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Pietro De Camilli - One of the best experts on this subject based on the ideXlab platform.

  • absence of sac2 inpp5f enhances the phenotype of a parkinson s disease mutation of synaptojanin 1
    Proceedings of the National Academy of Sciences of the United States of America, 2020
    Co-Authors: Mian Cao, Daehun Park, Pietro De Camilli
    Abstract:

    Numerous genes whose mutations cause, or increase the risk of, Parkinson's disease (PD) have been identified. An inactivating mutation (R258Q) in the Sac inositol Phosphatase domain of synaptojanin 1 (SJ1/PARK20), a Phosphoinositide Phosphatase implicated in synaptic vesicle recycling, results in PD. The gene encoding Sac2/INPP5F, another Sac domain-containing protein, is located within a PD risk locus identified by genome-wide association studies. Knock-In mice carrying the SJ1 patient mutation (SJ1RQKI) exhibit PD features, while Sac2 knockout mice (Sac2KO) do not have obvious neurologic defects. We report a "synthetic" effect of the SJ1 mutation and the KO of Sac2 in mice. Most mice with both mutations died perinatally. The occasional survivors had stunted growth, died within 3 wk, and showed abnormalities of striatal dopaminergic nerve terminals at an earlier stage than SJ1RQKI mice. The abnormal accumulation of endocytic factors observed at synapses of cultured SJ1RQKI neurons was more severe in double-mutant neurons. Our results suggest that SJ1 and Sac2 have partially overlapping functions and are consistent with a potential role of Sac2 as a PD risk gene.

  • absence of sac2 inpp5f enhances the phenotype of a parkinson s disease mutation of synaptojanin 1
    bioRxiv, 2020
    Co-Authors: Mian Cao, Daehun Park, Pietro De Camilli
    Abstract:

    Abstract Many genes whose mutations cause, or increase the risk of, Parkinson’s disease (PD) have been identified. An inactivating mutation (R258Q) in the Sac inositol Phosphatase domain of synaptojanin 1 (SJ1/PARK20), a Phosphoinositide Phosphatase implicated in synaptic vesicle recycling, results in PD. The gene encoding Sac2/INPP5F, another Sac domain containing protein, was identified as a PD risk locus by GWAS. Knock-In mice carrying the SJ1 patient mutation (SJ1RQKI) exhibit PD features, while Sac2 knockout mice (Sac2KO) do not have obvious neurological defects. We report a “synthetic” effect of the SJ1 mutation and the KO of Sac2 in mice. Most mice with both mutations died perinatally. The occasional survivors had stunted growth, died within 3 weeks, and showed abnormalities of striatal dopaminergic nerve terminals at an earlier stage than SJ1RQKI mice. The abnormal accumulation of endocytic factors observed at synapses of cultured SJ1RQKI neurons was more severe in double mutant. Our results suggest that SJ1 and Sac2 have partially overlapping functions and are consistent with a potential role of Sac2 as a PD risk gene.

  • regulation of postsynaptic ampa responses by synaptojanin 1
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Liang Wei Gong, Pietro De Camilli
    Abstract:

    Endocytosis of postsynaptic AMPA receptors is a mechanism through which efficiency of neurotransmission is regulated. We have genetically tested the hypothesis that synaptojanin 1, a Phosphoinositide Phosphatase implicated in the endocytosis of synaptic vesicles presynaptically, may also function in the endocytosis of AMPA receptors postsynaptically. Electrophysiological recordings of cultured hippocampal neurons showed that miniature excitatory postsynaptic current amplitudes were larger in synaptojanin 1 knockout (KO) neurons because of an increase of surface-exposed AMPA receptors. This change did not represent an adaptive response to decreased presynaptic release in KO cultures and was rescued by the expression of wild type, but not catalytically inactive synaptojanin 1, in the postsynaptic neuron. NMDA-induced internalization of pHluorin-tagged AMPA receptors (GluR2) was impaired in KO neurons. These results reveal a function of synaptojanin 1 in constitutive and triggered internalization of AMPA receptors and thus indicate a role for phosphatidylinositol(4,5)-bisphosphate metabolism in the regulation of postsynaptic AMPA responses.

  • cell and stimulus dependent heterogeneity of synaptic vesicle endocytic recycling mechanisms revealed by studies of dynamin 1 null neurons
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Mitsuko Hayashi, Andrea Raimondi, Chiara Collesi, Summer Paradise, Ottavio Cremona, Shawn M Ferguson, Pietro De Camilli, Eileen T Otoole
    Abstract:

    Mice lacking expression of dynamin 1, a GTPase implicated in the fission reaction of synaptic vesicle endocytosis, fail to thrive and exhibit severe activity-dependent endocytic defects at their synapses. Here, we have used electron tomography to investigate the massive increase in clathrin-coated pit abundance that is selectively observed at a subset of synapses in dynamin 1 KO primary neuron cultures under conditions of spontaneous network activity. This increase, leading to branched tubular plasma membrane invaginations capped by clathrin-coated buds, occurs selectively at inhibitory synapses. A similar massive increase of clathrin-coated profiles (in this case, of clathrin-coated vesicles) is observed at inhibitory synapses of neurons that lack expression of synaptojanin 1, a Phosphoinositide Phosphatase involved in clathrin-coated vesicle uncoating. Thus, although excitatory synapses are largely spared under these conditions, inhibitory synapses are uniquely sensitive to perturbation of endocytic proteins, probably as a result of their higher levels of tonic activity leading to a buildup of clathrin-coated intermediates in these synapses. In contrast, the predominant endocytic structures observed at the majority of dynamin 1 KO synapses after acute stimulation are endosome-like intermediates that originate by a dynamin 1-independent form of endocytosis. These findings reveal a striking heterogeneity in the mode of synaptic vesicle recycling in different synapses and functional states.

  • insp3 mediated intracellular calcium signalling is altered by expression of synaptojanin 1
    Biochemical Journal, 2004
    Co-Authors: Friedrich W Johenning, Markus R Wenk, Pietro De Camilli, Per Uhlen, Brenda Degray, Eunkyung Lee, Barbara E Ehrlich
    Abstract:

    Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] plays an important physiological role as a precursor for the InsP3-mediated intracellular calcium (Ca2+) signalling cascade. It also regulates membrane trafficking, actin function and transmembrane proteins. SJ-1 (synaptojanin-1), a Phosphoinositide Phosphatase, regulates the turnover of a PtdIns(4,5)P2 pool involved in clathrin and actin dynamics at the cell surface. We tested the interrelationship of this pool with PtdIns(4,5)P2 pools involved in Ca2+ signalling by expressing in Chinese-hamster ovary cells full-length SJ-1 or its 5-Pase (inositol 5-Phosphatase) domain. SJ-1 significantly attenuated the generation of Ca2+ oscillations induced by ATP and the 5-Pase domain mimicked this effect. These changes correlated with increased PtdIns(4,5)P2 Phosphatase activity of cellular extracts. Overexpression of the endoplasmic reticulum-anchored PtdIns(4)P Phosphatase Sac1 did not affect Ca2+ oscillations, although it increased the Ca2+ efflux rate from intracellular stores. The ability of SJ-1 to alter intracellular Ca2+ signalling indicates a close functional interrelationship between plasma membrane PtdIns(4,5)P2 pools that control actin and endocytosis and those involved in the regulation of specific spatio-temporal Ca2+ signals.

Yasushi Okamura - One of the best experts on this subject based on the ideXlab platform.

  • voltage sensitive Phosphoinositide Phosphatases of xenopus their tissue distribution and voltage dependence
    Journal of Cellular Physiology, 2011
    Co-Authors: William J Ratzan, Yasushi Okamura, Alexei V Evsikov, Laurinda A Jaffe
    Abstract:

    Voltage-sensitive Phosphatases (VSPs) are unique proteins in which membrane potential controls enzyme activity. They are comprised of the voltage sensor domain of an ion channel coupled to a lipid Phosphatase specific for Phosphoinositides, and for ascidian and zebrafish VSPs, the Phosphatase activity has been found to be activated by membrane depolarization. The physiological functions of these proteins are unknown, but their expression in testis and embryos suggests a role in fertilization or development. Here we investigate the expression pattern and voltage dependence of VSPs in two frog species, Xenopus laevis and Xenopus tropicalis, that are well suited for experimental studies of these possible functions. X. laevis has two VSP genes (Xl-VSP1 and Xl-VSP2), whereas X. tropicalis has only one gene (Xt-VSP). The highest expression of these genes was observed in testis, ovary, liver, and kidney. Our results show that while Xl-VSP2 activates only at positive membrane potentials outside of the physiological range, Xl-VSP1 and Xt-VSP Phosphatase activity is regulated in the voltage range that regulates sperm–egg fusion at fertilization. J. Cell. Physiol. 226: 2740–2746, 2011. 2011 Wiley-Liss, Inc. Voltage-sensitive Phosphatases (VSPs) are four-transmembrane proteins that contain the characteristic voltage sensor domain of an ion channel, but instead of controlling an ion pore, the voltage sensor is linked to a cytoplasmic Phosphoinositide Phosphatase (Murata et al., 2005). Studies of the VSPs of the ascidian Ciona intestinalis (Ci-VSP) and the zebrafish Danio rerio VSP (Dr-VSP) have shown that depolarization activates the Phosphatase activity (Murata and Okamura, 2007; Hossain et al., 2008). This has been proposed to occur because movement of the voltage sensor results in movement of the Phosphatase domain closer to the plasma membrane

  • crystal structure of the cytoplasmic Phosphatase and tensin homolog pten like region of ciona intestinalis voltage sensing Phosphatase provides insight into substrate specificity and redox regulation of the Phosphoinositide Phosphatase activity
    Journal of Biological Chemistry, 2011
    Co-Authors: Makoto Matsuda, Yasushi Okamura, Souhei Sakata, Kohei Takeshita, Tatsuki Kurokawa, Mamoru Suzuki, Eiki Yamashita, Atsushi Nakagawa
    Abstract:

    Abstract Ciona intestinalis voltage-sensing Phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the Phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a Phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wild-type (248–576), wild-type (236–576), and G365A mutant (248–576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.

  • a voltage sensing Phosphatase ci vsp which shares sequence identity with pten dephosphorylates phosphatidylinositol 4 5 bisphosphate
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Hirohide Iwasaki, Yoshimichi Murata, Jack E Dixon, Young Jun Kim, Md Israil Hossain, Carolyn A Worby, Thomas J Mccormack, Takehiko Sasaki, Yasushi Okamura
    Abstract:

    Phosphatidylinositol lipids play diverse physiological roles, and their concentrations are tightly regulated by various kinases and Phosphatases. The enzymatic activity of Ciona intestinalis voltage sensor-containing Phosphatase (Ci-VSP), recently identified as a member of the PTEN (Phosphatase and tensin homolog deleted on chromosome 10) family of phosphatidylinositol Phosphatases, is regulated by its own voltage-sensor domain in a voltage-dependent manner. However, a detailed mechanism of Ci-VSP regulation and its substrate specificity remain unknown. Here we determined the in vitro substrate specificity of Ci-VSP by measuring the Phosphoinositide Phosphatase activity of the Ci-VSP cytoplasmic Phosphatase domain. Despite the high degree of identity shared between the active sites of PTEN and Ci-VSP, Ci-VSP dephosphorylates not only the PTEN substrate, phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3], but also, unlike PTEN, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Enzymatic action on PI(4,5)P2 removes the phosphate at position 5 of the inositol ring, resulting in the production of phosphatidylinositol 4-phosphate [PI(4)P]. The active site Cys-X5-Arg (CX5R) sequence of Ci-VSP differs with that of PTEN only at amino acid 365 where a glycine residue in Ci-VSP is replaced by an alanine in PTEN. Ci-VSP with a G365A mutation no longer dephosphorylates PI(4,5)P2 and is not capable of inducing depolarization-dependent rundown of a PI(4,5)P2-dependent potassium channel. These results indicate that Ci-VSP is a PI(3,4,5)P3/PI(4,5)P2 Phosphatase that uniquely functions in the voltage-dependent regulation of ion channels through regulation of PI(4,5)P2 levels.

  • depolarization activates the Phosphoinositide Phosphatase ci vsp as detected in xenopus oocytes coexpressing sensors of pip2
    The Journal of Physiology, 2007
    Co-Authors: Yoshimichi Murata, Yasushi Okamura
    Abstract:

    Voltage-evoked signals play critical roles in neural activities, muscle contraction and exocytosis. Ciona voltage-sensor containing Phosphatase (Ci-VSP) consists of the transmembrane voltage sensor domain (VSD) and a cytoplasmic domain of Phosphoinositide Phosphatase, homologous to Phosphatase and tensin homologue deleted on chromosome 10 (PTEN). Previous experiments utilizing potassium channels as the sensor for Phosphoinositides have demonstrated that Phosphatase activities of Ci-VSP are voltage dependent. However, it still remained unclear whether enzyme activity is activated by depolarization or hyperpolarization. Further, a large gap in voltage dependency was found between the charge movement of the VSD and potassium channel-reporting Phosphatase activities. In this study, voltage-dependent dynamics of Phosphoinositides mediated by Ci-VSP were examined by confocal imaging and electrical measurements in Xenopus oocytes. Imaging of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) using green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains from phospholipase C δ subunit (PLC-δ) showed that PtdIns(4,5)P2 concentration is reduced during depolarization. In the presence of Ci-VSP, IRK1 channels with higher sensitivity to Phosphoinositide than GIRK2 channels decreased their magnitude during depolarization over 0 mV, indicating that the PtdIns(4,5)P2 level is reduced upon depolarization. KCNQ2/3 channels coexpressed with Ci-VSP exhibited voltage-dependent decay of the outward current that became sharper with higher depolarization in a voltage range up to 100 mV. These results indicate that Ci-VSP has an activity that depletes PtdIns(4,5)P2 unlike PTEN and that depolarization-activated voltage sensor movement is translated into activation of Phosphatase activity.

  • Phosphoinositide Phosphatase activity coupled to an intrinsic voltage sensor
    Nature, 2005
    Co-Authors: Yoshimichi Murata, Hirohide Iwasaki, Mari Sasaki, Kazuo Inaba, Yasushi Okamura
    Abstract:

    Changes in membrane potential affect ion channels and transporters, which then alter intracellular chemical conditions. Other signalling pathways coupled to membrane potential have been suggested1,2,3 but their underlying mechanisms are unknown. Here we describe a novel protein from the ascidian Ciona intestinalis that has a transmembrane voltage-sensing domain homologous to the S1–S4 segments of voltage-gated channels and a cytoplasmic domain similar to Phosphatase and tensin homologue. This protein, named C. intestinalis voltage-sensor-containing Phosphatase (Ci-VSP), displays channel-like ‘gating’ currents and directly translates changes in membrane potential into the turnover of Phosphoinositides. The activity of the Phosphoinositide Phosphatase in Ci-VSP is tuned within a physiological range of membrane potential. Immunocytochemical studies show that Ci-VSP is expressed in Ciona sperm tail membranes, indicating a possible role in sperm function or morphology. Our data demonstrate that voltage sensing can function beyond channel proteins and thus more ubiquitously than previously realized.

Markus R Wenk - One of the best experts on this subject based on the ideXlab platform.

  • synaptojanin 1 linked Phosphoinositide dyshomeostasis and cognitive deficits in mouse models of down s syndrome
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Sergey V Voronov, Silvia Giovedi, Markus R Wenk, Samuel G Frere, Elizabeth A Pollina, Christelle Borel, Hong Zhang, Cecilia Schmidt, Ellen C Akeson, Laurent Cimasoni
    Abstract:

    Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P(2)] is a signaling phospholipid implicated in a wide variety of cellular functions. At synapses, where normal PtdIns(4,5)P(2) balance is required for proper neurotransmission, the Phosphoinositide Phosphatase synaptojanin 1 is a key regulator of its metabolism. The underlying gene, SYNJ1, maps to human chromosome 21 and is thus a candidate for involvement in Down's syndrome (DS), a complex disorder resulting from the overexpression of trisomic genes. Here, we show that PtdIns(4,5)P(2) metabolism is altered in the brain of Ts65Dn mice, the most commonly used model of DS. This defect is rescued by restoring Synj1 to disomy in Ts65Dn mice and is recapitulated in transgenic mice overexpressing Synj1 from BAC constructs. These transgenic mice also exhibit deficits in performance of the Morris water maze task, suggesting that PtdIns(4,5)P(2) dyshomeostasis caused by gene dosage imbalance for Synj1 may contribute to brain dysfunction and cognitive disabilities in DS.

  • insp3 mediated intracellular calcium signalling is altered by expression of synaptojanin 1
    Biochemical Journal, 2004
    Co-Authors: Friedrich W Johenning, Markus R Wenk, Pietro De Camilli, Per Uhlen, Brenda Degray, Eunkyung Lee, Barbara E Ehrlich
    Abstract:

    Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] plays an important physiological role as a precursor for the InsP3-mediated intracellular calcium (Ca2+) signalling cascade. It also regulates membrane trafficking, actin function and transmembrane proteins. SJ-1 (synaptojanin-1), a Phosphoinositide Phosphatase, regulates the turnover of a PtdIns(4,5)P2 pool involved in clathrin and actin dynamics at the cell surface. We tested the interrelationship of this pool with PtdIns(4,5)P2 pools involved in Ca2+ signalling by expressing in Chinese-hamster ovary cells full-length SJ-1 or its 5-Pase (inositol 5-Phosphatase) domain. SJ-1 significantly attenuated the generation of Ca2+ oscillations induced by ATP and the 5-Pase domain mimicked this effect. These changes correlated with increased PtdIns(4,5)P2 Phosphatase activity of cellular extracts. Overexpression of the endoplasmic reticulum-anchored PtdIns(4)P Phosphatase Sac1 did not affect Ca2+ oscillations, although it increased the Ca2+ efflux rate from intracellular stores. The ability of SJ-1 to alter intracellular Ca2+ signalling indicates a close functional interrelationship between plasma membrane PtdIns(4,5)P2 pools that control actin and endocytosis and those involved in the regulation of specific spatio-temporal Ca2+ signals.

  • salmonella modulates vesicular traffic by altering Phosphoinositide metabolism
    Science, 2004
    Co-Authors: Lorraine D Hernandez, Markus R Wenk, Karsten Hueffer, Jorge E Galan
    Abstract:

    Salmonella enterica , the cause of food poisoning and typhoid fever, induces actin cytoskeleton rearrangements and membrane ruffling to gain access into nonphagocytic cells, where it can replicate and avoid innate immune defenses. Here, we found that SopB, a Phosphoinositide Phosphatase that is delivered into host cells by a type III secretion system, was essential for the establishment of Salmonella 9s intracellular replicative niche. SopB mediated the formation of spacious phagosomes following bacterial entry and was responsible for maintaining high levels of phosphatidylinositol-three-phosphate [PtdIns(3)P] in the membrane of the bacteria-containing vacuoles. Absence of SopB caused a significant defect in the maturation of the Salmonella -containing vacuole and impaired bacterial intracellular growth.

  • identification and characterization of a synaptojanin 2 splice isoform predominantly expressed in nerve terminals
    Journal of Biological Chemistry, 2001
    Co-Authors: Yasuo Nemoto, Niels Ringstad, Markus R Wenk, Laurie Daniell, Masami Watanabe, Tomoe Murakami, Hiroshi Yamada, Pietro De Camilli
    Abstract:

    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.

  • functional characterization of a mammalian sac1 and mutants exhibiting substrate specific defects in Phosphoinositide Phosphatase activity
    Journal of Biological Chemistry, 2000
    Co-Authors: Yasuo Nemoto, Markus R Wenk, Pietro De Camilli, Brian G Kearns, Hong Chen, Kensaku Mori, James G Alb, Vytas A Bankaitis
    Abstract:

    The Saccharomyces cerevisiae SAC1gene was identified via independent analyses of mutations that modulate yeast actin function and alleviate the essential requirement for phosphatidylinositol transfer protein (Sec14p) activity in Golgi secretory function. The SAC1 gene product (Sac1p) is an integral membrane protein of the endoplasmic reticulum and the Golgi complex. Sac1p shares primary sequence homology with a subfamily of cytosolic/peripheral membrane Phosphoinositide Phosphatases, the synaptojanins, and these Sac1 domains define novel Phosphoinositide Phosphatase modules. We now report the characterization of a rat counterpart of Sac1p. Rat Sac1 is a ubiquitously expressed 65-kDa integral membrane protein of the endoplasmic reticulum that is found at particularly high levels in cerebellar Purkinje cells. Like Sac1p, rat Sac1 exhibits intrinsic Phosphoinositide Phosphatase activity directed toward phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 3,5-bisphosphate substrates, and we identify mutant rat sac1 alleles that evoke substrate-specific defects in this enzymatic activity. Finally, rat Sac1 expression in Δsac1 yeast strains complements a wide phenotypes associated with Sac1p insufficiency. Biochemical and in vivo data indicate that rat Sac1 phosphatidylinositol-4-phosphate Phosphatase activity, but not its phosphatidylinositol-3-phosphate or phosphatidylinositol-3,5-bisphosphate Phosphatase activities, is essential for the heterologous complementation of Sac1p defectsin vivo. Thus, yeast Sac1p and rat Sac1 are integral membrane lipid Phosphatases that play evolutionary conserved roles in eukaryotic cell physiology.

Yasuo Nemoto - One of the best experts on this subject based on the ideXlab platform.

  • identification and characterization of a synaptojanin 2 splice isoform predominantly expressed in nerve terminals
    Journal of Biological Chemistry, 2001
    Co-Authors: Yasuo Nemoto, Niels Ringstad, Markus R Wenk, Laurie Daniell, Masami Watanabe, Tomoe Murakami, Hiroshi Yamada, Pietro De Camilli
    Abstract:

    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.

  • functional characterization of a mammalian sac1 and mutants exhibiting substrate specific defects in Phosphoinositide Phosphatase activity
    Journal of Biological Chemistry, 2000
    Co-Authors: Yasuo Nemoto, Markus R Wenk, Pietro De Camilli, Brian G Kearns, Hong Chen, Kensaku Mori, James G Alb, Vytas A Bankaitis
    Abstract:

    The Saccharomyces cerevisiae SAC1gene was identified via independent analyses of mutations that modulate yeast actin function and alleviate the essential requirement for phosphatidylinositol transfer protein (Sec14p) activity in Golgi secretory function. The SAC1 gene product (Sac1p) is an integral membrane protein of the endoplasmic reticulum and the Golgi complex. Sac1p shares primary sequence homology with a subfamily of cytosolic/peripheral membrane Phosphoinositide Phosphatases, the synaptojanins, and these Sac1 domains define novel Phosphoinositide Phosphatase modules. We now report the characterization of a rat counterpart of Sac1p. Rat Sac1 is a ubiquitously expressed 65-kDa integral membrane protein of the endoplasmic reticulum that is found at particularly high levels in cerebellar Purkinje cells. Like Sac1p, rat Sac1 exhibits intrinsic Phosphoinositide Phosphatase activity directed toward phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 3,5-bisphosphate substrates, and we identify mutant rat sac1 alleles that evoke substrate-specific defects in this enzymatic activity. Finally, rat Sac1 expression in Δsac1 yeast strains complements a wide phenotypes associated with Sac1p insufficiency. Biochemical and in vivo data indicate that rat Sac1 phosphatidylinositol-4-phosphate Phosphatase activity, but not its phosphatidylinositol-3-phosphate or phosphatidylinositol-3,5-bisphosphate Phosphatase activities, is essential for the heterologous complementation of Sac1p defectsin vivo. Thus, yeast Sac1p and rat Sac1 are integral membrane lipid Phosphatases that play evolutionary conserved roles in eukaryotic cell physiology.

  • Essential Role of Phosphoinositide Metabolism in Synaptic Vesicle Recycling
    Cell, 1999
    Co-Authors: Ottavio Cremona, Gilbert Di Paolo, Yasuo Nemoto, Kohji Takei, Markus R Wenk, Anita Lüthi, Warren T. Kim, Laurie Daniell, Stephen B. Shears, Richard A. Flavell
    Abstract:

    Abstract Growing evidence suggests that Phosphoinositides play an important role in membrane traffic. A polyPhosphoinositide Phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1–deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P 2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced Phosphoinositide Phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of Phosphoinositide metabolism in synaptic vesicle recycling.

Ottavio Cremona - One of the best experts on this subject based on the ideXlab platform.

  • recruitment of endophilin to clathrin coated pit necks is required for efficient vesicle uncoating after fission
    Neuron, 2011
    Co-Authors: Ira Milosevic, Andrea Raimondi, Chiara Collesi, Summer Paradise, Silvia Giovedi, Hongying Shen, Shawn M Ferguson, Eileen T Otoole, Ottavio Cremona
    Abstract:

    Summary Endophilin 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.

  • cell and stimulus dependent heterogeneity of synaptic vesicle endocytic recycling mechanisms revealed by studies of dynamin 1 null neurons
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Mitsuko Hayashi, Andrea Raimondi, Chiara Collesi, Summer Paradise, Ottavio Cremona, Shawn M Ferguson, Pietro De Camilli, Eileen T Otoole
    Abstract:

    Mice lacking expression of dynamin 1, a GTPase implicated in the fission reaction of synaptic vesicle endocytosis, fail to thrive and exhibit severe activity-dependent endocytic defects at their synapses. Here, we have used electron tomography to investigate the massive increase in clathrin-coated pit abundance that is selectively observed at a subset of synapses in dynamin 1 KO primary neuron cultures under conditions of spontaneous network activity. This increase, leading to branched tubular plasma membrane invaginations capped by clathrin-coated buds, occurs selectively at inhibitory synapses. A similar massive increase of clathrin-coated profiles (in this case, of clathrin-coated vesicles) is observed at inhibitory synapses of neurons that lack expression of synaptojanin 1, a Phosphoinositide Phosphatase involved in clathrin-coated vesicle uncoating. Thus, although excitatory synapses are largely spared under these conditions, inhibitory synapses are uniquely sensitive to perturbation of endocytic proteins, probably as a result of their higher levels of tonic activity leading to a buildup of clathrin-coated intermediates in these synapses. In contrast, the predominant endocytic structures observed at the majority of dynamin 1 KO synapses after acute stimulation are endosome-like intermediates that originate by a dynamin 1-independent form of endocytosis. These findings reveal a striking heterogeneity in the mode of synaptic vesicle recycling in different synapses and functional states.

  • Essential Role of Phosphoinositide Metabolism in Synaptic Vesicle Recycling
    Cell, 1999
    Co-Authors: Ottavio Cremona, Gilbert Di Paolo, Yasuo Nemoto, Kohji Takei, Markus R Wenk, Anita Lüthi, Warren T. Kim, Laurie Daniell, Stephen B. Shears, Richard A. Flavell
    Abstract:

    Abstract Growing evidence suggests that Phosphoinositides play an important role in membrane traffic. A polyPhosphoinositide Phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1–deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P 2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced Phosphoinositide Phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of Phosphoinositide metabolism in synaptic vesicle recycling.