP growth and maturation. The local synthesis of PI(4,5)P2 appears to be strictly limited to the nucleation stage of CME, as PIP5KIγ is neither found in CCVs [69] nor at nascent CCPs [59,64]. Furthermore, several PI(4,5)P2 5-phosphatases are recruited to maturing and late-stage CCPs. These include the p170 isoform of synaptojanin 1, an active 5- and 4-phosphatase that carries a C-terminal splice extension harboring binding sites for clathrin, AP-2, and Eps15. Synaptojaninp170 recruitment to CCPs parallels that of clathrin [70], suggesting that PI(4,5)P2 levels may possibly decline during CCP maturation. CCPs also contain the PI(3,4,5)P3- and PI(4,5)P2-specific 5-phosphatase srchomology 2 containing 5-phosphatase 2 (SHIP2). SHIP2 is recruited to CCPs by the scaffolding protein intersectin [71] during early stages, but leaves CCPs prior to scission. Depletion of SHIP2 decreases CCP lifetimes, a phenotype mimicked by acute rapamycin-induced PI(3,4,5)P3synthesis [71], suggesting a possible regulatory role of PI(3,4,5)P3 in CME. As SHIP2, in addition to PI(3,4,5)P3, can also hydrolyze PI(4,5)P2 it remains unclear whether altered CCP dynamics observed in SHIP2depleted cells are a consequence of elevated levels of PI(3,4,5)P3 or PI(4,5)P2. Taken together, these collective data open the possibility that the recruitment of PI phosphatases may cause PI(4,5)P2 levels to decline as CCPs mature. 1.2.4. PI(4,5)P2 in endocytic membrane fission Fission of late-stage CCPs from the plasma membrane, the final step of CME, is catalyzed by the mechanochemical GTPase dynamin. Besides an unstructured C-terminal proline-rich domain (PRD), by which dynamin associates with endocytic SH3 domain proteins such as


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Fig. 2. Domain organization and PI recognition of endocytic proteins AP-2, CALM, and SNX9. In all three examples of endocytic proteins binding to PIs, basic residues account for PI binding specificity. A. At the top, the structures of the AP-2 core in its closed conformation crystallized in complex with inositolhexakisphosphate (IP6) (PDB ID: 2VGL; Collins et al. Cell, 2002) and the open conformation (PDB ID: 2AX7; [41]), are shown. The PI binding sites in both structures are indicated with black circles. At the bottom left, the domain organization of the AP-2 subunits is depicted with the regions involved in PI binding indicated by gray lines. The bottom right panel shows a detailed view of the α-subunit (red) bound to IP6. B. The domain structure of CALM is depicted to the left of the CALM ANTH domain (red) bound to PI(4,5)P2 (PDB ID: 1HFA;[43]) and a detailed view of the PI binding site. C. The domain organization of SNX9 is depicted to the left of the structure of the SNX9 PX-BAR domain (PX domain in red) in complex with PI(3)P (PDB ID: 2RAK; [152]) and a detailed view of the PI binding site. Note that SNX9 can not only associate with PI(3)P but also with PI(3,4)P2 and to some degree also with PI(4,5)P2 [22,51]. However, no structural information for the latter interactions is available.

intersectin, amphiphysin, endophilin, or syndapin, dynamin comprises four additional domains: the GTPase domain, the bundle signaling element (BSE), the stalk and the PH domain. A number of intra- and intermolecular interactions between these domains are required for the helical assembly of dynamin and the regulation of its membrane remodeling activity [72–75]. The PH domain of dynamin binds to PIs with relatively broad specificity (i.e. PI(4,5)P2, PI(3,4,5)P3, and PI(3,4)P2) and with comparably low affinity [9,10], yet, is required for its function [13].

While the PH domain is dispensable for recruitment of dynamin to endocytic structures [76], its association with PI(4,5)P2 is required for dynamin-mediated fission [55,56]. Dynamin oligomerizes into a helical assembly around the neck of constricted, late stage endocytic pits [73–75]. In this structure, the PH domains of dynamin are positioned on the membrane, likely transmitting force generated by the extension, compaction, and twisting of the dynamin helix onto the membrane [72,77]. Moreover, insertion of bulky hydrophobic residues of the PH



domain of dynamin into the outer monolayer of the membrane might contribute to induction of membrane curvature en route to membrane fission [78]. These observations thus provide a molecular explanation for the critical role of the PH domain in dynamin-mediated membrane fission in vivo [55,56]. A similar mechanism of amphipathic helix (called helix H0) insertion has also been suggested to underlie a possible fission activity of the PI(4,5)P2-binding ENTH domain protein epsin [79]. Apart from regulating the function of dynamin, PI(4,5)P 2 has been suggested to serve additional roles in membrane fission relating to the creation of line tension. As discussed above, maturation of endocytic CCPs is accompanied by the recruitment of PI(4,5)P2-specific 5-phosphatases such as synaptojanin that catalyze PI(4,5)P2 hydrolysis. The assembly of a tight scaffold of BAR domain proteins at the vesicle neck likely will sequester acidic phospholipids including PI(4,5)P2 and thereby shield them from PI phosphatase-mediated hydrolysis. Because less PI(4,5)P2 is hydrolyzed on the tubule, a higher hydrogen bond density is created adjacent to the bud. The imbalance in electrostatic attraction from hydrogen bonds between the two adjacent regions (bud and tubule) results in a line tension encircling the neck [78], facilitating membrane fission. Such a scenario is consistent with the observation that synaptojanin 1 acts with membrane curvature generators/sensors, such as the BAR protein endophilin, to preferentially remove PI(4,5)P2 from curved membranes. These observations raise the possibility that geometry-based mechanisms may contribute to spatially restricting PI(4,5)P2 elimination during membrane internalization and suggests that the PI(4,5)P2-to-PI4P conversion by synaptojanin 1 at sites of high curvature may cooperate with dynamin to achieve membrane fission [80]. 1.2.5. PI(4,5)P2 hydrolysis after completion of CCV formation After fission of CCVs from the plasma membrane the clathrin coat is rapidly shed, recycling clathrin and adaptors for subsequent rounds of CME and allowing the newly formed endocytic vesicle to acquire fusion competence en route to early endosomes. The observation that synaptojanin 1-deficient mice display an accumulation of coated vesicles in their synaptic boutons was the first evidence for a requirement of PI(4,5)P2 hydrolysis during uncoating [33]. Indeed, the p145 isoform of synaptojanin is recruited to late stage CCPs in a burst coinciding with that of its main recruitment factor endophilin [81,82] and with dynamin [70]. Interestingly, although endophilin may contribute to membrane remodeling prior to dynamin-catalyzed fission, the loss of function phenotypes of synaptojanin and endophilin in worms, flies, and mammals is strikingly similar. Loss of synaptojanin 1 or of endophilins causes a defect in synaptic vesicle recycling due to impaired CCV uncoating [81–83]. In addition to synaptojanin-p145, the PI(3,4,5)P 3- and PI(4,5)P2 -specific 5-phosphatase oculocerebrorenal syndrome of Lowe (OCRL) is recruited to late stage CCPs [84]. In spite of OCRL interacting with AP-2 and with clathrin [85,86] the phosphatase is recruited to endocytic structures around or immediately after vesicle fission [84,87]. The significance of synaptojanin and potentially also OCRL activity during membrane fission is not entirely clear. As discussed above synaptojanin preferentially hydrolyzes PI(4,5)P2 in highly curved membranes, i.e. at the neck of CCPs, and, thus, may support dynaminmediated fission [80]. The striking accumulation of CCVs in nerve terminals of synaptojanin 1 KO mice, however, indicates that this role cannot be essential, while PI(4,5)P2 hydrolysis is necessary for uncoating [33,83]. When and to what extent PI(4,5)P2 conversion to PI(4)P mediated by synaptojanin-p170 and possibly SHIP-2 occurs during the late stages of CME remains to be resolved. For example, it is conceivable that depletion of PI(4,5)P2 prior or during the time of fission triggers the dissociation of PI(4,5)P2-binding adaptors (i.e. AP-2, CALM, epsins) before clathrin uncoating [83]. What is the signal that initiates disassembly of the clathrin lattice and final uncoating of the vesicle? Clathrin uncoating is catalyzed by the chaperone Hsc70 and its recruiting factor auxilin [28], a J-domain

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protein harboring a catalytically inactive domain with homology to the PI(3,4,5)P3- and PI(3,4)P2-specific 3-phosphatase domain of phosphatase and tensin homolog (PTEN). The timing of uncoating appears to be determined by the recruitment of auxilin, which interacts with clathrin and AP-2 [88,89], but is not present at significant levels during CCP growth and maturation [87,90]. Auxilin also interacts with dynamin [91], and dynamin activity is necessary for the burst of auxilin recruitment immediately after scission. However, a truncated auxilin mutant that retains all binding sites for clathrin, AP-2, and dynamin but lacks the PTEN-like domain fails to be recruited to late-stage CCPs. Evidence from PI dot blot assays suggests that the PTEN-like domain of auxilin may associate with lipids including PI(3)P and PI(3,4)P2 [90]. Hence, it appears likely that the timing of auxilin recruitment and, thus, of uncoating is determined by a dual key strategy involving its association with key endocytic proteins and with PIs characteristic of late-stage endocytic intermediates such as PI(3,4)P2 (see below) and possibly PI(3)P. Future studies will need to address this possibility in detail. 1.2.6. PI(3,4)P2 regulates maturation of CCPs The regulation of CME by PIs has long been believed to be restricted to the roles of PI(4,5)P2 in CCP nucleation and uncoating. Although the presence of PI3K C2α in CCVs was noted early on [92,93], the significance of this finding remained obscure. PI3K C2α is a member of the class II PI3Ks, large monomeric enzymes that share a conserved PI3K catalytic core with the heterodimeric p110/p85 class I PI3Ks. In addition, they contain a C-terminal PX- and C2-domain as well as an approximately 400 amino acid long N-terminal region predicted to be unstructured [94]. Binding of the clathrin terminal domain to a degenerate clathrin-box motif in the N-terminal region of PI3K C2α localizes the enzyme to clathrin-coated structures at the plasma membrane and to perinuclear recycling endosomes [21,95]. Remarkably, quantitative proteomic analysis of CCVs revealed PI3K C2α to be an abundant component of both AP-2- and AP-1-containing vesicles with an average of 10 copies per 40 clathrin triskelia, i.e. 25 copies for a 120–140 nm CCV [96]. Clathrin binding further stimulates its PI3K activity, likely by relieving PI3K C2α from auto-inhibition [95]. Collectively, these findings predicted the implication of 3-phosphorylated PIs in CME. Recently we have shown that depleting the plasma membrane of PI(3,4)P2 by expression of a constitutively membrane-associated PI(3,4)P2-4-phosphatase inhibits the CME of transferrin and causes an accumulation of stalled plasma membrane CCPs [22]. This effect is in contrast to PI(4,5)P2 depletion, which results in a loss of CCPs from the plasma membrane [37]. Thus, PI(4,5)P2 and PI(3,4)P2 exhibit distinct regulatory roles in CME. The endocytic phenotype of PI(3,4)P2 depletion is recapitulated in cells depleted of PI3K C2α. Mutants of PI3K C2α either lacking kinase activity or selectively producing PI(3)P but not PI(3,4)P2 failed to rescue CME [22]. Mechanistically, loss of PI3K C2α was found to result in a delay of CCP maturation characterized by the accumulation of deeply invaginated U-shaped CCPs prior to fission. Consistently, PI3K C2α is recruited to CCPs after clathrin but before the final burst of dynamin recruitment. Further biochemical and cell biological analyses identified the PX-BAR domain proteins SNX9 and SNX18 [97] as effectors of PI(3,4)P2 at CCPs. Indeed, recruitment of SNX9 to late stage CCPs is blocked in cells depleted of PI(3,4)P2 or lacking PI3K C2α [22]. These data suggest a model whereby clathrin-mediated recruitment of PI3K C2α regulates CCP maturation by synthesizing PI(3,4)P2, which serves to recruit or stabilize SNX9/SNX18 at endocytic intermediates. Formation of SNX9/SNX18 oligomers may be required to facilitate the constriction of CCPs, the final maturation step before dynamin can assemble around the narrow neck and catalyze scission [77]. How this occurs precisely remains to be investigated. The identification of PI3K C2α and its lipid product PI(3,4)P2 as regulators of CME leaves a number of unanswered questions. For example, it is unclear whether SNX9 and SNX18 are the only PI(3,4)P2-effectors in CME. In dynamin 2-depleted cells, late stage endocytic intermediates accumulate that display elongated tubular necks that are stabilized by


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assemblies of BAR-domain proteins, including SNX9, endophilin, as well as actin branch nucleators such as the ARP2/3 complex [98]. Depletion of PI(3,4)P2 or PI3K C2α in cells lacking dynamin 2 not only causes loss of SNX9 but also of ARP2/3 from arrested CCPs [22]. While these data suggest a possible connection between PI(3,4)P2 and the actin cytoskeleton, the underlying molecular mechanisms and their relationship to SNX9, which itself interacts with actin regulatory factors such as neural Wiskot–Aldrich syndrome protein (N-WASP) [52,99] remain to be established. PI(3,4)P2 may also play a role in coordinating the uncoating reaction [100]. As discussed above, disassembly of the clathrin cage is initiated by auxilin and indications exist that the PTEN-like domain of auxilin associates with PI(3)P and PI(3,4)P2 [90]. However, the lipid specificity of auxilin and the extent to which recruitment of auxilin depends on interactions with these PIs remains to be investigated.

1.2.7. Possible roles for PI(3,4,5)P3 in CME Apart from the constitutive endocytosis that takes place in most cell types, CME also participates in the internalization of signaling receptors, whose activation results in formation of PI(3,4,5)P3 by class I PI3Ks [94]. For example, internalization of β2-adrenergic receptors has been reported to be stimulated by class I PI3K p110γ-mediated PI(3,4,5)P3 formation [66]. An elegant study using membrane-permeable PI derivatives [101] showed that acute increases of PI(3,4,5)P3 in the absence of ligand-induced receptor activation trigger the internalization of receptor tyrosine kinases (RTKs). The epidermal growth factor receptor (EGFR) as well as the ephrinA4 receptor, but not GPCRs or the transferrin receptor, was internalized when the cellular concentration of PI(3,4,5)P3 was increased by either administration of exogenous PI(3,4,5)P3 or acute recruitment of class I PI3Ks to the plasma membrane [102]. EGFRs internalized via CME in response to PI(3,4,5)P3 were neither tyrosine-phosphorylated nor ubiquitylated and as a result did not undergo sorting to multivesicular bodies for degradation but

were recycled back to the cell surface. Subsequent analysis showed that PI(3,4,5)P3-induced endocytosis of EGFRs, apart from CME components, requires the PAR polarity complex and specifically the association of PAR3 with PI(3,4,5)P3 [102]. These findings suggest the possibility that PI(3,4,5)P3 may shunt internalized EGFRs back to the plasma membrane instead of their degradation via the multivesicular body/lysosomal sorting pathway. If and how this pathway operates under physiological conditions is currently unclear.

1.2.8. A comprehensive model of PI conversion during CME The study of the individual PI metabolizing enzymes participating in CME has led to an emerging overall picture of PI function in CME (Fig. 3). CCP nucleation depends on the presence of PI(4,5)P2. Numerous endocytic adaptor proteins recognize PI(4,5)P2 and the most abundant one, AP-2, drives initial stages of coat assembly through a feedforward loop by activating PIP5KIγ. PI(4,5)P2 can therefore be seen as a third interaction hub of the endocytic network (Fig. 1). Growth and maturation of CCPs are accompanied by recruitment of the 5-phosphatases synaptojanin-p170 and SHIP2. At this stage, the stabilizing clathrin lattice may reduce dependence of adaptor protein association on PI(4,5)P2 to an extent that could allow the balance to be shifted towards formation of PI(4)P from PI(4,5)P2. Clathrin-dependent recruitment of PI3K C2α enables conversion of PI(4)P present at the plasma membrane [18] or generated by synaptojanin-p170 or SHIP2-mediated PI(4,5)P2 hydrolysis into PI(3,4)P2 [71]. PI(3,4)P2 production by PI3K C2α facilitates the transition of open shallow CCPs to Ω-structures competent for fission. How precisely membrane remodeling during this transition occurs is unknown but it requires the PI(3,4)P2-mediated recruitment and assembly of SNX9/18 [22,77]. Along with the burst of dynamin and concomitant membrane fission recruitment of 5-phosphatases such as synaptojanin and OCRL leads to complete hydrolysis of PI(4,5)P2. We thus hypothesize that newly formed endocytic vesicles may be enriched in PI(3,4)P2 but largely

Fig. 3. PI conversion during clathrin-mediated endocytosis en route to endosomes. CME is nucleated by co-assembly of early acting endocytic proteins (FCHo, AP-2) with clathrin at PI(4,5) P2 (red)-rich plasma membrane sites generated by phosphatidylinositol 4-phosphate 5-kinase type I (PIPKI). CCP maturation is driven by partial PI conversion from PI(4,5)P2 (red) to PI(3,4)P2 (green) catalyzed by phosphatidylinositol 3-kinase C2α (PI3K C2α)-mediated phosphorylation of PI(4)P (yellow) to facilitate recruitment of the PI(3,4)P2-binding PX-BAR domain protein SNX9. PI(3,4)P2 synthesis may be accompanied by synaptojanin-p170 and SHIP-2-mediated dephosphorylation of PI(4,5)P2 to PI(4)P (yellow). In neurons and possibly other cell types uncoating during late stages of CME requires the activity of the short p145 isoform of synaptojanin to remove PI(4,5)P2. PI(3,4)P2 (green) may finally be converted to PI(3)P (blue) en route to endosomes by the PI(3,4)P2-specific 4-phosphatases INPP4A/B, effectors of the endosomal GTPase Rab5.



devoid of PI(4,5)P2. This postulated switch in lipid composition conceivably might also determine the onset of CCV uncoating by recruitment of auxilin and Hsc70-mediated clathrin disassembly. The progression of CCPs towards a fission-competent stage is thus coupled to depletion of PI(4,5)P2 and the concomitant formation of a 3-phosphorylated PI, PI(3,4)P2. We speculate that the generation of PI(3,4)P2 primes the nascent endocytic vesicle for adopting early endosomal PI(3)P identity. How and when this occurs is uncertain, but the PI(3,4)P2-specific inositol 4-phopshatase INPP4A/B, an effector of the endosomal GTPase Rab5 would be an excellent candidate to facilitate PI(3,4)P2 to PI(3)P conversion [103] (Fig. 3 and further discussed below). 1.3. PI regulation of clathrin-independent endocytic pathways Over the past decades it has become clear that apart from CME many cell types use a variety of mechanisms of clathrin-independent endocytosis (CIE) [24,104]. It has been estimated that up to 70% of endocytosed fluid in fibroblasts is internalized via clathrin-independent carriers (CLIC) [105], a pathway for high-capacity membrane turnover. On the mechanistic level our understanding of CIE is still far from complete. CIE frequently depends on cholesterol or sphingolipids [106] and on PIs [107], though in many cases their exact role remains poorly understood. For example, it has been shown that Arf6, a small GTPase that has been postulated to regulate both CME [35] and CIE [108,109], stimulates PI(4,5)P2 synthesis by direct binding and activation of PIP5KI [35,108,110,111]. 1.3.1. PIs in dynamin-dependent CIE: Caveolae and interleukin-2 receptor internalization Apart from its function in CME, dynamin 2 is also involved in the CIE of caveolae, flask-shaped membrane invaginations particularly abundant in endothelial cells, and in the interleukin 2 receptor (IL-2R) internalization pathway [24]. Caveolae are invaginations of the plasma membrane generated by caveolins, proteins with a membrane-integral hairpin anchor, and cavins, cytoplasmic proteins that are required for the stabilization of caveolae [112]. Although caveolae are internalized their function is not limited to a role as endocytic entry portals, but they rather serve as multi-functional platforms involved in mechanosensing, compartmentalized signaling, and lipid metabolism [112]. Whereas the core components of caveolae are not known to associate with PIs, the dynamin-related ATPase EHD2 binds PI(4,5)P2-rich membranes [113] and is recruited to caveolae. EHD2 functions as a negative regulator of internalization by retaining caveolae at the plasma membrane and requires its lipid-binding ability for localizing to these structures [114]. Interestingly, a study employing PI(4,5)P2 labeling with the PHdomain of phospholipase Cδ on freeze-fractured plasma membrane leaflets reported the accumulation of PI(4,5)P2 at the rim of caveolae [115]. The precise significance of the role of PIs in caveolin-mediated endocytosis thus remains elusive, yet caveolae do appear to be regulated by PI(4,5)P2. The IL-2R endocytic route is a so-far poorly characterized CIE pathway used by a group of interleukin receptors [24]. CIE of the IL-2R depends on actin-regulatory factors such as cortactin, N-WASP, and the ARP2/3 complex in addition to dynamin [116]. Engagement of the actin machinery is achieved through class I PI3K-mediated PI(3,4,5)P3 formation. Pharmacological inhibition of class I PI3Ks or smallinterfering RNA-mediated depletion of its regulatory p85 subunit inhibited IL-2R internalization [117]. PI(3,4,5)P3 likely triggers CIE by activating the small GTPase Rac1, which in turn induces actin polymerization [118]. 1.3.2. PIs in dynamin-independent CIE via the CLIC pathway The CLIC pathway has been postulated to represent the major mechanism of fluid phase endocytosis [105,119]. Glycosylphosphatidylinositol-

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anchored proteins (GPI-APs) are among the most prominent cargoes of the CLIC pathway, which hence has also been referred to as the GPI-AP-enriched endosomal compartments (GEECs) pathway. CLICs are characterized by a tubular and often ring-like ultrastructural appearance. Although the precise mechanism of CLIC endocytosis is not yet fully understood, several regulators of the pathway have emerged. Internalization of GPI-APs was shown to require the activity of the small GTPases Arf1, an activator of PIP5KI [111], and Cdc42 and concomitant actin polymerization [120–122]. Moreover, recent data show that CLIC biogenesis and internalization of CD44 depend on the association of galectin-3 with glycosphingolipids to drive membrane bending as a first step of CLIC formation [123]. This function may be assisted by BAR domain proteins such as GTPase regulator associated with focal adhesion kinase 1 (GRAF1), which was found to localize to tubular structures of the CLIC pathway and to be required for the internalization of fluid-phase markers. The subcellular localization of GRAF1 depends on its BAR-PH membrane interaction module, which specifically drives association with PI(4,5)P2-rich and preferentially curved membranes [124]. The effects of PI(4,5)P2 depletion on GRAF1 and the CLIC pathway have not been assessed yet, though PI(4,5)P2 may serve as a compartmental cue directing initiation of CLIC formation at glycosphingolipidcontaining plasma membrane sites. Whether PIs other than PI(4,5)P2 play a role in CIE via the CLIC pathway is unknown. A screen for regulators of the internalization of GPI-APs identified PI3K C2α to be required for dynamin-independent fluid phase and GPI-AP endocytosis [125]. Neither lipid-specificity, site of action, nor potential effector proteins of PI3K C2α function in this pathway have been addressed yet. Interestingly, the PI3K C2α effector in the context of CME, SNX9 [22], independently was found to regulate internalization of GPI-APs as well as fluid phase endocytosis, presumably along the CLIC pathway [99]. SNX9 has been proposed to couple membrane remodeling with actin dynamics via its function as an N-WASP and ARP2/3 activator. The implication of both PI3K C2α and SNX9 in GPI-AP and fluid phase endocytosis raises the possibility that PI(3,4)P2-mediated assembly of SNX9 is involved in membrane remodeling along the CLIC pathway, similar to its established function in CME. 1.3.3. PIs in macropinocytosis Macropinocytosis is a process for the engulfment of particles and larger amounts of extracellular fluid by membrane ruffles, i.e. sheetlike extensions of the plasma membrane formed by actin polymerization. When these fold back onto the plasma membrane and subsequently fuse with it, they typically generate 0.2 μm to 5 μm sized vacuolar structures called macropinosomes [126]. A detailed description of macropinocytosis exceeds the scope of this review and the closely related process of phagocytosis is covered in an accompanying review in this issue. We will therefore only briefly consider the central aspects of PI regulation of macropinocytosis. In most cells, macropinocytosis is initiated through the stimulation of membrane ruffle formation by growth factor signaling; only macrophages and dendritic cells undergo constitutive macropinocytosis. Growth factor stimulation leads to activation of several small GTPases, including Rac1, Cdc42, Arf6, and Rab5 by mechanisms that involve Ras-activation and synthesis of PI(3,4,5)P3 [126,127] via class I PI3Ks in response to growth-factor induction [128]. The remodeling of the actin cytoskeleton underlying membrane ruffling is largely driven by Rac1 [129] and Cdc42 [126,130] and revolves around PI(4,5)P2 as a modulator of actin dynamics [131]. During ruffle formation, Rac1 and possibly Arf6 recruit and stimulate PIP5KIα [111,132], resulting in increased PI(4,5)P2 levels on the nascent macropinocytic cup [133]. Cdc42 cooperates with PI(4,5)P2 in releasing N-WASP from its autoinhibited state, thereby stimulating expansion of the actin meshwork by activating ARP2/3 [134–136]. This early stage of cup formation may initially be triggered by PI(3,4,5)P3, but crucially depends on PI(4,5)P2. After ruffle closure, i.e. upon formation of a circular ruffle that only retains a small opening, PI(4,5)P2 levels are observed to sharply decline


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[133]. The observed drop in PI(4,5)P2 levels involves two enzymatic activities: (i) class I PI3K-mediated phosphorylation to yield PI(3,4,5)P3, (ii) and phospholipase C γ (PLCγ)-mediated hydrolysis to diacylglycerol and inositol-1,4,5-trisphosphate [133,137]. From studies on the closely related process of phagocytosis it can be inferred that PI(4,5)P2 depletion directs actin depolymerization, which is necessary for macropinosome release into the cell interior [138]. Consistently, completion of macropinosome formation depends on PI3K activity and levels of PI(3,4,5)P3 peak around that time [133,139]. It appears that the actual trigger for the final increase in PI(3,4,5)P3 levels is the morphological change of ruffle closure that restricts diffusion of PI(3,4,5)P3 out of the ruffled membrane region. This PI conversion from PI(4,5)P2 to PI(3,4,5)P3 in the macropinocytic cup correlates with and is required for increased Rac1 activity and is believed to initiate cup closure and macropinosome formation [140]. The precise mechanism of membrane constriction and fission of macropinosomes is unclear. Accumulating evidence suggests that several, possibly sequential PI conversions are required for this process. The formation of circular dorsal ruffles, a specialized form of macropinocytosis that occurs at the dorsal surface rather than the leading edge of cells, depends on SHIP2-mediated formation of PI(3,4)P2 from PI(3,4,5)P3 [141]. Furthermore, a recent detailed analysis of PIs during macropinocytosis showed that in addition to SHIP2 the PI(3,4)P2-specific 4-phosphatase INPP4B as well as myotubularinrelated 3-phosphatases MTMR6 and 9 are essential for completion of macropinosome formation, but not for membrane ruffling [142]. Consistently, it has been found that both PI(3)P and Rab5 associate with macropinosomes prior to cup closure [140]. MTMR6 and 9 were proposed to act on the macropinocytic cup rather than on the internalized macropinosome, though their precise function remains to be defined. PI(3)P produced by INPP4B-mediated hydrolysis of PI(3,4)P2 was shown to increase conductance of the Ca2+-activated K+-channel KCa3.1. Inhibition of KCa3.1 or expression of a PI(3)P-activation deficient mutant of KCa3.1 both interfere with macropinocytosis [142], suggesting that MTMR6/9 may be required to restrict PI(3)P levels and KCa3.1 opening on the macropinocytic cup before macropinosome formation. In summary, while we have obtained detailed insight into the PI conversions during macropinocytosis the effectors that bind to these PIs and their role in macropinosome formation remain largely elusive. 1.4. Outlook: PI conversion en route to endosomes & implications for disease Even after 20 years of intense research the field of PIs in endocytic membrane trafficking continues to thrive. While the role of PI(4,5)P2 and of PI(3)P as denominators of plasma membrane and endosomal identity are well established the question of how PI conversion from the cell surface en route to endosomes occurs remains poorly understood. Recently, molecular connections between the machinery for endocytosis and early endosomes have emerged. For example, as described above the PI(4,5)P2 phosphatase OCRL visits late-stage, endocytic clathrin-coated pits and binds to both clathrin and the Rab5 effector APPL1 on peripheral early endosomes. These data suggest that OCRL functions as a link between late stages of endocytosis, possibly related to uncoating, and the acquisition of endosomal PI(3)P identity [84,86]. Dysfunction of OCRL is associated with endocytic defects in oculocerebrorenal syndrome of Lowe and in Dent's disease [84,86], inherited kidney disorders as well as with defective cytokinesis [143]. Trisomy for the OCRL-related brain-enriched endocytic PI(4,5)P2 phosphatase synaptojanin 1 is linked to early endosomal defects and mental retardation in Down syndrome [144] and in Parkinson's disease [145,146]. Conversely, accumulation of Aβ leads to decreased PI(4,5)P2 levels, a phenotype rescued by synaptojanin 1 haploinsufficiency in mice [147,148]. These findings together with the observation that low hippocampal PI(4,5)P2 levels contribute to impaired cognition in aged

mice [149] open the intriguing possibility that dyshomeostasis of PI(4,5)P2 metabolism may underlie some of the cognitive defects in neurodegenerative disorders and during aging. In addition to loss of PI(4,5)P2 the conversion of compartmental identity from the plasma membrane to endosomes also requires the activity of PI 3-kinases. How precisely this is accomplished remains largely unknown but it is tempting to speculate that plasma membrane synthesis of PI(3,4)P2 by class II PI3K C2α and subsequent hydrolysis of the 4-phosphate moiety by the PI(3,4)P2-specific 4-phosphatases INPP4A/B provides such a PI conversion shunt to generate endosomal PI(3)P (Fig. 3). Indeed, recent data indicate that endosomal PI(3)P levels do not only depend on synthesis of PI(3)P from PI by class III PI 3-kinase hVps34, but also by class II PI 3-kinases C2α and C2β [20,21]. Whether this class II-dependent pool of PI(3)P is the result of direct class II PI3K-mediated PI(3)P synthesis from PI [21] or is made indirectly by conversion of plasma membrane PI(3,4)P2 remains to be clarified. Based on these data it is conceivable that endosomes comprise distinct pools of PI(3)P akin to the distinct plasma membrane PI(4,5)P2 pools involved in various physiological functions ranging from CME to ion channel gating [14]. This hypothesis is supported by the observation that depletion of class III PI 3-kinase hVps34 primarily causes defects within the degradative branch of the endolysosomal system rather than a general loss of early endosomes [20,150]. Lastly, little is known regarding the mechanism of PI conversion from the endosomal system to the plasma membrane, e.g. during endocytic recycling of transferrin and other receptors. We postulate that similar mechanisms must operate at the level of endosomes to facilitate PI(3)P hydrolysis and acquisition of PI(4,5)P2 identity as membranes exocytotically fuse with the cell surface. This PI conversion may involve myotubularin 3-phosphatases, a family of enzymes linked to a number of human diseases such as Charcot Marie Tooth disease and myotubular myopathy [151]. Future studies will need to address these questions as well as the precise role of PIs and the mechanisms underlying PI conversion in clathrinindependent endocytic pathways in detail. Acknowledgements Work in the authors' laboratory is supported by grants from the DFG (SFB740/C08 and SFB958/A07 to V.H.) and by a long-term fellowship from the European Molecular Biology Organization (EMBO) to M.E.G. We thank Andrea L. Marat for critical reading of the article. References [1] P.W. Majerus, T.S. Ross, T.W. Cunningham, K.K. Caldwell, A.B. Jefferson, V.S. Bansal, Recent insights in phosphatidylinositol signaling, Cell 63 (1990) 459–465. [2] R.H. Michell, Inositol lipids in cellular signalling mechanisms, Trends Biochem. Sci. 17 (1992) 274–276. [3] P.V. Schu, K. Takegawa, M.J. Fry, J.H. Stack, M.D. Waterfield, S.D. Emr, Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting, Science 260 (1993) 88–91. [4] K.A. Beck, H. Keen, Interaction of phosphoinositide cycle intermediates with the plasma membrane-associated clathrin assembly protein AP-2, J. Biol. Chem. 266 (1991) 4442–4447. [5] S.M. Voglmaier, J.H. Keen, J.-e. Murphy, C.D. Ferris, G.D. Prestwlch, S.H. Snyder, A.B. Theibert, Inositol hexakisphosphate receptor identified as the clathrin assembly protein AP-2, Biochem. Biophys. Res. Commun. 187 (1992) 158–163. [6] F.A. Norris, E. Ungewickell, P.W. Majerus, Inositol hexakisphosphate binds to clathrin assembly protein 3 (AP-3/AP180) and inhibits clathrin cage assembly in vitro, J. Biol. Chem. 270 (1995) 214–217. [7] I. Gaidarov, Q. Chen, J.R. Falck, K.K. Reddy, J.H. Keen, A functional phosphatidylinositol 3,4,5-trisphosphate/phosphoinositide binding domain in the clathrin adaptor AP-2 α subunit. Implications for the endocytic pathway, J. Biol. Chem. 271 (1996) 20922–20929. [8] M. Jost, F. Simpson, J.M. Kavran, M.A. Lemmon, S.L. Schmid, Phosphatidylinositol4,5-bisphosphate is required for endocytic coated vesicle formation, Curr. Biol. 8 (1998) 1399–1402. [9] K. Salim, M.J. Bottomley, E. Querfurthl, M.J. Zvelebill, I. Gout, R. Scaife, R.L. Margolis, R. Gigg, C.E. Smith, P.C. Driscoll, M.D. Waterfieldl, G. Panayotou, Distinct specificity in the recognition of phosphoinositides by the pleckstrin homology domains of dynamin and Bruton's tyrosine kinase, EMBO J. 15 (1996) 6241–6250. [10] J. Zheng, S.M. Cahill, M.A. Lemmon, D. Fushman, J. Schlessinger, D. Cowburn, Identification of the binding site for acidic phospholipids on the pH domain of



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Phosphoinositides, kinases and adaptors coordinating endocytosis in Trypanosoma brucei.

Phosphoinositides regulate ion channels.

The role of phosphoinositides in synapse function.

Role of phosphoinositides in transmembrane signaling.

Detection and manipulation of phosphoinositides.

Analysis, regulation, and roles of endosomal phosphoinositides.

Mirror image phosphoinositides regulate autophagy.

Phosphoinositides: Key modulators of energy metabolism.

Inositol phosphates and phosphoinositides activate insulin-degrading enzyme, while phosphoinositides also mediate binding to endosomes.

Endocytosis.

Phosphoinositides in the regulation of actin cortex and cell migration.

Phosphoinositides, Major Actors in Membrane Trafficking and Lipid Signaling Pathways.

Interaction of protein kinase C with phosphoinositides.

Systems dynamics in endocytosis.

Inositol phosphates and phosphoinositides in rat liver nodules.

Requirement of Phosphoinositides Containing Stearic Acid To Control Cell Polarity.

Calcium channels for endocytosis.

Neuronal signaling through endocytosis.

Exploiting endocytosis for nanomedicines.

Endocytosis and Enamel Formation.

Plant phosphoinositides-complex networks controlling growth and adaptation.

Homotypic vacuole fusion requires VTI11 and is regulated by phosphoinositides.

The nutritional importance of inositol and the phosphoinositides.

Endocytosis, signaling, and beyond.