Receptor-mediated Endocytosis 8 Utilizes an N-terminal Phosphoinositide-binding Motif to Regulate Endosomal Clathrin Dynamics

Receptor-mediated endocytosis 8 (RME-8) is a DnaJ domain containing protein implicated in translocation of Hsc70 to early endosomes for clathrin removal during retrograde transport. Previously, we have demonstrated that RME-8 associates with early endosomes in a phosphatidylinositol 3-phosphate (PI(3)P)-dependent fashion. In this study, we have now identified amino acid determinants required for PI(3)P binding within a region predicted to adopt a pleckstrin homology-like fold in the N terminus of RME-8. The ability of RME-8 to associate with PI(3)P and early endosomes is largely abolished when residues Lys¹⁷, Trp²⁰, Tyr²⁴, or Arg²⁶ are mutated resulting in diffuse cytoplasmic localization of RME-8 while maintaining the ability to interact with Hsc70. We also provide evidence that RME-8 PI(3)P binding regulates early endosomal clathrin dynamics and alters the steady state localization of the cation-independent mannose 6-phosphate receptor. Interestingly, RME-8 endosomal association is also regulated by the PI(3)P-binding protein SNX1, a member of the retromer complex. Wild type SNX1 restores endosomal localization of RME-8 W20A, whereas a SNX1 variant deficient in PI(3)P binding disrupts endosomal localization of wild type RME-8. These results further highlight the critical role for PI(3)P in the RME-8-mediated organizational control of various endosomal activities, including retrograde transport.     

Sequential actions of myotubularin lipid phosphatases regulate endosomal PI(3)P and growth factor receptor trafficking

Two different human diseases, X-linked myotubular myopathy and Charcot-Marie-Tooth disease, result from mutant MTM1 or MTMR2 lipid phosphatases. Although events involved in endosomal PI(3)P and PI(3,5)P(2) synthesis are well established and pivotal in receptor signaling and degradation, enzymes involved in phosphoinositide degradation and their roles in trafficking are incompletely characterized. Here, we dissect the functions of the MTM1 and MTMR2 myotubularins and establish how they contribute to endosomal PI(3)P homeostasis. By mimicking loss of function in disease through siRNA-mediated depletion of the myotubularins, excess PI(3)P accumulates on early (MTM1) and late (MTMR2) endosomes. Surprisingly, the increased PI(3)P blocks the egress of epidermal growth factor receptors from early or late endosomes, suggesting that the accumulation of signaling receptors in distinct endosomes may contribute to the unique disease etiologies when MTM1 or MTMR2 are mutant. We further demonstrate that direct myotubularin binding to the type III PI 3-kinase complex hVps34/hVps15 leads to phosphatase inactivation. The lipid kinase-phosphatase interaction also precludes interaction of the PI 3-kinase with Rab GTPase activators. Thus, unique molecular complexes control kinase and phosphatase activation and locally regulate PI(3)P on discrete endosome populations, thereby providing a molecular rationale for related human myo- and neuropathies.     

Endosomal phosphoinositides and human diseases

Phosphoinositides (PIs) are lipid second messengers implicated in signal transduction and membrane trafficking. Seven distinct PIs can be synthesized by phosphorylation of the inositol ring of phosphatidylinositol (PtdIns), and their metabolism is accurately regulated by PI kinases and phosphatases. Two of the PIs, PtdIns3 P and PtdIns(3,5) P ₂, are present on intracellular endosomal compartments, and several studies suggest that they have a role in membrane remodeling and trafficking. We refer to them as 'endosomal PIs'. An increasing number of human genetic diseases including myopathy and neuropathies are associated to mutations in enzymes regulating the turnover of these endosomal PIs. The PtdIns3 P and PtdIns(3,5) P ₂ 3-phosphatase myotubularin gene is mutated in X-linked centronuclear myopathy, whereas its homologs MTMR2 and MTMR13 and the PtdIns(3,5) P ₂ 5-phosphatase SAC3/FIG4 are implicated in Charcot–Marie–Tooth peripheral neuropathies. Mutations in the gene encoding the PtdIns3 P 5-kinase PIP5K3/PIKfyve have been found in patients affected with François–Neetens fleck corneal dystrophy. This review presents the roles of the endosomal PIs and their regulators and proposes defects of membrane remodeling as a common pathological mechanism for the corresponding diseases.     

Myotubularin lipid phosphatase binds the hVPS15/hVPS34 lipid kinase complex on endosomes

Myotubularins constitute a ubiquitous family of phosphatidylinositol (PI) 3-phosphatases implicated in several neuromuscular disorders. Myotubularin [myotubular myopathy 1 (MTM1)] PI 3-phosphatase is shown associated with early and late endosomes. Loss of endosomal phosphatidylinositol 3-phosphate [PI(3)P] upon overexpression of wild-type MTM1, but not a phosphatase-dead MTM1C375S mutant, resulted in altered early and late endosomal PI(3)P levels and rapid depletion of early endosome antigen-1. Membrane-bound MTM1 was directly complexed to the hVPS15/hVPS34 [vacuolar protein sorting (VPS)] PI 3-kinase complex with binding mediated by the WD40 domain of the hVPS15 (p150) adapter protein and independent of a GRAM-domain point mutation that blocks PI(3,5)P(2) binding. The WD40 domain of hVPS15 also constitutes the binding site for Rab7 and, as shown previously, contributes to Rab5 binding. In vivo, the hVPS15/hVPS34 PI 3-kinase complex forms mutually exclusive complexes with the Rab GTPases (Rab5 or Rab7) or with MTM1, suggesting a competitive binding mechanism. Thus, the Rab GTPases together with MTM1 likely serve as molecular switches for controlling the sequential synthesis and degradation of endosomal PI(3)P. Normal levels of endosomal PI(3)P and PI(3,5)P(2) are crucial for both endosomal morphology and function, suggesting that disruption of endosomal sorting and trafficking in skeletal muscle when MTM1 is mutated may be a key factor in precipitating X-linked MTM.     

The phosphoinositide-3-phosphatase MTMR2 associates with MTMR13, a membrane-associated pseudophosphatase also mutated in type 4B Charcot-Marie-Tooth disease

Charcot-Marie-Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by distinctive, focally folded myelin sheaths. CMT4B is caused by recessively inherited mutations in either myotubularin-related 2 (MTMR2) or MTMR13 (also called SET-binding factor 2). MTMR2 encodes a member of the myotubularin family of phosphoinositide-3-phosphatases, which dephosphorylate phosphatidylinositol 3-phosphate (PI(3)P) and bisphosphate PI(3,5)P2. MTMR13 encodes a large, uncharacterized member of the myotubularin family. The MTMR13 phosphatase domain is catalytically inactive because the essential Cys and Arg residues are absent. Given the genetic association of both MTMR2 and MTMR13 with CMT4B, we investigated the biochemical relationship between these two proteins. We found that the endogenous MTMR2 and MTMR13 proteins are associated in human embryonic kidney 293 cells. MTMR2-MTMR13 association is mediated by coiled-coil sequences present in each protein. We also examined the cellular localization of MTMR2 and MTMR13 using fluorescence microscopy and subcellular fractionation. We found that (i) MTMR13 is a predominantly membrane-associated protein; (ii) MTMR2 and MTMR13 cofractionate in both a light membrane fraction and a cytosolic fraction; and (iii) MTMR13 membrane association is mediated by the segment of the protein which contains the pseudophosphatase domain. This work, which describes the first cellular or biochemical investigation of the MTMR13 pseudophosphatase protein, suggests that MTMR13 functions in association with MTMR2. Loss of MTMR13 function in CMT4B2 patients may lead to alterations in MTMR2 function and subsequent alterations in 3-phosphoinositide signaling. Such a mechanism would explain the strikingly similar phenotypes of patients with recessive mutations in either MTMR2 or MTMR13.     

Phosphatidylinositol-5-phosphate activation and conserved substrate specificity of the myotubularin phosphatidylinositol 3-phosphatases

Phosphoinositides control many different processes required for normal cellular function. Myotubularins are a family of Phosphatidylinositol 3-phosphate (PtdIns3P) phosphatases identified by the positional cloning of the MTM1 gene in patients suffering from X-linked myotubular myopathy and the MTMR2 gene in patients suffering from the demyelinating neuropathy Charcot-Marie-Tooth disease type 4B. MTM1 is a phosphatidylinositol phosphatase with reported specificity toward PtdIns3P, while the related proteins MTMR2 and MTMR3 hydrolyze both PtdIns3P and PtdIns(3,5)P2. We have investigated MTM1 and MTMR6 and find that they use PtdIns(3,5)P2 in addition to PtdIns3P as a substrate in vitro. The product of PtdIns(3,5)P2 hydrolysis, PtdIns5P, causes MTM1 to form a heptameric ring that is 12.5 nm in diameter, and it is a specific allosteric activator of MTM1, MTMR3, and MTMR6. A disease-causing mutation at arginine 69 of MTM1 falling within a putative pleckstrin homology domain reduces the ability of the enzyme to respond to PtdIns5P. We propose that the myotubularin family of enzymes utilize both PtdIns3P and PtdIns(3,5)P2 as substrates, and that PtdIns5P functions in a positive feedback loop controlling their activity. These findings highlight the importance of regulated phosphatase activity for the control of phosphoinositide metabolism.     

Specificity of the myotubularin family of phosphatidylinositol-3-phosphatase is determined by the PH/GRAM domain

Myotubularins (MTM) are a large subfamily of lipid phosphatases that specifically dephosphorylate at the D3 position of phosphatidylinositol 3-phosphate (PI(3)P) in PI(3)P and PI(3,5)P2. We recently found that MTMR6 specifically inhibits the Ca2+-activated K+ channel, KCa3.1, by dephosphorylating PI(3)P. We now show that inhibition is specific for MTMR6 and other MTMs do not inhibit KCa3.1. By replacing either or both of the coiled-coil (CC) and pleckstrin homology/GRAM (PH/G) domains of MTMs that failed to inhibit KCa3.1 with the CC and PH/G domains of MTMR6, we found that chimeric MTMs containing both the MTMR6 CC and PH/G domains functioned like MTMR6 to inhibit KCa3.1 channel activity, whereas chimeric MTMs containing either domain alone did not. Immunofluorescent microscopy demonstrated that both the MTMR6 CC and PH/G domains are required to co-localize MTMR6 to the plasma membrane with KCa3.1. These findings support a model in which two specific low affinity interactions are required to co-localize MTMR6 with KCa3.1: 1) between the CC domains on MTMR6 and KCa3.1 and (2) between the PH/G domain and a component of the plasma membrane. Our inability to detect significant interaction of the MTMR6 G/PH domain with phosphoinositides suggests that this domain may bind a protein. Identifying the specific binding partners of the CC and PH/G domains on other MTMs will provide important clues to the specific functions regulated by other MTMs as well as the mechanism(s) whereby loss of some MTMs lead to disease.     

Crystal Structure of Human Myotubularin-Related Protein 1 Provides Insight into the Structural Basis of Substrate Specificity

Myotubularin-related protein 1 (MTMR1) is a phosphatase that belongs to the tyrosine/dual-specificity phosphatase superfamily. MTMR1 has been shown to use phosphatidylinositol 3-monophosphate (PI(3)P) and/or phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) as substrates. Here, we determined the crystal structure of human MTMR1. The refined model consists of the Pleckstrin homology (PH)-GRAM and phosphatase (PTP) domains. The overall structure was highly similar to the previously reported MTMR2 structure. Interestingly, two phosphate molecules were coordinated by strictly conserved residues located in the C(X)₅R motif of the active site. Additionally, our biochemical studies confirmed the substrate specificity of MTMR1 for PI(3)P and PI(3,5)P₂ over other phosphatidylinositol phosphates. Our structural and enzymatic analyses provide insight into the catalytic mechanism and biochemical properties of MTMR1.     

SWIP mediates retromer-independent membrane recruitment of the WASH complex

The pentameric WASH complex facilitates endosomal protein sorting by activating Arp2/3, which in turn leads to the formation of F-actin patches specifically on the endosomal surface. It is generally accepted that WASH complex attaches to the endosomal membrane via the interaction of its subunit FAM21 with the retromer subunit VPS35. However, we observe the WASH complex and F-actin present on endosomes even in the absence of VPS35. We show that the WASH complex binds to the endosomal surface in both a retromer-dependent and a retromer-independent manner. The retromer-independent membrane anchor is directly mediated by the subunit SWIP. Furthermore, SWIP can interact with a number of phosphoinositide species. Of those, our data suggest that the interaction with phosphatidylinositol-3,5-bisphosphate (PI(3,5)P₂) is crucial to the endosomal binding of SWIP. Overall, this study reveals a new role of the WASH complex subunit SWIP and highlights the WASH complex as an independent, self-sufficient trafficking regulator.     

The role of the phosphoinositides at the Golgi complex

The phosphorylated derivatives of phosphatidylinositol (PtdIns), known as the polyphosphoinositides (PIs), represent key membrane-localized signals in the regulation of fundamental cell processes, such as membrane traffic and cytoskeleton remodelling. The reversible production of the PIs is catalyzed through the combined activities of a number of specific phosphoinositide phosphatases and kinases that can either act separately or in concert on all the possible combinations of the 3, 4, and 5 positions of the inositol ring. So far, seven distinct PI species have been identified in mammalian cells and named according to their site(s) of phosphorylation: PtdIns 3-phosphate (PI3P); PtdIns 4-phosphate (PI4P); PtdIns 5-phosphate (PI5P); PtdIns 3,4-bisphosphate (PI3,4P2); PtdIns 4,5-bisphosphate (PI4,5P2); PtdIns 3,5-bisphosphate (PI3,5P2); and PtdIns 3,4,5-trisphosphate (PI3,4,5P3). Over the last decade, accumulating evidence has indicated that the different PIs serve not only as intermediates in the synthesis of the higher phosphorylated phosphoinositides, but also as regulators of different protein targets in their own right. These regulatory actions are mediated through the direct binding of their protein targets. In this way, the PIs can control the subcellular localization and activation of their various effectors, and thus execute a variety of cellular responses. To exert these functions, the metabolism of the PIs has to be finely regulated both in time and space, and this is achieved by controlling the subcellular distribution, regulation, and activation states of the enzymes involved in their synthesis and removal (kinases and phosphatases). These exist in many different isoforms, each of which appears to have a distinctive intracellular localization and regulation. As a consequence of this subcompartimentalized PI metabolism, a sort of "PI-fingerprint" of each cell membrane compartment is generated. When combined with the targeted recruitment of their protein effectors and the different intracellular distributions of other lipids and regulatory proteins (such as small GTPases), these factors can maintain and determine the identity of the cell organelles despite the extensive membrane flux []. Here, we provide an overview of the regulation and roles of different phosphoinositide kinases and phosphatases and their lipid products at the Golgi complex.     

The phosphatidylinositol 3-phosphate phosphatase myotubularin- related protein 6 (MTMR6) is a negative regulator of the Ca2+-activated K+ channel KCa3.1

Myotubularins (MTMs) belong to a large subfamily of phosphatases that dephosphorylate the 3' position of phosphatidylinositol 3-phosphate [PI(3)P] and PI(3,5)P(2). MTM1 is mutated in X-linked myotubular myopathy, and MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth syndrome. However, little is known about the general mechanism(s) whereby MTMs are regulated or the specific biological processes regulated by the different MTMs. We identified a Ca(2+)-activated K channel, K(Ca)3.1 (also known as KCa4, IKCa1, hIK1, or SK4), that specifically interacts with the MTMR6 subfamily of MTMs via coiled coil (CC) domains on both proteins. Overexpression of MTMR6 inhibited K(Ca)3.1 channel activity, and this inhibition required MTMR6's CC and phosphatase domains. This inhibition is specific; MTM1, a closely related MTM, did not inhibit K(Ca)3.1. However, a chimeric MTM1 in which the MTM1 CC domain was swapped for the MTMR6 CC domain inhibited K(Ca)3.1, indicating that MTM CC domains are sufficient to confer target specificity. K(Ca)3.1 was also inhibited by the PI(3) kinase inhibitors LY294002 and wortmannin, and this inhibition was rescued by the addition of PI(3)P, but not other phosphoinositides, to the patch pipette solution. PI(3)P also rescued the inhibition of K(Ca)3.1 by MTMR6 overexpression. These data, when taken together, indicate that K(Ca)3.1 is regulated by PI(3)P and that MTMR6 inhibits K(Ca)3.1 by dephosphorylating the 3' position of PI(3)P, possibly leading to decreased PI(3)P in lipid microdomains adjacent to K(Ca)3.1. K(Ca)3.1 plays important roles in controlling proliferation by T cells, vascular smooth muscle cells, and some cancer cell lines. Thus, our findings not only provide unique insights into the regulation of K(Ca)3.1 channel activity but also raise the possibility that MTMs play important roles in the negative regulation of T cells and in conditions associated with pathological cell proliferation, such as cancer and atherosclerosis.     

Myotubularin regulates the function of the late endosome through the gram domain-phosphatidylinositol 3,5-bisphosphate interaction

Myotubularin and related proteins constitute a large and highly conserved family possessing phosphoinositide 3-phosphatase activity, although not all members possess this activity. This family contains a conserved region called the GRAM domain that is found in a variety of proteins associated with membrane-coupled processes and signal transduction. Mutations of myotubularin are found in X-linked myotubular myopathy, a severe muscle disease. Mutations in the GRAM domain are responsible for this condition, suggesting crucial roles for this region. Here, we show that the GRAM domain of myotubularin binds to phosphoinositide with the highest affinity to phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)). In patients with myotubular myopathy, mutations in the myotubularin GRAM domain eliminate this binding, indicating that the PtdIns(3,5)P(2) binding ability of the GRAM (glucosyltransferases, Rablike GTPase activators and myotubularin) domain is crucial for the functions of myotubularin in vivo. Stimulation of epidermal growth factor recruits myotubularin to the late endosomal compartment in a manner dependent on the phosphoinositide binding. Overexpression of myotubularin inhibits epidermal growth factor receptor trafficking from late endosome to lysosome and induces the large endosomal vacuoles. Thus, our data suggest that myotubularin phosphatase physiologically functions in late endosomal trafficking and vacuolar morphology through interaction with PtdIns(3,5)P(2).     

Endosomal targeting of the phosphoinositide 3-phosphatase MTMR2 is regulated by an N-terminal phosphorylation site

MTMR2 is a member of the myotubularin family of inositol lipid phosphatases, a large protein-tyrosine phosphatase subgroup that is conserved from yeast to humans. Furthermore, the peripheral neuromuscular disease Charcot-Marie Tooth disease type 4B has been attributed to mutations in the mtmr2 gene. Because the molecular mechanisms regulating MTMR2 have been poorly defined, we investigated whether reversible phosphorylation might regulate MTMR2 function. We used mass spectrometry-based methods to identify a high stoichiometry phosphorylation site on serine 58 of MTMR2. Phosphorylation at Ser(58), or a phosphomimetic S58E mutation, markedly decreased MTMR2 localization to endocytic vesicular structures. In contrast, a phosphorylation-deficient MTMR2 mutant (S58A) displayed constitutive localization to early endocytic structures. This localization pattern was accompanied by displacement of a PI(3)P-specific sensor protein and an increase in signal transduction pathways. Thus, MTMR2 phosphorylation is likely to be a critical mechanism by which MTMR2 access to its lipid substrate(s) is temporally and spatially regulated, thereby contributing to the control of downstream endosome maturation events.     

PI5P migrates out of the PIP shadow

In a recent study published in EMBO reports, the group of Jorgen Wesche identified a new role for PI5P as a positive regulator of cell migration, most likely by facilitating actin cytoskeletal rearrangements.EMBO reports (2013) 14 1, 57–64 doi:10.1038/embor.2012.183The dynamic nature of membrane phospholipid turnover was first described by Hokin and Hokin during their studies on exocrine tissue stimulation (reviewed in [1]). Subsequently, phosphatidylinositol (PI) and its phosphorylated products, called phosphoinositides, were shown to have a fundamental role in regulating membrane–cytosol interfaces in several contexts, including regulating signal transduction, membrane traffic and permeability, the cytoskeleton, nuclear events and transport [1]. Seven phosphoinositides have been identified and are defined by the phosphorylation state of the inositol headgroup at the 3′-, 4′- and 5′-position, which is regulated reversibly by several lipid kinases and phosphatases [1]. Of the seven lipid species, phosphatidylinositol-5-phosphate (PI5P) was most recently discovered by the Cantley group and remains the most enigmatic of the phosphoinositide family [2]. PI5P levels have been shown to change in response to stimuli such as thrombin, histamine, insulin and oxidative stress, and have been suggested to regulate various processes, including signalling pathways, nuclear functions, vesicular transport and cytoskeletal organization [3,4,5]. In a recent issue of EMBO reports, Oppelt and colleagues identified a new role for PI5P as a positive regulator of cell migration probably by facilitating actin cytoskeletal rearrangements [6].Unlike other phosphoinositides (PI3P and PI4P) that can be produced through the direct phosphorylation of PI, there is no evidence that PI5P can be produced through the direct in vivo phosphorylation of PI. Instead, PI4P 5-kinases generate PI(4,5)P₂ from PI4P and PIKfyve generates PI(3,5)P₂ from PI3P, and the products PI(3,5)P₂ and PI(4,5)P₂ are dephosphorylated by myotubularin-related proteins (that is, MTMR3) and inositol 4-phosphatases (PI(4,5)P₂ phosphatases type I and II), respectively, to produce PI5P (Fig 1). Recent in vivo evidence indicates that the PIKfyve–MTMR pathway is responsible for most PI5P production [7]. The study from the Wesche lab shows that production of PI5P through the MTMR3 pathway is important for cell migration [6]. After initially observing that depletion of the class III PI(3)K Vps34 and its lipid product, PI3P, impairs cell migration, the authors performed a small interfering RNA (siRNA) screen to identify FYVE or PX-domain-containing PI3P effectors that regulate this process. In addition to factors already known to promote migration—for example, Cdc42, FGD1, FGD2 and Hrs—PIKfyve and MTMR3 were identified as new candidates. PIKfyve has a PI3P-binding FYVE domain and, as mentioned above, synthesizes PI(3,5)P₂, whilst MTMR3, a member of the myotubularin family containing a PI3P-binding PH-GRAM domain, is a phosphatase that dephosphorylates PI(3,5)P₂ to PI5P. Cells depleted of PIKfyve or MTMR3 showed impaired cell polarization and defects in orienting actin filaments and the Golgi complex towards the growth factor stimulus. In addition, siRNA experiments and pharmacological inhibition of PIKfyve decreased migration velocity and persistence. These findings were confirmed in vivo when ablation of MTMR3 in border cells inhibited border cell migration in Drosophila during oogenesis. Taken together, the authors demonstrate that the lipid enzymes Vps34, PIKfyve and MTMR3 regulate the PI3P–PI(3,5)P₂–PI5P phosphoinositide interconversion pathway that mediates cell migration [6].Open in a separate windowFigure 1Pathways of PI5P synthesis and turnover. Pathways demonstrated in vivo are shown in bold text and arrows, whereas in vitro, putative pathways are represented in italics and broken arrows. Thick arrows highlight the PI5P production pathway responsible for cell migration as reported in reference [6]. IpgD, invasion plasmid gene D; MTM, myotubularin; PI, phosphatidylinositol; PI(3)K, phosphoinositide 3-kinase; PI5P, phosphatidylinositol-5-phosphate; PIKfyve, phosphoinositide kinase, FYVE finger containing; PTPMP1, protein tyrosine phosphatase, mitochondrial 1.The low abundancy, dynamic turnover and tight spatial restriction of the phosphoinositides enable them to mediate acute responses within cells [1]. Part of the initial difficulty in identifying PI5P was due to the fact that it is the phosphoinositide of lowest abundance—approximately 1–2% of PI4P—and that earlier high-performance liquid chromatography (HPLC) techniques had erroneously measured PI5P as its PI4P isomer [8]. By using improved biochemical techniques, the authors measured phosphoinoside levels during cell migration and found specifically that levels of PI5P rise acutely in response to migratory stimulation by fibroblast growth factor 1 (FGF1). Cells treated with PIKfyve and MTMR3 siRNA failed to produce PI5P on FGF1 stimulation, again suggesting that PIKfyve and MTMR3 constitute the interconversion pathway responsible for the production of PI5P during migration. Importantly, Oppelt and colleagues showed convincingly that PI5P is a relevant signalling mediator of cell migration [6]—when exogenous PI5P was added to MTMR3 and PIKfyve siRNA-treated cells, migration was partly rescued. To further confirm this finding, the authors showed that overexpression of the Shigella flexneri virulence factor IpgD, a PI(4,5)P₂ 4-phosphatase that converts PI(4,5)P₂ to PI5P [9], and production of endogenous PI5P in MTMR3 siRNA-treated cells also rescue migration defects. In fact, this production of PI5P from PI(4,5)P₂ proved that PI5P is a relevant migratory signal and not merely an intermediate to the production of PI(4,5)P₂, which has been implicated previously in regulating actin dynamics and, by contrast, requires PI4P synthesis—another potential intermediate for its signalling effects [1]. Interestingly, manipulation of PI5P levels in control cells does not initiate cell migration in the absence of stimulation, suggesting that PI5P is not sufficient to promote migration; instead, the pathway works together with others to promote migration and there is an inhibitory regulatory pathway that acts downstream from PI5P. This clearly demonstrates that it is PI5P and not PI3P, PI(3,5)P₂ or PI(4,5)P₂ that regulates cell migration.Preliminary evidence from Oppelt and colleagues suggests that PI5P might help to regulate actin cytoskeletal remodelling during migration [6]. For instance, silencing of MTMR3 and PIKfyve resulted in actin fibres that are unable to orient properly and form knots when cell migration was stimulated. These findings complement previous work indicating that PI5P regulates cytoskeletal organization in other contexts. For instance, PI5P production in response to insulin stimulation or on overexpression of the bacterial virulence factor IpgD induces disassembly of actin stress fibres [9,10]. The molecular mechanisms underlying PI5P''s regulation of actin polymerization and disassembly remain unclear. Additionally, the subcellular location of PI5P production in response to these agonists remains largely undetermined; however, membrane fractionation and HPLC analysis has localized PI5P to the nucleus, endoplasmic reticulum, Golgi and plasma membrane under basal conditions [8]. Standard tools used to study phosphoinositide localization have yet to be developed for PI5P. PI5P-binding domains are beginning to be identified and include the PHD domain of ING2, and the PH domain of Dok proteins [4], but it will be important to develop PI5P probes as well as continue to identify additional effectors to better understand PI5P function.The PI5P field is at a naive, yet exciting, stage in which studies such as that of Oppelt and colleagues advance our understanding of PI5P multiple roles in cellular function. Although this study has concentrated largely on identifying Vps34, PIKfyve and MTMR3 in the production of PI5P in response to migration stimuli, it will be essential to explore how the activities of these enzymes are regulated. Moreover, the mechanism of PI5P catabolism in this context and others, as well as the precise molecular basis for PI5P actions in the cellular contexts, have to be further elucidated. Finally, the crosstalk between PI5P and other phosphoinositide-mediated signalling cascades, such as the PI(3,4,5)P₃–Akt pathway, will have to be closely examined in the context of cell migration, based on previous studies showing that PI5P is a positive regulator of class IA PI(3)Ks [3].     

Phosphatidylinositol 4‐kinase IIα function at endosomes is regulated by the ubiquitin ligase Itch

Phosphatidylinositol (PI) 4‐phosphate (PI(4)P) and its metabolizing enzymes serve important functions in cell signalling and membrane traffic. PI 4‐kinase type IIα (PI4KIIα) regulates Wnt signalling, endosomal sorting of signalling receptors, and promotes adaptor protein recruitment to endosomes and the trans‐Golgi network. Here we identify the E3 ubiquitin ligase Itch as binding partner and regulator of PI4KIIα function. Itch directly associates with and ubiquitinates PI4KIIα, and both proteins colocalize on endosomes containing Wnt‐activated frizzled 4 (Fz4) receptor. Depletion of PI4KIIα or Itch regulates Wnt signalling with corresponding changes in Fz4 internalization and degradative sorting. These findings unravel a new molecular link between phosphoinositide‐regulated endosomal membrane traffic, ubiquitin and the modulation of Wnt signalling.     

Drosophila Mtm and class II PI3K coregulate a PI(3)P pool with cortical and endolysosomal functions

Reversible phosphoinositide phosphorylation provides a dynamic membrane code that balances opposing cell functions. However, in vivo regulatory relationships between specific kinases, phosphatases, and phosphoinositide subpools are not clear. We identified myotubularin (mtm), a Drosophila melanogaster MTM1/MTMR2 phosphoinositide phosphatase, as necessary and sufficient for immune cell protrusion formation and recruitment to wounds. Mtm-mediated turnover of endosomal phosphatidylinositol 3-phosphate (PI(3)P) pools generated by both class II and III phosphatidylinositol 3-kinases (Pi3K68D and Vps34, respectively) is needed to down-regulate membrane influx, promote efflux, and maintain endolysosomal homeostasis. Endocytosis, but not endolysosomal size, contributes to cortical remodeling by mtm function. We propose that Mtm-dependent regulation of an endosomal PI(3)P pool has separable consequences for endolysosomal homeostasis and cortical remodeling. Pi3K68D depletion (but not Vps34) rescues protrusion and distribution defects in mtm-deficient immune cells and restores functions in other tissues essential for viability. The broad interactions between mtm and class II Pi3K68D suggest a novel strategy for rebalancing PI(3)P-mediated cell functions in MTM-related human disease.     

The DnaJ-domain protein RME-8 functions in endosomal trafficking

Through a proteomic analysis of clathrin-coated vesicles from rat liver we identified the mammalian homolog of receptor-mediated endocytosis 8 (RME-8), a DnaJ domain-containing protein originally identified in a screen for endocytic defects in Caenorhabditis elegans. Mammalian RME-8 has a broad tissue distribution, and affinity selection assays reveal the ubiquitous chaperone Hsc70, which regulates protein conformation at diverse membrane sites as the major binding partner for its DnaJ domain. RME-8 is tightly associated with microsomal membranes and co-localizes with markers of the endosomal system. Small interfering RNA-mediated knock down of RME-8 has no influence on transferrin endocytosis but causes a reduction in epidermal growth factor internalization. Interestingly, and consistent with a localization to endosomes, knock down of RME-8 also leads to alterations in the trafficking of the cation-independent mannose 6-phosphate receptor and improper sorting of the lysosomal hydrolase cathepsin D. Our data demonstrate that RME-8 functions in intracellular trafficking and provides the first evidence of a functional role for a DnaJ domain-bearing co-chaperone on endosomes.     

Structural basis for endosomal targeting by FYVE domains

The FYVE domain is a conserved protein motif characterized by its ability to bind with high affinity and specificity to phosphatidylinositol 3-phosphate (PI3P), a phosphoinositide highly enriched in early endosomes. The PI3P polar head group contacts specific amino acid residues that are conserved among FYVE domains. Despite full conservation of these residues, the ability of different FYVE domains to bind to endosomes in cells is highly variable. Here we show that the endosomal localization in intact cells absolutely requires structural features intrinsic to the FYVE domain in addition to the PI3P binding pocket. These features are involved in FYVE domain dimerization and in interaction with the membrane bilayer. These interactions, which are determined by non-conserved residues, are likely to be essential for the temporal and spatial control of protein associations at the membrane-cytosol interface within the endocytic pathway.     

Snx10 and PIKfyve are required for lysosome formation in osteoclasts

Bone resorption and organelle homeostasis in osteoclasts require specialized intracellular trafficking. Sorting nexin 10 (Snx10) is a member of the sorting nexin family of proteins that plays crucial roles in cargo sorting in the endosomal pathway by its binding to phosphoinositide(3)phosphate (PI3P) localized in early endosomes. We and others have shown previously that the gene encoding sorting Snx10 is required for osteoclast morphogenesis and function, as osteoclasts from humans and mice lacking functional Snx10 are dysfunctional. To better understand the role and mechanisms by which Snx10 regulates vesicular transport, the aim of the present work was to study PIKfyve, another PI3P-binding protein, which phosphorylates PI3P to PI(3,5)P2. PI(3,5)P2 is known to be required for endosome/lysosome maturation, and the inhibition of PIKfyve causes endosome enlargement. Overexpression of Snx10 also induces accumulation of early endosomes suggesting that both Snx10 and PIKfyve are required for normal endosome/lysosome transition. Apilimod is a small molecule with specific, nanomolar inhibitory activity on PIKfyve but only in the presence of key osteoclast factors CLCN7, OSTM1, and Snx10. This observation suggests that apilimod's inhibitory effects are mediated by endosome/lysosome disruption. Here we show that both Snx10 and PIKfyve colocalize to early endosomes in osteoclasts and coimmunoprecipitate in vesicle fractions. Treatment with 10 nM apilimod or genetic deletion of PIKfyve in cells resulted in the accumulation of early endosomes, and in the inhibition of osteoclast differentiation, lysosome formation, and secretion of TRAP from differentiated osteoclasts. Snx10 and PIKfyve also colocalized in gastric zymogenic cells, another cell type impacted by Snx10 mutations. Apilimod-specific inhibition of PIKfyve required Snx10 expression, as it did not inhibit lysosome biogenesis in Snx10-deficient osteoclasts. These findings suggest that Snx10 and PIKfyve are involved in the regulation of endosome/lysosome homeostasis via the synthesis of PI(3,5)P2 and may point to a new strategy to prevent bone loss.