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Neurons use and recycle synaptic vesicles continuously during synaptic transmission. The mechanisms of synaptic vesicle release and recycling have been a subject of much study. There are several known mechanisms of vesicle endocytosis, and recent work suggests that ultrafast endocytosis (UFE) occurs under physiologic conditions at a much faster timescale than previously thought. In this process, endocytosis of membrane occurs in a clathrin-independent manner on the order of 100ms in both C. elegans and mammalian mouse neurons. Following the retrieval of membrane, endocytosed vesicles fuse to an endosome, from which clathrin coated pits bud off, and regenerate synaptic vesicles within 30-300ms. In spite of its rapid time-scale, separation from the plasma membrane in UFE is dynamin-dependent and requires actin. The other proteins that are required to orchestrate the steps of UFE at its characteristic rapid speed are not yet known. Here we show that synaptojanin and endophilin play critical roles in allowing ultrafast endocytosis to achieve its rapid time-scale. Specifically, we show that endophilin and synaptojanin are each necessary for UFE to occur. In this manuscript, we go one step further to address these proteins��� specific roles in this process. We show that UFE requires a functional 5-phosphatase domain but not Sac-1 domain of synaptojanin to occur at its characteristic rapid timescale and that endophilin is required for neck formation in UFE. We also show that endophilin A1 and A2 can both fill the endophilin requirement for UFE and reacidification. By using ���flash-and- freeze��� electron microscopy, we examine the trafficking of membranes in the absence or modification of endophilin and synaptojanin. Our fluorescence imaging experiments allow us to observe corresponding effects on protein trafficking and vesicle acidification. Taken together, our data are the first to suggest a specific role for synaptojanin and endophilin in UFE, and allow us to better understand this mechanism of vesicle recycling., Wa��hrend der synaptischen U��bertragung werden kontinuierlich synaptische Vesikel verwendet und recycelt. Die dabei involvierten Mechanismen sind der Gegenstand vieler Untersuchungen und mehrere Mechanismen sind bereits bekannt. Ju��ngste Ergebnisse legen nahe, dass eine ultraschnelle Endozytose (UFE) unter physiologischen Bedingungen viel schneller auftritt als bisher angenommen. Bei diesem Prozess la��uft die Endozytose in einer Clathrin-unabha��ngigen Weise innerhalb von grob 100ms ab, dies wurde sowohl in C. elegans und Sa��ugetier-Ma��use-Neuronen gezeigt. Nach der Ru��ckholung der Membran, fusionieren endozytierte Vesikel zu einem Endosom, vom dem sich Clathrin-beschichtete Pits ablo��sen und synaptische Vesikel innerhalb von 30-300ms regenerieren. Trotz des raschen Ablaufs dieses Prozesses ist die Trennung der Plasmamembran im Rahmen der UFE Dynamin-abha��ngig, und beno��tigt Aktin. Die anderen Proteine, die erforderlich sind, um die Schritte der UFE mit ihrer charakteristischen schnellen Geschwindigkeit zu orchestrieren, sind noch nicht bekannt. Wir zeigen, dass Synaptojanin und Endophilin einen entscheidenden Beitrag zu diesem raschen Ablauf der ultraschnellen Endozytose leisten. Insbesondere zeigen wir, dass sowohl Endophilin als auch Synaptojanin fu��r das Auftreten von UFE notwendig sind. In diesem Manuskript gehen wir auf die spezifischen Rollen dieser Proteine im Prozess der UFE ein. Wir zeigen, dass UFE eine funktionelle 5-Phosphatase-Doma��ne, aber keine Sac-1-Doma��ne von Synaptojanin beno��tigt, um ultraschnelle Endozytose zu erlauben, und dass Endophilin fu��r die Hals-bildung der Vesikel in UFE erforderlich ist. Wir zeigen auch, dass Endophilin A1 und A2 sowohl den Endophilin-Bedarf fu��r UFE als auch fu��r die Widerherstellung eines sauren Milieus innerhalb der Vesikel erfu��llt. Mit der "Flash-and-Freeze" -Elektronenmikroskopie untersuchen wir den Austausch der Membranen in Abwesenheit, oder mit einer Modifikation, von Endophilin und Synaptojanin. Unsere Fluoreszenz-Bildgebungs- Experimente erlauben uns, entsprechende Auswirkungen auf den Proteinverkehr und die Ansa��uerung der Vesikel zu beobachten. Unsere Daten weisen erstmals auf eine spezifische Rolle von Synaptojanin und Endophilin im Rahmen der UFE hin und erlauben ein besseres Versta��ndnis der Mechanismen des Vesikel-recyclings.

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis. Cellular membranes have distinct lipid compositions despite intermixing, and it is unclear why plasma membrane lipids do not accumulate on endosomes. Here, the authors use the C. elegans embryo to identify lipid transfer proteins and phosphatases that are critical for endosomal lipid homeostasis.

Neurons use multiple modes of endocytosis, including clathrin-mediated endocytosis (CME) and activity-dependent bulk endocytosis (ADBE), during mild and intense neuronal activity, respectively, to maintain stable neurotransmission. While molecular players modulating CME are well characterized, factors regulating ADBE and mechanisms coordinating CME and ADBE activations remain poorly understood. Here we report that Minibrain/DYRK1A (Mnb), a kinase mutated in autism and up-regulated in Down’s syndrome, plays a novel role in suppressing ADBE. We demonstrate that Mnb, together with calcineurin, delicately coordinates CME and ADBE by controlling the phosphoinositol phosphatase activity of synaptojanin (Synj) during varying synaptic demands. Functional domain analyses reveal that Synj’s 5′-phosphoinositol phosphatase activity suppresses ADBE, while SAC1 activity is required for efficient ADBE. Consequently, Parkinson’s disease mutation in Synj’s SAC1 domain impairs ADBE. These data identify Mnb and Synj as novel regulators of ADBE and further indicate that CME and ADBE are differentially governed by Synj’s dual phosphatase domains.

Presynaptic terminals are metabolically active and accrue damage through continuous vesicle cycling. How synapses locally regulate protein homeostasis is poorly understood. We show that the presynaptic lipid phosphatase synaptojanin is required for macroautophagy, and this role is inhibited by the Parkinson's disease mutation R258Q. Synaptojanin drives synaptic endocytosis by dephosphorylating PI(4,5)P2, but this function appears normal in Synaptojanin RQ knock‐in flies. Instead, R258Q affects the synaptojanin SAC1 domain that dephosphorylates PI(3)P and PI(3,5)P2, two lipids found in autophagosomal membranes. Using advanced imaging, we show that Synaptojanin RQ mutants accumulate the PI(3)P/PI(3,5)P2‐binding protein Atg18a on nascent synaptic autophagosomes, blocking autophagosome maturation at fly synapses and in neurites of human patient induced pluripotent stem cell‐derived neurons. Additionally, we observe neurodegeneration, including dopaminergic neuron loss, in Synaptojanin RQ flies. Thus, synaptojanin is essential for macroautophagy within presynaptic terminals, coupling protein turnover with synaptic vesicle cycling and linking presynaptic‐specific autophagy defects to Parkinson's disease.

PCTAIRE-1 (also known as cyclin-dependent protein kinase (CDK) 16), is a Ser/Thr kinase that has been implicated in many cellular processes, including cell cycle, spermatogenesis, neurite outgrowth, and vesicle trafficking. Most recently, it has been proposed as a novel X-linked intellectual disability (XLID) gene, where loss-of-function mutations have been identified in human patients. The precise molecular mechanisms that regulate PCTAIRE-1 remained largely obscure, and only a few cellular targets/substrates have been proposed with no clear functional significance. We and others recently showed that cyclin Y binds and activates PCTAIRE-1 via phosphorylation and 14-3-3 binding. In order to understand the physiological role that PCTAIRE-1 plays in brain, we have performed a chemical genetic screen in vitro using an engineered PCTAIRE-1/cyclin Y complex and mouse brain extracts. Our screen has identified potential PCTAIRE-1 substrates (AP2-Associated Kinase 1 (AAK1), dynamin 1, and synaptojanin 1) in brain that have been shown to regulate crucial steps of receptor endocytosis, and are involved in control of neuronal synaptic transmission. Furthermore, mass spectrometry and protein sequence analyses have identified potential PCTAIRE-1 regulated phosphorylation sites on AAK1 and we validated their PCTAIRE-1 dependence in a cellular study and/or brain tissue lysates. Our results shed light onto the missing link between PCTAIRE-1 regulation and proposed physiological functions, and provide a basis upon which to further study PCTAIRE-1 function in vivo and its potential role in neuronal/brain disorders.

In Hedgehog (Hh) signalling, Hh ligand concentration gradient is effectively translated into a spatially distinct transcriptional program to give precisely controlled context dependent developmental outcomes. In the absence of Hh, the receptor Patched (Ptc) inhibits the signal transducer Smoothened (Smo) by maintaining low phosphatidylinositol 4-phosphate (PI(4)P) levels. Binding of Hh to its receptor Ptc promotes PI(4)P production, which in turn activates Smo. Using wingdiscs of Drosophila melanogaster, this study shows that Synaptojanin (Synj), a dual phosphatase, modulates PI(4)P levels and affects Smo activation, and thereby functions as an additional regulatory step in the Hh pathway. Reducing the levels of Synj in the wing-discs caused enhancement of a Hh dominant gain-of-function Moonrat phenotype in the adult wings. Synj downregulation augmented Hh signalling, which was associated with elevated PI(4)P levels and Smo activation. Synj did not control the absolute pathway activity but rather fine-tuned the response since its downregulation increased expression of decapentaplegic (dpp), a low-threshold target of the pathway while the high-threshold targets remained unaffected. This is the first report that identifies Synj as a negative regulator of Hh signalling, implying its importance and an additional regulatory step in Hh signal transduction.

Hyperosmotic shock drives a reorganization of calcineurin and Inp53/synaptojanin. Calcineurin accumulates at sites of polarized growth, dephosphorylates Inp53 to maintain normal membrane and actin cytoskeleton morphology, and causes it to dissociate from clathrin and bind to endocytic proteins Bsp1, Bzz1, and Sla1., During hyperosmotic shock, Saccharomyces cerevisiae adjusts to physiological challenges, including large plasma membrane invaginations generated by rapid cell shrinkage. Calcineurin, the Ca2+/calmodulin–dependent phosphatase, is normally cytosolic but concentrates in puncta and at sites of polarized growth during intense osmotic stress; inhibition of calcineurin-activated gene expression suggests that restricting its access to substrates tunes calcineurin signaling specificity. Hyperosmotic shock promotes calcineurin binding to and dephosphorylation of the PI(4,5)P2 phosphatase synaptojanin/Inp53/Sjl3 and causes dramatic calcineurin-dependent reorganization of PI(4,5)P2-enriched membrane domains. Inp53 normally promotes sorting at the trans-Golgi network but localizes to cortical actin patches in osmotically stressed cells. By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells. In response to hyperosmotic shock and calcineurin-dependent regulation, Inp53 shifts from associating predominantly with clathrin to interacting with endocytic proteins Sla1, Bzz1, and Bsp1, suggesting that Inp53 mediates stress-specific endocytic events. This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion. We propose that activation of Ca2+/calcineurin and PI(4,5)P2 signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.

Neuronal signaling depends on the exocytic fusion and subsequent endocytic retrieval and reformation of neurotransmitter-containing synaptic vesicles at synapses. Recent findings have uncovered surprising roles of presynaptic endocytic proteins in the formation and transport of autophagosomes. These include functions of the membrane remodelling protein endophilin and its downstream effector, the phosphoinositide phosphatase synaptojanin, in autophagosome formation and in Parkinson's disease, the endocytic sorting adaptor CALM in protein degradation via the autophagy/lysosomal pathway in Alzheimer's disease, and the clathrin adaptor complex AP-2 in retrograde transport of signaling autophagosomes to prevent neurodegeneration. These findings reveal unanticipated connections between the machineries for synaptic neurotransmission and neuronal proteostasis and identify presynaptic endocytic proteins as potential targets to treat neurodegenerative diseases.

Synj2 (synaptojanin 2) encodes an inositol polyphosphate phosphatase that functions in recycling neurotransmitter vesicles and is implicated in spermatogenesis. Transcription of Synj2 is thought to occur from one of two promoters based on analysis of a variable 5′ untranslated region. Clustering all known mouse Synj2 transcripts led us to uncover a novel subset of transcripts that appears to derive from a region located within intron 7. We identified two alternate splice variants emanating from use of this promoter. These alternate splice variants manifest developmental stage specificity and somatic versus gametic differences in expression.

The four mammalian phosphatidylinositol 4-kinases, together with the PI(4,5)P(2) depleting 5-phosphatases of the oculocerebrorenal syndrome of Lowe and synaptojanin families, modulate neuronal pools of PI4P lipid and regulate intracellular membrane trafficking in the endocytic and secretory pathways. Dysfunctions in these enzymes have been associated with a broad spectrum of disorders including schizophrenia, bipolar disorder, Lowe syndrome, age-related neurodegeneration, Alzheimer's disease and Down syndrome. Recent work has shown that reduced expression of individual phosphatidylinositol 4-kinase isozymes is associated with impaired survival of specific neuronal populations within the CNS. Furthermore, alterations to the concentrations of different phosphoinositide lipid species in the brain and, in particular, the ratio of PI4P to PI(4,5)P(2) can have deleterious effects on clathrin-dependent membrane trafficking both in the Golgi-endosomal pathway and at the plasma membrane. In this article, we focus on the cell biology, biochemistry and neuronal functions of the phosphatidylinositol 4-kinases and their emerging roles in psychiatric and neurological pathologies.

Although neurons are highly polarized, how neuronal polarity is generated remains poorly understood. An evolutionarily conserved inositol-producing enzyme myo-inositol monophosphatase (IMPase) is essential for polarized localization of synaptic molecules in Caenorhabditis elegans and can be inhibited by lithium, a drug for bipolar disorder. The synaptic defect of IMPase mutants causes defects in sensory behaviors including thermotaxis. Here we show that the abnormalities of IMPase mutants can be suppressed by mutations in two enzymes, phospholipase Cβ or synaptojanin, which presumably reduce the level of membrane phosphatidylinositol 4,5-bisphosphate (PIP2). We also found that mutations in phospholipase Cβ conferred resistance to lithium treatment. Our results suggest that reduction of PIP2 on plasma membrane is a major cause of abnormal synaptic polarity in IMPase mutants and provide the first in vivo evidence that lithium impairs neuronal PIP2 synthesis through inhibition of IMPase. We propose that the PIP2 signaling regulated by IMPase plays a novel and fundamental role in the synaptic polarity.

SummaryEndophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.

The importance of phosphoinositides (phosphorylated phosphatidyl inositol derivatives, PIs) for normal cellular function cannot be overstated. Although they represent a small fraction of the total phospholipid within the cell, they are essential regulators of many cellular functions. They direct membrane trafficking by functioning as recruitment factors for vesicular trafficking components, they can modulate ion channel activity through direct binding within cellular membranes, and their hydrolysis generates second messenger signaling molecules. Despite an explosion of information regarding the importance of these lipids in cellular biology, their precise roles in vertebrate retinal photoreceptors has not been established. This review summarizes the literature on potential roles for different phosphoinositides and their regulators in vertebrate rods and cones. A brief description of the importance of PI signaling in other photosensitive cells is also presented. The highly specialized functions of the vertebrate photoreceptor, combined with the established importance of phosphoinositides, promise significant future discoveries in this field.

Summary Ultrafast endocytosis generates vesicles from the plasma membrane as quickly as 50 ms in hippocampal neurons following synaptic vesicle fusion. The molecular mechanism underlying the rapid maturation of these endocytic pits is not known. Here we demonstrate that synaptojanin-1, and its partner endophilin-A, function in ultrafast endocytosis. In the absence of synaptojanin or endophilin, the membrane is rapidly invaginated, but pits do not become constricted at the base. The 5-phosphatase activity of synaptojanin is involved in formation of the neck, but 4-phosphatase is not required. Nevertheless, these pits are eventually cleaved into vesicles; within a 30-s interval, synaptic endosomes form and are resolved by clathrin-mediated budding. Then synaptojanin and endophilin function at a second step to aid with the removal of clathrin coats from the regenerated vesicles. These data together suggest that synaptojanin and endophilin can mediate membrane remodeling on a millisecond timescale during ultrafast endocytosis.

Synaptojanin 1 (SynJ1) is a polyphosphoinositide phosphatase involved in clathrin-mediated endocytosis in conventional synapses. Studies with the zebrafish mutant nrc have revealed that loss of SynJ1 also affects cone photoreceptor ribbon synapses, causing pronounced morphological and functional abnormalities. In this study we continue to examine the role of SynJ1 in photoreceptors. Using a newly generated antibody specific for zebrafish SynJ1, we localized this protein predominantly to cone photoreceptors. We then used blastula stage transplantation experiments to demonstrate that rods from nrc mutants lacking SynJ1 develop normally and do not have the pronounced morphological defects detected in cones. Given the known involvement of SynJ1 in synaptic vesicle endocytosis, we hypothesize that rods and cones use distinct mechanisms for vesicle recycling.

Summary Synaptic vesicle endocytosis is critical for maintaining synaptic communication during intense stimulation. Here we describe Tweek, a conserved protein that is required for synaptic vesicle recycling. tweek mutants show reduced FM1-43 uptake, cannot maintain release during intense stimulation, and harbor larger than normal synaptic vesicles, implicating it in vesicle recycling at the synapse. Interestingly, the levels of a fluorescent PI(4,5)P 2 reporter are reduced at tweek mutant synapses, and the probe is aberrantly localized during stimulation. In addition, various endocytic adaptors known to bind PI(4,5)P 2 are mislocalized and the defects in FM1-43 dye uptake and adaptor localization are partially suppressed by removing one copy of the phosphoinositide phosphatase synaptojanin , suggesting a role for Tweek in maintaining proper phosphoinositide levels at synapses. Our data implicate Tweek in regulating synaptic vesicle recycling via an action mediated at least in part by the regulation of PI(4,5)P 2 levels or availability at the synapse.

Dynamin I and at least five other nerve terminal proteins, amphiphysins I and II, synaptojanin, epsin and eps15 (collectively called dephosphins), are coordinately dephosphorylated by calcineurin during endocytosis of synaptic vesicles. Here we have identified a new dephosphin, the essential endocytic protein AP180. Blocking dephosphorylation of the dephosphins is known to inhibit endocytosis, but the role of phosphorylation has not been determined. We show that the protein kinase C (PKC) antagonists Ro 31-8220 and Go 7874 block the rephosphorylation of dynamin I and synaptojanin that occurs during recovery from an initial depolarizing stimulus (S1). The rephosphorylation of AP180 and amphiphysins 1 and 2, however, were unaffected by Ro 31-8220. Although these dephosphins share a single phosphatase, different protein kinases phosphorylated them after nerve terminal stimulation. The inhibitors were used to selectively examine the role of dynamin I and/or synaptojanin phosphorylation in endocytosis. Ro 31-8220 and Go 7874 did not block the initial S1 cycle of endocytosis, but strongly inhibited endocytosis following a second stimulus (S2). Therefore, phosphorylation of a subset of dephosphins, which includes dynamin I and synaptojanin, is required for the next round of stimulated synaptic vesicle retrieval.

Hsc70 with its cochaperone, either auxilin or GAK, not only uncoats clathrin-coated vesicles but also acts as a chaperone during clathrin-mediated endocytosis. However, because synaptojanin is also involved in uncoating, it is not clear whether GAK is an essential gene. To answer this question, GAK conditional knockout mice were generated and then mated to mice expressing Cre recombinase under the control of the nestin, albumin, or keratin-14 promoters, all of which turn on during embryonic development. Deletion of GAK from brain, liver, or skin dramatically altered the histology of these tissues, causing the mice to die shortly after birth. Furthermore, by expressing a tamoxifen-inducible promoter to express Cre recombinase we showed that deletion of GAK caused lethality in adult mice. Mouse embryonic fibroblasts in which the GAK was disrupted showed a lack of clathrin-coated pits and a complete block in clathrin-mediated endocytosis. We conclude that GAK deletion blocks development and causes lethality in adult animals by disrupting clathrin-mediated endocytosis.

Recently, a pathway involving the highly choreographed recruitment of endocytic proteins to sites of clathrin/actin-mediated endocytosis has been revealed in budding yeast. Here, we investigated possible roles for candidate disassembly factors in regulation of the dynamics of the endocytic coat proteins Sla2p, Ent1p, Ent2p, Sla1p, Pan1p and End3p, each of which has mammalian homologues. Live cell imaging analysis revealed that in addition to the synaptojanin, Sjl2p, the Ark1p and Prk1p protein kinases, the putative Arf GTPase-activating protein, Gts1p and the Arf GTPase-interacting protein, Lsb5p, also arrive at endocytic sites late in the internalization pathway, consistent with roles in coat disassembly. Analysis of coat dynamics in various mutant backgrounds revealed that multiple pathways, including the ones mediated by an Arf guanosine triphosphatase and a synaptojanin, facilitate efficient disassembly of different endocytic coat proteins. In total, at least four separate processes are important for disassembly of endocytic complexes and efficient downstream trafficking of endocytic cargo.

The lipid polyunsaturated fatty acids are highly enriched in synaptic membranes, including synaptic vesicles, but their precise function there is unknown. Caenorhabditis elegans fat-3 mutants lack long-chain polyunsaturated fatty acids (LC-PUFAs); they release abnormally low levels of serotonin and acetylcholine and are depleted of synaptic vesicles, but the mechanistic basis of these defects is unclear. Here we demonstrate that synaptic vesicle endocytosis is impaired in the mutants: the synaptic vesicle protein synaptobrevin is not efficiently retrieved after synaptic vesicles fuse with the presynaptic membrane, and the presynaptic terminals contain abnormally large endosomal-like compartments and synaptic vesicles. Moreover, the mutants have abnormally low levels of the phosphoinositide phosphatase synaptojanin at release sites and accumulate the main synaptojanin substrate phosphatidylinositol 4,5-bisphosphate at these sites. Both synaptobrevin and synaptojanin mislocalization can be rescued by providing exogenous arachidonic acid, an LC-PUFA, suggesting that the endocytosis defect is caused by LC-PUFA depletion. By showing that the genes fat-3 and synaptojanin act in the same endocytic pathway at synapses, our findings suggest that LC-PUFAs are required for efficient synaptic vesicle recycling, probably by modulating synaptojanin localization at synapses.

Synaptojanin1 degrades the signaling lipid phosphatidylinositol-4,5-bisphosphate and facilitates compensatory endocytosis, clathrin-coat disassembly, and vesicle reavailability at active synapses. In this issue of Neuron , Mani et al. provide new information about the separate roles of synaptojanin's two phosphatase domains and its interactions with endophilin in regulating these important aspects of the vesicle cycle.

Huntingtin-interacting protein 1 (HIP1) was identified through its interaction with htt (huntingtin), the Huntington's disease (HD) protein. HIP1 is an endocytic protein that influences transport and function of AMPA and NMDA receptors in the brain. However, little is known about its contribution to neuronal dysfunction in HD.We report that theCaenorhabditis elegansHIP1 homologhipr-1modulates presynaptic activity and the abundance of synaptobrevin, a protein involved in synaptic vesicle fusion. Presynaptic function was also altered in hippocampal brain slices of HIP1−/−mice demonstrating delayed recovery from synaptic depression and a reduction in paired-pulse facilitation, a form of presynaptic plasticity. Interestingly, neuronal dysfunction in transgenic nematodes expressing mutant N-terminal huntingtin was specifically enhanced byhipr-1loss of function. A similar effect was observed with several other mutant proteins that are expressed at the synapse and involved in endocytosis, such asunc-11/AP180,unc-26/synaptojanin, andunc-57/endophilin.Thus, HIP1 is involved in presynaptic nerve terminal activity and modulation of mutant polyglutamine-induced neuronal dysfunction. Moreover, synaptic proteins involved in endocytosis may protect neurons against amino acid homopolymer expansion.

SummarySynaptojanin is a lipid phosphatase required to degrade phosphatidylinositol 4,5 bisphosphate (PIP2) at cell membranes during synaptic vesicle recycling [1, 2]. Synaptojanin mutants in C. elegans are severely uncoordinated and are depleted of synaptic vesicles, possibly because of accumulation of PIP2[2]. To identify proteins that act downstream of PIP2 during endocytosis, we screened for suppressors of synaptojanin mutants in the nematode C. elegans. A class of uncoordinated mutants called “fainters” partially suppress the locomotory, vesicle depletion, and electrophysiological defects in synaptojanin mutants. These suppressor loci include the genes for the NCA ion channels [3], which are homologs of the vertebrate cation leak channel NALCN [4], and a novel gene called unc-80. We demonstrate that unc-80 encodes a novel, but highly conserved, neuronal protein required for the proper localization of the NCA-1 and NCA-2 ion channel subunits. These data suggest that activation of the NCA ion channel in synaptojanin mutants leads to defects in recycling of synaptic vesicles.

Myosin 1E is one of two “long-tailed” human Class I myosins that contain an SH3 domain within the tail region. SH3 domains of yeast and amoeboid myosins I interact with activators of the Arp2/3 complex, an important regulator of actin polymerization. No binding partners for the SH3 domains of myosins I have been identified in higher eukaryotes. In the current study, we show that two proteins with prominent functions in endocytosis, synaptojanin-1 and dynamin, bind to the SH3 domain of human Myo1E. Myosin 1E co-localizes with clathrin- and dynamin-containing puncta at the plasma membrane and this co-localization requires an intact SH3 domain. Expression of Myo1E tail, which acts in a dominant-negative manner, inhibits endocytosis of transferrin. Our findings suggest that myosin 1E may contribute to receptor-mediated endocytosis.

Eisosomes are multiprotein structures that generate linear invaginations at the plasma membrane of yeast cells. The core component of eisosomes, the BAR domain protein Pil1, generates these invaginations through direct binding to lipids including phosphoinositides. Eisosomes promote hydrolysis of phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) by functioning with synaptojanin, but the cellular processes regulated by this pathway have been unknown. Here, we found that PI(4,5)P2 regulation by eisosomes inhibits the cell integrity pathway, a conserved MAPK signal transduction cascade. This pathway is activated by multiple environmental conditions including osmotic stress in the fission yeast Schizosaccharomyces pombe. Activation of the MAPK Pmk1 was impaired by mutations in the phosphatidylinositol (PI) 5-kinase Its3, but this defect was suppressed by removal of eisosomes. Using fluorescent biosensors, we found that osmotic stress induced the formation of PI(4,5)P2 clusters that were spatially organized by eisosomes in both fission yeast and budding yeast cells. These cortical clusters contained the PI 5-kinase Its3 and did not assemble in the its3-1 mutant. The GTPase Rho2, an upstream activator of Pmk1, also co-localized with PI(4,5)P2 clusters under osmotic stress, providing a molecular link between these novel clusters and MAPK activation. Our findings have revealed that eisosomes regulate activation of MAPK signal transduction through the organization of cortical lipid-based microdomains.

Rhythmicity is prevalent in the cortical dynamics of diverse single and multicellular systems. Current models of cortical oscillations focus primarily on cytoskeleton-based feedbacks, but information on signals upstream of the actin cytoskeleton is limited. In addition, inhibitory mechanisms--especially local inhibitory mechanisms, which ensure proper spatial and kinetic controls of activation--are not well understood. Here, we identified two phosphoinositide phosphatases, synaptojanin 2 and SHIP1, that function in periodic traveling waves of rat basophilic leukemia (RBL) mast cells. The local, phase-shifted activation of lipid phosphatases generates sequential waves of phosphoinositides. By acutely perturbing phosphoinositide composition using optogenetic methods, we showed that pulses of PtdIns(4,5)P2 regulate the amplitude of cyclic membrane waves while PtdIns(3,4)P2 sets the frequency. Collectively, these data suggest that the spatiotemporal dynamics of lipid metabolism have a key role in governing cortical oscillations and reveal how phosphatidylinositol 3-kinases (PI3K) activity could be frequency-encoded by a phosphatase-dependent inhibitory reaction.

Synaptojanin and endophilin represent a classic pair of endocytic proteins that exhibit coordinated action during rapid synaptic vesicle endocytosis. Current models suggest that synaptojanin activity is tightly associated with endophilin through high-affinity binding between the synaptojanin proline-rich domain (PRD) and the endophilin SH3 domain. Surprisingly, we find that truncated synaptojanin lacking the PRD domain sustains normal synaptic transmission, indicating that synaptojanin's core function in vivo resides in the remaining two domains that contain phosphoinositide-phosphatase activities: an N-terminal Sac1 phosphatase domain and a 5-phosphatase domain. We further show that the Sac1 domain plays an unexpected role in targeting synaptojanin to synapses. The requirement for Sac1 is bypassed by tethering the synaptojanin 5-phophatase to the endophilin membrane-bending Bin–Amphiphysin–Rvs (BAR) domain. Together, our results uncover an unexpected role for the Sac1 domain in vivo in supporting coincident action between synaptojanin and endophilin at synapses. DOI: http://dx.doi.org/10.7554/eLife.05660.001, eLife digest Nerve cells called neurons can rapidly carry information around the body. Each neuron forms connections called synapses with several other cells to build networks for information exchange. At most synapses, electrical activity in one neuron results in the release of chemicals called neurotransmitters from storage compartments called synaptic vesicles. The neurotransmitters leave the cell and cross the gap between the two neurons to activate the next cell. After the neurotransmitters have been released, the synaptic vesicles need to be regenerated via a recycling process called endocytosis. This recycling process is very important for synapses to work properly, but it is not clear exactly how it occurs. Two of the proteins involved are called synaptojanin and endophilin. Synaptojanin is made up of three structural units (or ‘domains’), including the proline-rich domain and the Sac1 domain. It has been proposed that interactions between endophilin and the proline-rich domain of synaptojanin are essential for vesicle recycling. Here, Dong et al. studied nematode worms that carry mutant forms of synaptojanin. The experiments show that the Sac1 domain, but not the proline-rich domain, is required for the synapses to work properly. However, the Sac1 domain is not required if synaptojanin is artificially linked to endophilin. Dong et al.'s findings suggest that synaptojanin uses its Sac1 domains to work with endophilin. A future challenge will be to understand the details of how this cooperative action occurs. DOI: http://dx.doi.org/10.7554/eLife.05660.002

Amplified HER2, which encodes a member of the epidermal growth factor receptor (EGFR) family, is a target of effective therapies against breast cancer. In search for similarly targetable genomic aberrations, we identified copy number gains in SYNJ2, which encodes the 5'-inositol lipid phosphatase synaptojanin 2, as well as overexpression in a small fraction of human breast tumors. Copy gain and overexpression correlated with shorter patient survival and a low abundance of the tumor suppressor microRNA miR-31. SYNJ2 promoted cell migration and invasion in culture and lung metastasis of breast tumor xenografts in mice. Knocking down SYNJ2 impaired the endocytic recycling of EGFR and the formation of cellular lamellipodia and invadopodia. Screening compound libraries identified SYNJ2-specific inhibitors that prevented cell migration but did not affect the related neural protein SYNJ1, suggesting that SYNJ2 is a potentially druggable target to block cancer cell migration.

Synaptojanin is a phosphoinositide phosphatase that plays a crucial role in synaptic vesicle recycling. The focus of this review is on the major direct interacting partners of synaptojanin with respect to recognition motifs in synaptojanin isoforms and splice variants. Those proteins that bind synaptojanin are mainly involved in endocytosis and function at the interface between actin and the endocytic machinery. Some interaction partners underline synaptojanin's emerging role in signaling and pathophysiological processes.

Sorting nexin 9 (SNX9, also referred to as SH3PX1) is a binding partner for the non-receptor and Cdc42-associated kinase (ACK) in Drosophila and mammals. ACK1 is known to bind clathrin and influence EGF receptor endocytosis. SNX9 comprises an N-terminal Src homology domain 3 (SH3), a central PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. In order to investigate SNX9 further we have made use of a novel in vivo biotinylation system to label various GST-SH3 domains and perform blot overlays, thereby identifying synaptojanin-1 as a partner for SNX9. Biotinylated SH3 domains were also used for specific identification of target proline-rich sequences in synaptojanin and ACK1 on synthetic peptides arrays. Direct assessment of SH3 binding efficiencies at different positions within the extensive proline-rich regions of these proteins were thus determined. While SNX9 targets a number of sequences within the proline-rich regions of synaptojanin, a single site was identified in human ACK1. By testing the association of various truncations of ACK1 with SNX9 we confirmed the dominant SNX9 binding domain in human ACK1 (residues 920–955). In the presence of SNX9 we find that synaptojanin is able to colocalize with distinct ACK1 containing vesicles, indicating that this tyrosine kinase is linked to many components involved in vesicle dynamics including clathrin, AP2 and synaptojanin-1.

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

Visual, vestibular, and auditory neurons rely on ribbon synapses for rapid continuous release and recycling of synaptic vesicles. Molecular mechanisms responsible for the properties of ribbon synapses are mostly unknown. The zebrafish vision mutantnrchas unanchored ribbons and abnormal synaptic transmission at cone photoreceptor synapses. We used positional cloning to identify thenrcmutation as a premature stop codon in thesynaptojanin1(synj1) gene. Synaptojanin 1 (Synj1) is undetectable innrcextracts, and biochemical activities associated with it are reduced. Furthermore, morpholinos directed againstsynj1phenocopy thenrcmutation. Synj1 is a polyphosphoinositide phosphatase important at conventional synapses for clathrin-mediated endocytosis and actin cytoskeletal rearrangement. In thenrccone photoreceptor pedicle, not only are ribbons unanchored, but synaptic vesicles are reduced in number, abnormally distributed, and interspersed within a dense cytoskeletal matrix. Our findings reveal a new role for Synj1 and link phosphoinositide metabolism to ribbon architecture and function at the cone photoreceptor synapse.

Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin. In contrast, Ca2+ and Cu2+ are inhibitory. We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5. The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4. Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective SPsynaptojanin and SHIP2 inhibitors for pharmacological investigation.

Dap160/Intersectin is a multidomain adaptor protein that colocalizes with endocytic machinery in the periactive zone at the Drosophila NMJ. We have generated severe loss-of-function mutations that eliminate Dap160 protein from the NMJ. dap160 mutant synapses have decreased levels of essential endocytic proteins, including dynamin, endophilin, synaptojanin, and AP180, while other markers of the active zone and periactive zone are generally unaltered. Functional analyses demonstrate that dap160 mutant synapses are unable to sustain high-frequency transmitter release, show impaired FM4-64 loading, and show a dramatic increase in presynaptic quantal size consistent with defects in synaptic vesicle recycling. The dap160 mutant synapse is grossly malformed with abundant, highly ramified, small synaptic boutons. We present a model in which Dap160 scaffolds both endocytic machinery and essential synaptic signaling systems to the periactive zone to coordinately control structural and functional synapse development.

Endophilin 1 (Endo1) participates in synaptic vesicle biogenesis through interactions of its Src homology 3 domain with the polyphosphoinositide phosphatase Synaptojanin and the GTPase Dynamin. Endo1 has also been reported to affect endocytosis by converting membrane curvature via its lysophosphatidic acid acyltransferase activity. Here we report that a closely related isoform of Endo1, Endo3, inhibits clathrin-mediated endocytosis. Mutational analyses showed that the variable region of Endo3 is important in regulating transferrin endocytosis. In the brain, Endo3 is co-localized with dopamine D2 receptor in olfactory nerve terminals and inhibits its clathrin-mediated endocytosis in COS-7 cells. Furthermore, overexpression of Endo3 in an olfactory epithelium-derived cell line suppressed dopamine D2 receptor-mediated endocytosis and therefore accelerated its dopamine-induced differentiation. These results indicate that Endo3 may act as a negative regulator of clathrin-mediated endocytosis in brain neurons.

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.

Yeast TGN resident proteins that frequently cycle between the TGN and endosomes are much more slowly transported to the prevacuolar/late endosomal compartment (PVC) than other proteins. However, TGN protein transport to the PVC is accelerated in mutants lacking function of Inp53p. Inp53p contains a SacI polyphosphoinositide phosphatase domain, a 5-phosphatase domain, and a proline-rich domain. Here we show that all three domains are required to mediate “slow delivery” of TGN proteins into the PVC. Although deletion of the proline-rich domain did not affect general membrane association, it caused localization to become less specific. The proline-rich domain was shown to bind to two proteins, including clathrin heavy chain, Chc1p. Unlike chc1 mutants, inp53 mutants do not mislocalize TGN proteins to the cell surface, consistent with the idea that Chc1p and Inp53p act at a common vesicular trafficking step but that Chc1p is used at other steps also. Like mutations in the AP-1 adaptor complex, mutations in INP53 exhibit synthetic growth and transport defects when combined with mutations in the GGA proteins. Taken together with other recent studies, our results suggest that Inp53p and AP-1/clathrin act together in a TGN-to-early endosome pathway distinct from the direct TGN-to-PVC pathway mediated by GGA/clathrin.

Endophilin-1, a cytoplasmic Src homology 3 (SH3) domain-containing protein, localises in brain presynaptic nerve termini. Endophilin dimerises through its N-terminus, and participates at multiple stages in clathrin-coated endocytosis, from early membrane invagination to synaptic vesicle uncoating. Both its C-terminal SH3 domain and N-terminus are required for endocytosis. Through its SH3 domain, endophilin bound to proline-rich domains (PRDs) in other endocytic proteins, including synaptojanin and dynamin. The N-terminal region possesses unique functions affecting lipid membrane curvature, through lysophosphatidic acid acyl transferase (LPAAT) activity and direct binding and tubulating activity. In addition to synaptic vesicle formation, endophilin-1 complexes with signalling molecules, including cell surface receptors, metalloprotease disintegrins and germinal centre kinase-like kinase (GLK). Therefore, endophilin-1 may serve to couple vesicle biogenesis with intracellular signalling cascades.

Phosphorylation has emerged as a crucial regulatory mechanism in the nervous system to integrate the dynamic signalling required for proper synaptic development, function and plasticity, particularly during changes in neuronal activity. Here we present evidence that Minibrain (Mnb; also known as Dyrk1A), a serine/threonine kinase implicated in autism spectrum disorder and Down syndrome, is required presynaptically for normal synaptic growth and rapid synaptic vesicle endocytosis at the Drosophila neuromuscular junction (NMJ). We find that Mnb-dependent phosphorylation of Synaptojanin (Synj) is required, in vivo, for complex endocytic protein interactions and to enhance Synj activity. Neuronal stimulation drives Mnb mobilization to endocytic zones and triggers Mnb-dependent phosphorylation of Synj. Our data identify Mnb as a synaptic kinase that promotes efficient synaptic vesicle recycling by dynamically calibrating Synj function at the Drosophila NMJ, and in turn endocytic capacity, to adapt to conditions of high synaptic activity.

Stable compartments of the plasma membrane promote a wide range of cellular functions. In yeast cells, cytosolic structures called eisosomes generate prominent cortical invaginations of unknown function. Through a series of genetic screens in fission yeast, we found that the eisosome proteins Pil1 and Sle1 function with the synaptojanin-like lipid phosphatase Syj1 and its ligand Tax4. This genetic pathway connects eisosome function with the hydrolysis of phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] in cells. Defects in PI(4,5)P2 regulation led to eisosome defects, and we found that the core eisosome protein Pil1 can bind to and tubulate liposomes containing PI(4,5)P2. Mutations in components of the Pil1–Sle1–Syj1–Tax4 pathway suppress the growth and morphology defects of TORC2 mutants, indicating that eisosome-dependent regulation of PI(4,5)P2 feeds into signal transduction pathways. We propose that the geometry of membrane invaginations generates spatial and temporal signals for lipid-mediated signaling events in cells.

We have previously identified synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of synaptojanin 2 that shares several properties with synaptojanin 1. This isoform, synaptojanin 2B, undergoes further alternative splicing to generate synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners synaptojanin 1, bind synaptojanin 2B2, whereas only amphiphysin binds synaptojanin 2B1. Sequence similar to the endophilin-binding site in synaptojanin 1 is present only in synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of synaptojanin 2, but not of synaptojanin 1. These results suggest that synaptojanin 2B has a partially overlapping function with synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.

Localization of resident membrane proteins to the yeasttrans-Golgi network (TGN) involves both their retrieval from a prevacuolar/endosomal compartment (PVC) and a “slow delivery” mechanism that inhibits their TGN-to-PVC transport. A screen for genes required for the slow delivery mechanism uncoveredINP53, a gene encoding a phosphoinositide phosphatase. A retrieval-defective model TGN protein, A(F→A)-ALP, was transported to the vacuole in inp53 mutants approximately threefold faster than in wild type. Inp53p appears to function in a process distinct from PVC retrieval because combining inp53 with mutations that block retrieval resulted in a much stronger phenotype than either mutation alone. In vps27 strains defective for both anterograde and retrograde transport out of the PVC, a loss of Inp53p function markedly accelerated the rate of transport of TGN residents A-ALP and Kex2p into the PVC. Inp53p function is cargo specific because a loss of Inp53p function had no effect on the rate of Vps10p transport to the PVC in vps27 cells. The rate of early secretory pathway transport appeared to be unaffected ininp53 mutants. Cell fractionation experiments suggested that Inp53p associates with Golgi or endosomal membranes. Taken together, these results suggest that a phosphoinositide signaling event regulates TGN-to-PVC transport of select cargo proteins.

The phosphoinositide lipid PI(4,5)P(2) is now established as a key cofactor in signaling to the actin cytoskeleton and in vesicle trafficking. PI(4,5)P(2) accumulates at membrane rafts and promotes local co-recruitment and activation of specific signaling components at the cell membrane. PI(4,5)P(2) rafts may thus be platforms for local regulation of morphogenetic activity at the cell membrane. Raft PI(4,5)P(2) is regulated by lipid kinases (PI5-kinases) and lipid phosphatases (e.g. synaptojanin). In addition, GAP43-like proteins have recently emerged as a group of PI(4,5)P(2) raft-modulating proteins. These locally abundant proteins accumulate at inner leaflet plasmalemmal rafts where they bind to and co-distribute with PI(4,5)P(2), and promote actin cytoskeleton accumulation and dynamics. In keeping with their proposed role as positive modulators of PI(4,5)P(2) raft function, GAP43-like proteins confer competence for regulated morphogenetic activity on cells that express them. Their function has been investigated extensively in the nervous system, where their expression promotes neurite outgrowth, anatomical plasticity and nerve regeneration. Extrinsic signals and intrinsic factors may thus converge to modulate PI(4,5)P(2) rafts, upstream of regulated activity at the cell surface.

Amphiphysin I and II, proteins enriched in nerve terminals, form heterodimers and interact with dynamin and synaptojanin through their Src homology 3 (SH3) domain. In order to study the expression profile of Amphs in cells and tissues and the interaction state with other cellular molecules, we have prepared specific monoclonal antibodies (mAbs) designed to bait N-terminus, middle part, and C-terminus domains of Amph I, respectively by immunizing with the expressed smaller domain molecules using the GST gene fusion system. The expression of Amphs was found to be most abundant in PC12 cells, followed by B103 cells and vascular smooth muscle cells. Western blot analysis showed a relatively high level expression of Amphs that were found in both mouse and rat brain. There appeared to be some species difference in the expression pattern, i.e. Amphs are present more in the testis than in the lungs in rats, however, they are reversed in mice. Characterization of the mAbs revealed that clone 14-23 precipitated Amph I and II, whereas clone 8-2 could only precipitate Amph I. In addition, clathrin and dynamin in a complex with Amph were captured in the precipitate formed by mAbs and identified by the Western blot analysis. Cellular distribution of Amph was visualized with confocal immunofluorescence microscopy performed using the labeled-mAbs. Taken together, these results demonstrated that mAbs provided an excellent measure for studying Amphs' expression profile and their interacting proteins.

The Ser–Thr kinase Akt is activated in epithelial cells by Salmonella enterica serovar typhimurium. The bacterial effector SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. Here, we investigated the inositol phospholipid substrate preferences of SigD. Recombinant SigD preferentially dephosphorylated phosphatidylinositol 3,5-biphosphate and phosphatidylinositol 3,4,5-triphosphate over other phosphatidylinositol lipids. Phosphatidylinositol 3-phosphate was not a substrate, suggesting the 5′ phosphate moiety is one of the preferred substrates. Database searches revealed that SigD bears a small region of homology to the mammalian type II inositol 5-phosphatase synaptojanin. Mutation of two conserved residues in this region, Lys527 and Lys530, decreased or abrogated phosphatase activity, respectively. The Shigella flexneri SigD homologue, IpgD, displayed a similar activity in vitro and also activated Akt when used to complement a ΔsigD Salmonella strain. A mutation in IpgD at Lys507, analogous to Lys530 of SigD, also failed to activate Akt. Thus, we have characterized a region near the carboxyl-terminus of SigD which is important for phosphatase activity. We discuss how dephosphorylation of inositol phospholipids by SigD in vivo might contribute to the activation of Akt.

Advances in our understanding of the roles of phosphatidylinositol phosphates in controlling cellular functions such as endocytosis, exocytosis and the actin cytoskeleton have included new insights into the phosphatases that are responsible for the interconversion of these lipids. One of these is an entirely novel class of phosphatase domain found in a number of well characterized proteins. Proteins containing this Sac phosphatase domain include the yeast Saccharomyces cerevisiae proteins Sac1p and Fig4p. The Sac phosphatase domain is also found within the mammalian phosphoinositide 5-phosphatase synaptojanin and the yeast synaptojanin homologues Inp51p, Inp52p and Inp53p. These proteins therefore contain both Sac phosphatase and 5-phosphatase domains. This review describes the Sac phosphatase domain-containing proteins and their actions, with particular reference to the genetic and biochemical insights provided by study of the yeast Saccharomyces cerevisiae.

EEN, identified initially as a fusion partner to the mixed-lineage leukaemia gene in human leukaemia, and its related members, EEN-B1 and EEN-B2, have recently been shown to interact with two endocytic molecules, dynamin and synaptojanin, as well as with the huntingtin protein. In the present study, we show that the expression of the EEN gene-family members is differentially regulated. Multiple-spliced variants were identified for EEN-B2. In the brain, EEN-B1 and EEN-B2 mRNA are preferentially expressed in the cerebellar Purkinje and granule cells, dentate gyrus cells, hippocampal pyramidal neurons and cerebral granule cells. The expression patterns of EEN-B1 and EEN-B2 mRNA in the brain overlap with those of dynamin-I/III, synaptojanin-I and huntingtin, whereas the ubiquitous expression of EEN is consistent with that of dynamin-II. In testes, members of the EEN family are co-expressed with testis-type dynamin and huntingtin in Sertoli cells and germ cells respectively. Our results on the overlapping expression patterns are consistent with the proposed interaction of EEN family members with dynamin, synaptojanin and huntingtin protein in vivo. Although all three EEN family members bind to dynamin and synaptojanin, EEN-B1 has the highest affinity for binding, followed by EEN and EEN-B2. We also demonstrate that amphiphysin, a major synaptojanin-binding protein in brain, can compete with the EEN family for binding to synaptojanin and dynamin. We propose that recruitment of the EEN family by dynamin/synaptojanin to clathrin-coated pits can be regulated by amphiphysin.

Eps15 homology (EH) domains interact with proteins involved in endocytosis and signal transduction. EH domains bind to Asn-Pro-Phe (NPF) consensus motifs of target proteins. A few EH domains, such as the third EH domain (EH(3)) of human Eps15, prefer to bind Phe-Trp (FW) sequences. The structure of EH(3) has been solved by nuclear magnetic resonance (NMR) spectroscopy and is the first of an FW- and NPF-binding EH domain. Both FW and NPF sequences bind in the same hydrophobic pocket as shown by heteronuclear chemical shift mapping. EH(3) contains the dual EF-hand fold characteristic of the EH domain family, but it binds calcium with high affinity in the first EF-hand rather than the usual coordination in the second EF-hand. Point mutations were designed based on differences in the EH(3) and the second EH domain (EH(2)) of human Eps15 that alter the affinity of the domains for FW or NPF motif peptides. Peptides that mimic binding sites in the potential EH(3) targets Rab, synaptojanin, and the cation-dependent mannose 6-phosphate receptor were used to explore wild-type and mutant affinities. Characterization of the structure and binding properties of an FW- and NPF-binding EH domain and comparison to an NPF-specific EH domain provide important insights into the mechanisms of EH domain ligand recognition.