Publications (14)101.36 Total impact
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Article: Phosphorylation of syndapin I F-BAR domain at two helix-capping motifs regulates membrane tubulation.
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ABSTRACT: Syndapin I (PACSIN 1) is a synaptically enriched membrane tubulating protein that plays important roles in activity-dependent bulk endocytosis and neuronal morphogenesis. While syndapin I is an in vitro phosphoprotein, it is not known to be phosphorylated in neurons. Here, we report the identification of two phosphorylation sites, S76 and T181, of syndapin I from nerve terminals. Both residues are located at the N-terminal helix-capping motifs (N-Cap) of different α-helices in the F-BAR domain, important for F-BAR homodimer curvature and dimer-dimer filament assembly, respectively. Phospho-mimetic mutations of these residues regulate lipid-binding and tubulation both in vitro and in cells. Neither phosphosite regulated syndapin I function in activity-dependent bulk endocytosis. Rather, T181 phosphorylation was developmentally regulated and inhibited syndapin I function in neuronal morphogenesis. This suggests a novel mechanism for phosphorylation control of an F-BAR function through the regulation of α-helix interactions and stability within the folded F-BAR domain.Proceedings of the National Academy of Sciences 03/2012; 109(10):3760-5. · 9.68 Impact Factor -
Article: Regulation of AMPA receptor trafficking and synaptic plasticity.
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ABSTRACT: AMPA receptors (AMPARs) mediate the majority of fast excitatory synaptic transmission in the brain. Dynamic changes in neuronal synaptic efficacy, termed synaptic plasticity, are thought to underlie information coding and storage in learning and memory. One major mechanism that regulates synaptic strength involves the tightly regulated trafficking of AMPARs into and out of synapses. The life cycle of AMPARs from their biosynthesis, membrane trafficking, and synaptic targeting to their degradation are controlled by a series of orchestrated interactions with numerous intracellular regulatory proteins. Here we review recent progress made toward the understanding the regulation of AMPAR trafficking, focusing on the roles of several key intracellular AMPAR interacting proteins.Current opinion in neurobiology 01/2012; 22(3):461-9. · 7.21 Impact Factor -
Article: Regulation of AMPA receptor function by the human memory-associated gene KIBRA.
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ABSTRACT: KIBRA has recently been identified as a gene associated with human memory performance. Despite the elucidation of the role of KIBRA in several diverse processes in nonneuronal cells, the molecular function of KIBRA in neurons is unknown. We found that KIBRA directly binds to the protein interacting with C-kinase 1 (PICK1) and forms a complex with α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs), the major excitatory neurotransmitter receptors in the brain. KIBRA knockdown accelerates the rate of AMPAR recycling following N-methyl-D-aspartate receptor-induced internalization. Genetic deletion of KIBRA in mice impairs both long-term depression and long-term potentiation at hippocampal Schaffer collateral-CA1 synapses. Moreover, KIBRA knockout mice have severe deficits in contextual fear learning and memory. These results indicate that KIBRA regulates higher brain function by regulating AMPAR trafficking and synaptic plasticity.Neuron 09/2011; 71(6):1022-9. · 14.74 Impact Factor -
Article: Gain-of-function glutamate receptor interacting protein 1 variants alter GluA2 recycling and surface distribution in patients with autism.
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ABSTRACT: Glutamate receptor interacting protein 1 (GRIP1) is a neuronal scaffolding protein that interacts directly with the C termini of glutamate receptors 2/3 (GluA2/3) via its PDZ domains 4 to 6 (PDZ4-6). We found an association (P<0.05) of a SNP within the PDZ4-6 genomic region with autism by genotyping autistic patients (n=480) and matched controls (n=480). Parallel sequencing identified five rare missense variants within or near PDZ4-6 only in the autism cohort, resulting in a higher cumulative mutation load (P=0.032). Two variants correlated with a more severe deficit in reciprocal social interaction in affected sibling pairs from proband families. These variants were associated with altered interactions with GluA2/3 and faster recycling and increased surface distribution of GluA2 in neurons, suggesting gain-of-function because GRIP1/2 deficiency showed opposite phenotypes. Grip1/2 knockout mice exhibited increased sociability and impaired prepulse inhibition. These results support a role for GRIP in social behavior and implicate GRIP1 variants in modulating autistic phenotype.Proceedings of the National Academy of Sciences 03/2011; 108(12):4920-5. · 9.68 Impact Factor -
Article: PICK1 loss of function occludes homeostatic synaptic scaling.
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ABSTRACT: Homeostatic synaptic scaling calibrates neuronal excitability by adjusting synaptic strengths during prolonged changes in synaptic activity. The molecular mechanisms that regulate the trafficking of AMPA receptors (AMPARs) during synaptic scaling are largely unknown. Here, we show that chronic activity blockade reduces PICK1 protein level on a time scale that coincides with the accumulation of surface AMPARs. PICK1 loss of function alters the subunit composition and the abundance of GluA2-containing AMPARs. Due to aberrant trafficking of these receptors, the increase in synaptic strength in response to synaptic inactivity is occluded in neurons generated from PICK1 knock-out mouse. In agreement with electrophysiological recordings, no defect of AMPAR trafficking is observed in PICK1 knock-out neurons in response to elevated neuronal activity. Overall, our data reveal an important role of PICK1 in inactivity-induced synaptic scaling by regulating the subunit composition, abundance, and trafficking of GluA2-containing AMPARs.Journal of Neuroscience 02/2011; 31(6):2188-96. · 7.11 Impact Factor -
Article: Phosphorylation of dynamin II at serine-764 is associated with cytokinesis.
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ABSTRACT: Calcineurin is a phosphatase that is activated at the last known stage of mitosis, abscission. Among its many substrates, it dephosphorylates dynamin II during cytokinesis at the midbody of dividing cells. However, dynamin II has several cellular roles including clathrin-mediated endocytosis, centrosome cohesion and cytokinesis. It is not known whether dynamin II phosphorylation plays a role in any of these functions nor have the phosphosites involved in cytokinesis been directly identified. We now report that dynamin II from rat lung is phosphorylated to a low stoichiometry on a single major site, Ser-764, in the proline-rich domain. Phosphorylation on Ser-764 also occurred in asynchronously growing HeLa cells and was greatly increased upon mitotic entry. Tryptic phospho-peptides isolated by TiO(2) chromatography revealed only a single phosphosite in mitotic cells. Mitotic phosphorylation was abolished by roscovitine, suggesting the mitotic kinase is cyclin-dependent kinase 1. Cyclin-dependent kinase 1 phosphorylated full length dynamin II and Glutathione-S-Transferase-tagged-dynamin II-proline-rich domain in vitro, and mutation of Ser-764 to alanine reduced proline-rich domain phosphorylation by 80%, supporting that there is only a single major phosphosite. Ser-764 phosphorylation did not affect clathrin-mediated endocytosis or bulk endocytosis using penetratin-based phospho-deficient or phospho-mimetic peptides or following siRNA depletion/rescue experiments. Phospho-dynamin II was enriched at the mitotic centrosome, but this targeting was unaffected by the phospho-deficient or phospho-mimetic peptides. In contrast, the phospho-mimetic peptide displaced endogenous dynamin II, but not calcineurin, from the midbody and induced cytokinesis failure. Therefore, phosphorylation of dynamin II primarily occurs on a single site that regulates cytokinesis downstream of calcineurin, rather than regulating endocytosis or centrosome function.Biochimica et Biophysica Acta 12/2010; 1813(10):1689-99. · 4.66 Impact Factor -
Article: PICK1 regulates incorporation of calcium-permeable AMPA receptors during cortical synaptic strengthening.
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ABSTRACT: While AMPA-type glutamate receptors (AMPARs) found at principal neuron excitatory synapses typically contain the GluR2 subunit, several forms of behavioral experience have been linked to the de novo synaptic insertion of calcium-permeable (CP) AMPARs, defined by their lack of GluR2. In particular, whisker experience drives synaptic potentiation as well as the incorporation of CP-AMPARs in the neocortex. Previous studies implicate PICK1 (protein interacting with C kinase-1) in activity-dependent internalization of GluR2, suggesting one potential mechanism leading to the subsequent accumulation of synaptic CP-AMPARs and increased synaptic strength. Here we test this hypothesis by using a whisker stimulation paradigm in PICK1 knock-out mice. We demonstrate that PICK1 facilitates the surface expression of CP-AMPARs and is indispensable for their experience-dependent synaptic insertion. However, the failure to incorporate CP-AMPARs in PICK1 knock-outs does not preclude sensory-induced enhancement of synaptic currents. Our results indicate that synaptic strengthening in the early postnatal cortex does not require PICK1 or the addition of GluR2-lacking AMPARs. Instead, PICK1 permits changes in AMPAR subunit composition to occur in conjunction with synaptic potentiation.Journal of Neuroscience 05/2010; 30(18):6360-6. · 7.11 Impact Factor -
Article: Identification of a small-molecule inhibitor of the PICK1 PDZ domain that inhibits hippocampal LTP and LTD.
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ABSTRACT: Proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) domains play key roles in the assembly and regulation of cellular signaling pathways and represent putative targets for new pharmacotherapeutics. Here we describe the first small-molecule inhibitor (FSC231) of the PDZ domain in protein interacting with C kinase 1 (PICK1) identified by a screening of approximately 44,000 compounds in a fluorescent polarization assay. The inhibitor bound the PICK1 PDZ domain with an affinity similar to that observed for endogenous peptide ligands (K(i) approximately 10.1 microM). Mutational analysis, together with computational docking of the compound in simulations starting from the PDZ domain structure, identified the binding mode of FSC231. The specificity of FSC231 for the PICK1 PDZ domain was supported by the lack of binding to PDZ domains of postsynaptic density protein 95 (PSD-95) and glutamate receptor interacting protein 1 (GRIP1). Pretreatment of cultured hippocampal neurons with FSC231 inhibited coimmunopreciptation of the AMPA receptor GluR2 subunit with PICK1. In agreement with inhibiting the role of PICK1 in GluR2 trafficking, FSC231 accelerated recycling of pHluorin-tagged GluR2 in hippocampal neurons after internalization in response to NMDA receptor activation. FSC231 blocked the expression of both long-term depression and long-term potentiation in hippocampal CA1 neurons from acute slices, consistent with inhibition of the bidirectional function of PICK1 in synaptic plasticity. Given the proposed role of the PICK1/AMPA receptor interaction in neuropathic pain, excitotoxicity, and cocaine addiction, FSC231 might serve as a lead in the future development of new therapeutics against these conditions.Proceedings of the National Academy of Sciences 12/2009; 107(1):413-8. · 9.68 Impact Factor -
Article: The phospho-dependent dynamin-syndapin interaction triggers activity-dependent bulk endocytosis of synaptic vesicles.
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ABSTRACT: Synaptic vesicles (SVs) are retrieved by more than one mode in central nerve terminals. During mild stimulation, the dominant SV retrieval pathway is classical clathrin-mediated endocytosis (CME). During elevated neuronal activity, activity-dependent bulk endocytosis (ADBE) predominates, which requires activation of the calcium-dependent protein phosphatase calcineurin. We now report that calcineurin dephosphorylates dynamin I in nerve terminals only above the same activity threshold that triggers ADBE. ADBE was arrested when the two major phospho-sites on dynamin I were perturbed, suggesting that dynamin I dephosphorylation is a key step in its activation. Dynamin I dephosphorylation stimulates a specific dynamin I-syndapin I interaction. Inhibition of this interaction by competitive peptides or by site-directed mutagenesis exclusively inhibited ADBE but did not affect CME. The results reveal that the phospho-dependent dynamin-syndapin interaction recruits ADBE to massively increase SV endocytosis under conditions of elevated neuronal activity.Journal of Neuroscience 07/2009; 29(24):7706-17. · 7.11 Impact Factor -
Chapter: Septins and the Synapse
11/2008: pages 247 - 267; , ISBN: 9780470779705 -
Article: Styryl dye-based synaptic vesicle recycling assay in cultured cerebellar granule neurons.
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ABSTRACT: Neurons transmit information by exocytosis of synaptic vesicles (SV), which contain neurotransmitter. Exocytosis is followed by efficient retrieval of the plasma membrane by endocytosis to generate a new SV. SV retrieval supports multiple cycles of synaptic transmission. Over the years, styryl dyes have been widely used to probe the mechanism of SV recycling in the processes of cultured neurons. The styryl dye method is complementary to electrophysiological measurements or genetic reporter approaches. Owing to their ease to culture, cerebellar granule neurons provide a robust neuronal model system for the assay. These cells are readily transfected with various DNA constructs to study the function of exocytic or endocytic proteins in SV recycling.Methods in molecular biology (Clifton, N.J.) 02/2008; 457:333-45. -
Article: Syndapin I and endophilin I bind overlapping proline-rich regions of dynamin I: role in synaptic vesicle endocytosis.
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ABSTRACT: Dynamin I mediates vesicle fission during synaptic vesicle endocytosis (SVE). Its proline-rich domain (PRD) binds the Src-homology 3 (SH3) domain of a subset of proteins that can deform membranes. Syndapin I, amphiphysin I, and endophilin I are its major partners implicated in SVE. Syndapin binding is controlled by phosphorylation at Ser-774 and Ser-778 in the dynamin phospho-box. We now define syndapin and endophilin-binding sites by peptide competition and site-directed mutagenesis. Both bound the same region of the dynamin PRD and both exhibited unusual bidirectional binding modes around core PxxP motifs, unlike amphiphysin which employed a class II binding mode. Endophilin binds to tandem PxxP motifs in the sequence (778)SPTPQRRAPAVPPARPGSR(796) in dynamin, with SPTPQ being an overhang sequence. In contrast, syndapin binding involves two components in the region (772)RRSPTSSPTPQRRAPAVPPARPGSR(796). It required a single PxxP core and a non-PxxP N-terminally anchored extension which bridges the phospho-box and may contribute to binding specificity and affinity. Syndapin binding is exquisitely sensitive to the introduction of negative charges almost anywhere along this region, explaining why it is a highly tuned phospho-sensor. Over-expression of dynamin point mutants that fail to bind syndapin or endophilin inhibit SVE in cultured neurons. Due to overlapping binding sites the interactions between dynamin and syndapin or endophilin were mutually exclusive. Because syndapin acts as a phospho-sensor, this supports its role in depolarization-induced SVE at the synapse, which involves dynamin dephosphorylation. We propose syndapin and endophilin function either at different stages during SVE or in mechanistically distinct types of SVE.Journal of Neurochemistry 09/2007; 102(3):931-43. · 4.06 Impact Factor -
Article: The in vivo phosphorylation sites of rat brain dynamin I.
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ABSTRACT: Dynamin I (dynI) is phosphorylated in synaptosomes at Ser(774) and Ser(778) by cyclin-dependent kinase 5 to regulate recruitment of syndapin I for synaptic vesicle endocytosis, and in PC12 cells on Ser(857). Hierarchical phosphorylation of Ser(774) precedes phosphorylation of Ser(778). In contrast, Thr(780) phosphorylation by cdk5 has been reported as the sole site (Tomizawa, K., Sunada, S., Lu, Y. F., Oda, Y., Kinuta, M., Ohshima, T., Saito, T., Wei, F. Y., Matsushita, M., Li, S. T., Tsutsui, K., Hisanaga, S. I., Mikoshiba, K., Takei, K., and Matsui, H. (2003) J. Cell Biol. 163, 813-824). To resolve the discrepancy and to better understand the biological roles of dynI phosphorylation, we undertook a systematic identification of all phosphorylation sites in rat brain nerve terminal dynI. Using phosphoamino acid analysis, exclusively phospho-serine residues were found. Thr(780) phosphorylation was not detectable. Mutation of Ser(774), Ser(778), and Thr(780) confirmed that Thr(780) phosphorylation is restricted to in vitro conditions. Mass spectrometry of (32)P-labeled phosphopeptides separated by two-dimensional mapping revealed seven in vivo phosphorylation sites: Ser(774), Ser(778), Ser(822), Ser(851), Ser(857), Ser(512), and Ser(347). Quantification of (32)P radiation in each phosphopeptide showed that Ser(774) and Ser(778) were the major sites (up to 69% of the total), followed by Ser(851) and Ser(857) (12%), and Ser(853) (2%). Phosphorylation of Ser(851) and Ser(857) was restricted to the long tail splice variant dynIxa and was not hierarchical. Co-purified, (32)P-labeled dynIII was phosphorylated at Ser(759), Ser(763), and Ser(853). Ser(853) is homologous to Ser(851) in dynIxa. The results identify all major and several minor phosphorylation sites in dynI and provide the first measure of their relative abundance and relative responses to depolarization. The multiple phospho-sites suggest subtle regulation of synaptic vesicle endocytosis by new protein kinases and new protein-protein interactions. The homologous dynI and dynIII phosphorylation indicates a high mechanistic similarity. The results suggest a unique role for the long splice variants of dynI and dynIII in nerve terminals.Journal of Biological Chemistry 06/2007; 282(20):14695-707. · 4.77 Impact Factor -
Article: Syndapin I is the phosphorylation-regulated dynamin I partner in synaptic vesicle endocytosis.
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ABSTRACT: Dynamin I is dephosphorylated at Ser-774 and Ser-778 during synaptic vesicle endocytosis (SVE) in nerve terminals. Phosphorylation was proposed to regulate the assembly of an endocytic protein complex with amphiphysin or endophilin. Instead, we found it recruits syndapin I for SVE and does not control amphiphysin or endophilin binding in rat synaptosomes. After depolarization, syndapin showed a calcineurin-mediated interaction with dynamin. A peptide mimicking the phosphorylation sites disrupted the dynamin-syndapin complex, not the dynamin-endophilin complex, arrested SVE and produced glutamate release fatigue after repetitive stimulation. Pseudophosphorylation of Ser-774 or Ser-778 inhibited syndapin binding without affecting amphiphysin recruitment. Site mutagenesis to alanine arrested SVE in cultured neurons. The effects of the sites were additive for syndapin I binding and SVE. Thus syndapin I is a central component of the endocytic protein complex for SVE via stimulus-dependent recruitment to dynamin I and has a key role in synaptic transmission.Nature Neuroscience 07/2006; 9(6):752-60. · 15.53 Impact Factor
Top co-authors
Top Journals
Institutions
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2010–2012
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Johns Hopkins University
- Department of Neuoscience
Baltimore, MD, USA -
University of Sydney
- Children's Medical Research Institute (CMRI)
Sydney, New South Wales, Australia
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2009–2012
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Howard Hughes Medical Institute
Chevy Chase, MD, USA
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2006–2008
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Children's Medical Research Institute
Sydney, New South Wales, Australia
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2007
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Cell Signaling Technology
Beverly, MA, USA
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