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By admin, on May 16th, 2012
J Neurosci. 2012 May 9; 32(19): 6587-6599
Takano T, Tomomura M, Yoshioka N, Tsutsumi K, Terasawa Y, Saito T, Kawano H, Kamiguchi H, Fukuda M, Hisanaga SI
Axonal outgrowth is a coordinated process of cytoskeletal dynamics and membrane trafficking; however, little is known about proteins responsible for regulating the membrane supply. LMTK1 (lemur kinase 1)/AATYK1 (apoptosis-associated tyrosine kinase 1) is a serine/threonine kinase that is highly expressed in neurons. We recently reported that LMTK1 plays a role in recycling endosomal trafficking in CHO-K1 cells. Here we explore the role of LMTK1 in axonal outgrowth and its regulation by Cdk5 using mouse brain cortical neurons. LMTK1 was expressed and was phosphorylated at Ser34, the Cdk5 phosphorylation site, at the time of axonal outgrowth in culture and colocalized with Rab11A, the small GTPase that regulates recycling endosome traffic, at the perinuclear region and in the axon. Overexpression of the unphosphorylated mutant LMTK1-S34A dramatically promoted axonal outgrowth in cultured neurons. Enhanced axonal outgrowth was diminished by the inactivation of Rab11A, placing LMTK1 upstream of Rab11A. Unexpectedly, the downregulation of LMTK1 by knockdown or gene targeting also significantly enhanced axonal elongation. Rab11A-positive vesicles were transported anterogradely more quickly in the axons of LMTK1-deficient neurons than in those of wild-type neurons. The enhanced axonal outgrowth was reversed by LMTK1-WT or the LMTK1-S34D mutant, which mimics the phosphorylated state, but not by LMTK1-S34A. Thus, LMTK1 can negatively control axonal outgrowth by regulating Rab11A activity in a Cdk5-dependent manner, and Cdk5-LMTK1-Rab11 is a novel signaling pathway involved in axonal outgrowth.
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LMTK1/AATYK1 Is a Novel Regulator of Axonal Outgrowth That Acts via Rab11 in a Cdk5-Dependent Manner.
By admin, on May 14th, 2012
Comp Biochem Physiol A Mol Integr Physiol. 2012 May 2;
Notch EG, Chapline C, Flynn E, Lameyer T, Lowell A, Sato D, Shaw JR, Stanton BA
The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies of environmental toxicants and osmoregulation. Previous research in our laboratory has shown that acute acclimation to seawater is mediated by an increase in SGK1. SGK1 promotes the trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane of the gill within the first hour in seawater resulting in increased chloride secretion. Although we have shown that the increase in gill SGK1 does not require activation of the glucocorticoid receptor, the mechanisms that mediate the rise SGK1 during acute acclimation is unknown. To test the hypothesis that mitogen activated protein kinase (MAPK14) is responsible for the rise in SGK1 we identified the coding sequence of killifish MAPK14-1 and designed a translational blocking vivo-morpholino targeting MAPK14-1. Injection of the MAPK14-1 vivo-morpholino resulted in a 30% reduction of MAPK14-1 and a 45% reduction in phosphorylated-MAPK14-1 protein in the gill of killifish transitioned from freshwater to seawater. Knock down of phosphorlyated-MAPK14-1 completely blocked the rise in SGK1 mRNA and protein in the killifish gill, providing the first direct and in vivo evidence that MAPK14-1 is necessary for acute seawater acclimation.
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Mitogen activated protein kinase 14-1 regulates serum glucocorticoid kinase 1 during seawater acclimation in Atlantic killifish, Fundulus heteroclitus.
By admin, on May 11th, 2012
By admin, on May 11th, 2012
By admin, on April 29th, 2012
J Neurosci. 2012 Apr 25; 32(17): 6014-23
Cheung G, Cousin MA
Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes and that both brefeldin A knockdown and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor protein 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor proteins 1 and 3 indicated that they generated the same population of synaptic vesicles. Thus, adaptor protein complexes 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis.
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Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes.
By admin, on April 29th, 2012
J Neurosci. 2012 Apr 25; 32(17): 6014-23
Cheung G, Cousin MA
Activity-dependent bulk endocytosis is the dominant synaptic vesicle retrieval mode during high intensity stimulation in central nerve terminals. A key event in this endocytosis mode is the generation of new vesicles from bulk endosomes, which replenish the reserve vesicle pool. We have identified an essential requirement for both adaptor protein complexes 1 and 3 in this process by employing morphological and optical tracking of bulk endosome-derived synaptic vesicles in rat primary neuronal cultures. We show that brefeldin A inhibits synaptic vesicle generation from bulk endosomes and that both brefeldin A knockdown and shRNA knockdown of either adaptor protein 1 or 3 subunits inhibit reserve pool replenishment from bulk endosomes. Conversely, no plasma membrane function was found for adaptor protein 1 or 3 in either bulk endosome formation or clathrin-mediated endocytosis. Simultaneous knockdown of both adaptor proteins 1 and 3 indicated that they generated the same population of synaptic vesicles. Thus, adaptor protein complexes 1 and 3 play an essential dual role in generation of synaptic vesicles during activity-dependent bulk endocytosis.
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Adaptor protein complexes 1 and 3 are essential for generation of synaptic vesicles from activity-dependent bulk endosomes.
By admin, on April 26th, 2012
PLoS One. 2012; 7(4): e35674
Taniuchi K, Yokotani K, Saibara T
A novel function for the binder of Arl two (BART) molecule in pancreatic cancer cells is reported. BART inhibits invasiveness of pancreatic cancer cells through binding to a Ca(2+)-dependent, phosphorylated, guanosine triphosphatase (GTPase) membrane fusion protein, annexin7 (ANX7). A tumor suppressor function for ANX7 was previously reported based on its prognostic role in human cancers and the cancer-prone mouse phenotype ANX7(+/-). Further investigation demonstrated that the BART-ANX7 complex is transported toward cell protrusions in migrating cells when BART supports the binding of ANX7 to the protein kinase C (PKC) isoform PKCα. Recent evidence has suggested that phosphorylation of ANX7 by PKC significantly potentiates ANX7-induced fusion of phospholipid vesicles; however, the current data suggest that the BART-ANX7 complex reduces PKCα activity. Knocking down endogenous BART and ANX7 increases activity of PKCα, and specific inhibitors of PKCα significantly abrogate invasiveness induced by BART and ANX7 knockdown. These results imply that BART contributes to regulating PKCα activity through binding to ANX7, thereby affecting the invasiveness of pancreatic cancer cells. Thus, it is possible that BART and ANX7 can distinctly regulate the downstream signaling of PKCα that is potentially relevant to cell invasion by acting as anti-invasive molecules.
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BART Inhibits Pancreatic Cancer Cell Invasion by PKCα Inactivation through Binding to ANX7.
By admin, on April 26th, 2012
PLoS One. 2012; 7(4): e35674
Taniuchi K, Yokotani K, Saibara T
A novel function for the binder of Arl two (BART) molecule in pancreatic cancer cells is reported. BART inhibits invasiveness of pancreatic cancer cells through binding to a Ca(2+)-dependent, phosphorylated, guanosine triphosphatase (GTPase) membrane fusion protein, annexin7 (ANX7). A tumor suppressor function for ANX7 was previously reported based on its prognostic role in human cancers and the cancer-prone mouse phenotype ANX7(+/-). Further investigation demonstrated that the BART-ANX7 complex is transported toward cell protrusions in migrating cells when BART supports the binding of ANX7 to the protein kinase C (PKC) isoform PKCα. Recent evidence has suggested that phosphorylation of ANX7 by PKC significantly potentiates ANX7-induced fusion of phospholipid vesicles; however, the current data suggest that the BART-ANX7 complex reduces PKCα activity. Knocking down endogenous BART and ANX7 increases activity of PKCα, and specific inhibitors of PKCα significantly abrogate invasiveness induced by BART and ANX7 knockdown. These results imply that BART contributes to regulating PKCα activity through binding to ANX7, thereby affecting the invasiveness of pancreatic cancer cells. Thus, it is possible that BART and ANX7 can distinctly regulate the downstream signaling of PKCα that is potentially relevant to cell invasion by acting as anti-invasive molecules.
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BART Inhibits Pancreatic Cancer Cell Invasion by PKCα Inactivation through Binding to ANX7.
By admin, on April 21st, 2012
PLoS One. 2012; 7(4): e34424
Liapis A, Chen FW, Davies JP, Wang R, Ioannou YA
MLN64 is an integral membrane protein localized to the late endosome and plasma membrane that is thought to function as a mediator of cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria. The protein consists of two distinct domains: an N-terminal membrane-spanning domain that shares homology with the MENTHO protein and a C-terminal steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain that binds cholesterol. To further characterize the MLN64 protein, full-length and truncated proteins were overexpressed in cells and the effects on MLN64 trafficking and endosomal morphology were observed. To gain insight into MLN64 function, affinity chromatography and mass spectrometric techniques were used to identify potential MLN64 interacting partners. Of the 15 candidate proteins identified, 14-3-3 was chosen for further characterization. We show that MLN64 interacts with 14-3-3 in vitro as well as in vivo and that the strength of the interaction is dependent on the 14-3-3 isoform. Furthermore, blocking the interaction through the use of a 14-3-3 antagonist or MLN64 mutagenesis delays the trafficking of MLN64 to the late endosome and also results in the dispersal of endocytic vesicles to the cell periphery. Taken together, these studies have determined that MLN64 is a novel 14-3-3 binding protein and indicate that 14-3-3 plays a role in the endosomal trafficking of MLN64. Furthermore, these studies suggest that 14-3-3 may be the link by which MLN64 exerts its effects on the actin-mediated endosome dynamics.
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MLN64 Transport to the Late Endosome Is Regulated by Binding to 14-3-3 via a Non-canonical Binding Site.
By admin, on April 21st, 2012
PLoS One. 2012; 7(4): e34424
Liapis A, Chen FW, Davies JP, Wang R, Ioannou YA
MLN64 is an integral membrane protein localized to the late endosome and plasma membrane that is thought to function as a mediator of cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria. The protein consists of two distinct domains: an N-terminal membrane-spanning domain that shares homology with the MENTHO protein and a C-terminal steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain that binds cholesterol. To further characterize the MLN64 protein, full-length and truncated proteins were overexpressed in cells and the effects on MLN64 trafficking and endosomal morphology were observed. To gain insight into MLN64 function, affinity chromatography and mass spectrometric techniques were used to identify potential MLN64 interacting partners. Of the 15 candidate proteins identified, 14-3-3 was chosen for further characterization. We show that MLN64 interacts with 14-3-3 in vitro as well as in vivo and that the strength of the interaction is dependent on the 14-3-3 isoform. Furthermore, blocking the interaction through the use of a 14-3-3 antagonist or MLN64 mutagenesis delays the trafficking of MLN64 to the late endosome and also results in the dispersal of endocytic vesicles to the cell periphery. Taken together, these studies have determined that MLN64 is a novel 14-3-3 binding protein and indicate that 14-3-3 plays a role in the endosomal trafficking of MLN64. Furthermore, these studies suggest that 14-3-3 may be the link by which MLN64 exerts its effects on the actin-mediated endosome dynamics.
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MLN64 Transport to the Late Endosome Is Regulated by Binding to 14-3-3 via a Non-canonical Binding Site.
By admin, on April 6th, 2012
Biochemistry. 2012 Apr 4;
Joung MJ, Mohan SK, Yu C
Synaptotagmin I (Syt I) is a synaptic vesicle membrane protein that serves as a multifunctional regulator during the exocytosis of neurotransmitter release. It contains C2A and C2B domains. The binding of Ca2+ to the C2A domain activates the exocytosis of secretory vesicles, while the binding of inositol polyphosphates (IP4, IP5 and IP6) to the C2B domain inhibits this process. To understand the IP6-induced inhibition of exocytosis of secretory vesicles, we solved the 3D structure of the C2B-IP6 complex by NMR. In this study, we have determined the binding constant by isothermal titration calorimetry. The CD measurements demonstrated that IP6 can stabilize the C2B molecule. We identified the binding site using 1H-15N HSQC titration data and solved the C2B-IP6 complex structure using multi-dimensional NMR studies. This information will aid in the design of better pharmacological treatments for neurological disorders.
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Molecular Level Interaction of Inositol Hexaphosphate with C2B domain of the Human Synaptotagmin I.
By admin, on April 6th, 2012
Biochemistry. 2012 Apr 4;
Joung MJ, Mohan SK, Yu C
Synaptotagmin I (Syt I) is a synaptic vesicle membrane protein that serves as a multifunctional regulator during the exocytosis of neurotransmitter release. It contains C2A and C2B domains. The binding of Ca2+ to the C2A domain activates the exocytosis of secretory vesicles, while the binding of inositol polyphosphates (IP4, IP5 and IP6) to the C2B domain inhibits this process. To understand the IP6-induced inhibition of exocytosis of secretory vesicles, we solved the 3D structure of the C2B-IP6 complex by NMR. In this study, we have determined the binding constant by isothermal titration calorimetry. The CD measurements demonstrated that IP6 can stabilize the C2B molecule. We identified the binding site using 1H-15N HSQC titration data and solved the C2B-IP6 complex structure using multi-dimensional NMR studies. This information will aid in the design of better pharmacological treatments for neurological disorders.
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Molecular Level Interaction of Inositol Hexaphosphate with C2B domain of the Human Synaptotagmin I.
By admin, on April 6th, 2012
PLoS One. 2012; 7(3): e33962
Valdes VJ, Athie A, Salinas LS, Navarro RE, Vaca L
Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane “cholesterol recognition/interaction amino acid consensus” (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals.
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CUP-1 Is a Novel Protein Involved in Dietary Cholesterol Uptake in Caenorhabditis elegans.
By admin, on April 6th, 2012
PLoS One. 2012; 7(3): e33962
Valdes VJ, Athie A, Salinas LS, Navarro RE, Vaca L
Sterols transport and distribution are essential processes in all multicellular organisms. Survival of the nematode Caenorhabditis elegans depends on dietary absorption of sterols present in the environment. However the general mechanisms associated to sterol uptake in nematodes are poorly understood. In the present work we provide evidence showing that a previously uncharacterized transmembrane protein, designated Cholesterol Uptake Protein-1 (CUP-1), is involved in dietary cholesterol uptake in C. elegans. Animals lacking CUP-1 showed hypersensitivity to cholesterol limitation and were unable to uptake cholesterol. A CUP-1-GFP fusion protein colocalized with cholesterol-rich vesicles, endosomes and lysosomes as well as the plasma membrane. Additionally, by FRET imaging, a direct interaction was found between the cholesterol analog DHE and the transmembrane “cholesterol recognition/interaction amino acid consensus” (CRAC) motif present in C. elegans CUP-1. In-silico analysis identified two mammalian homologues of CUP-1. Most interestingly, CRAC motifs are conserved in mammalian CUP-1 homologous. Our results suggest a role of CUP-1 in cholesterol uptake in C. elegans and open up the possibility for the existence of a new class of proteins involved in sterol absorption in mammals.
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CUP-1 Is a Novel Protein Involved in Dietary Cholesterol Uptake in Caenorhabditis elegans.
By admin, on April 5th, 2012
Curr Protoc Neurosci. 2012 Apr; Chapter 2: Unit2.7
Digiovanni J, Sun T, Sheng ZH
Cloning and characterization of synaptic vesicle proteins and their binding counterparts on the presynaptic plasma membrane have greatly advanced our understanding of the molecular mechanisms involved in the synaptic vesicle cycle and neurotransmitter release. This unit discusses multidisciplinary approaches to characterize proteins from synaptosome-enriched subcellular fractions and localize them within cultured neurons. The first approach regroups methods used to isolate synaptic vesicles from rat brain synaptosomal preparations, allowing for specific biochemical investigation of synaptic vesicle proteins. The second is a detailed procedure for pre-embedding immunogold staining and electron microscopic observation, which permits the morphological identification of proteins in individual vesicles at intact synapses. Additionally, this chapter proposes methods for light microscopic examination of hippocampal neurons. It includes procedures for embryonic and postnatal hippocampal neuron culture and describes an immunocytochemical staining protocol used to investigate synaptic vesicle protein localization with respect to other proteins or subcellular structures. Curr. Protoc. Neurosci. 59:2.7.1-2.7.22. © 2012 by John Wiley & Sons, Inc.
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Characterizing synaptic vesicle proteins using synaptosomal fractions and cultured hippocampal neurons.
By admin, on April 5th, 2012
Curr Protoc Neurosci. 2012 Apr; Chapter 2: Unit2.7
Digiovanni J, Sun T, Sheng ZH
Cloning and characterization of synaptic vesicle proteins and their binding counterparts on the presynaptic plasma membrane have greatly advanced our understanding of the molecular mechanisms involved in the synaptic vesicle cycle and neurotransmitter release. This unit discusses multidisciplinary approaches to characterize proteins from synaptosome-enriched subcellular fractions and localize them within cultured neurons. The first approach regroups methods used to isolate synaptic vesicles from rat brain synaptosomal preparations, allowing for specific biochemical investigation of synaptic vesicle proteins. The second is a detailed procedure for pre-embedding immunogold staining and electron microscopic observation, which permits the morphological identification of proteins in individual vesicles at intact synapses. Additionally, this chapter proposes methods for light microscopic examination of hippocampal neurons. It includes procedures for embryonic and postnatal hippocampal neuron culture and describes an immunocytochemical staining protocol used to investigate synaptic vesicle protein localization with respect to other proteins or subcellular structures. Curr. Protoc. Neurosci. 59:2.7.1-2.7.22. © 2012 by John Wiley & Sons, Inc.
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Characterizing synaptic vesicle proteins using synaptosomal fractions and cultured hippocampal neurons.
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