- Email: [email protected]
Dimension of GluAs reconstituted in lipid bilayer
Abstracts / Neuroscience Research 71S (2011) e108–e415
characterizations of this non-neuronal tissue may pave the way for novel perspectives on the central nervous system physiology and pathology. We have demonstrated previously that choroid plexus epithelial cells (CPECs) express multiple primary cilia, and that the cilia modulate the cellular transport activity across the epithelial layer. Since the mode of ciliary expression is highly unusual, we performed a proteomic analysis of CPEC cilia to elucidate the molecular basis of extraordinariness. CPEC cilia were isolated and purified from swine choroid plexus tissue. The subsequent proteomic analysis identified ∼1,100 proteins. Among them, we found several molecules implicated in ciliary motility. Real-time PCR also validated the expression of these molecules in CPECs. Immunostaining for one of them, radial spoke head 9 homolog (Rsph9), demonstrated that the molecule localized to some, but not all, swine CPEC cilia. Surprisingly, live imaging of mouse choroid plexus demonstrated that CPEC cilia could beat at the neonatal stage. The beating pattern appeared to be different from that of nodal cilia. By contrast, motile cilia were not found in adult choroid plexus. In conclusion, our CPEC ciliome revealed a novel heterogeneous nature of CPECs in terms of ciliary motility and the subsequent analyses demonstrated striking postnatal transition of this property. These results also suggested that CPEC cilia could be classified as either hybrid or mosaic type. We propose to rearrange the concept of ciliary subtype classifications. Research fund: KAKENHI 22770190. doi:10.1016/j.neures.2011.07.1391
P4-b06 Putative function of TRPV4 on the choroid plexus primary cilia Shohei Sasamoto , Keishi Narita, Sen Takeda Department of Anatomy and Cell Biology, Faculty of Medicine, University of Yamanashi Primary cilia are eukaryotic cell organelle projecting from the cell surface. It has been shown that primary cilia, in general, function as a biosensor to sense diverse mechanical and chemical stimuli from extracellular environment. However, the detailed molecular mechanisms of cilia-mediated sensing as well as its diversity among different cell types have not been fully characterized. Recently, we performed a proteomic analysis of primary cilia isolated from choroid plexus epithelial cells (CPECs), which produce the cerebrospinal fluid (CSF) of the ventricular system in vertebrate brain, and identified the transient receptor potential channel vanilloid superfamily type 4 (TRPV4), a divalent cation channel activated by various mechanical and chemical stresses. In the present study, we hypothesized that TRPV4 in CPEC primary cilia may play a role in regulation of CSF production. We demonstrated that TRPV4 was localized in primary cilia of CPECs by immunocytochemistry. TRPV4 was present in 75% of total CPEC primary cilia. The intracellular Ca2+ levels in CPECs were elevated by GSK1016790A, a TRPV4 agonist, with the EC50 value of 34 nM, validating the function of TRPV4 in CPECs. A 24-hr treatment with 4 mM chloral hydrate, a potent deciliation reagent, reduced the number of cilia-positive cells as well as the TRPV4 protein levels in cultured CPECs. In addition, we found that GSK1016790A increased the basal-to-apical fluid transcytosis, which reflects CSF production activities in vitro. In conclusion, our study revealed that functional TRPV4 is expressed in CPECs, and that its activity is abolished by deciliation. Our study also suggested that TRPV4 in CPEC primary cilia may play an important role in the regulation of CSF homeostasis. doi:10.1016/j.neures.2011.07.1392
P4-b07 Identification and characterization of a novel splicing variant, short form sigma-1 receptor Norifumi Shioda , Kiyoshi Ishikawa, Kohji Fukunaga Dept. Pharmacol., Tohoku University Grad. Sch. Pharm. Sci., Sendai, Japan Sigma-1 receptor (Sigma-1R) is characterized a endoplasmic reticulum (ER)associated chaperone protein, regulating ER-mitochondrial interorganellar Ca2+ mobilization through type-3 inositol 1,4,5-trisphosphate receptors (IP3 R3) and promoting cell survival. We identified a novel splice variant of Sigma-1R, termed short form Sigma-1R (Sigma-1SR) from mouse brain cDNA libraries. Sigma-1SR lacks 50 ribonucleotides that encode the second transmembrane domain, resulting in a frame shift and giving rise to a novel 4 amino acid sequence in the C-terminus of the protein that not seen in Sigma-1R reported previously. The Sigma-1SR protein expression level is 30–40% of Sigma-1R in mouse cortex, hippocampus and striatum. When Sigma-1SR was expressed in neuroblastoma Neuro-2a cells, it localized on ER and golgi apparatus, and interacted with Sigma-1R, but not with IP3 R3. The stable expression of Sigma-1SR reduced the efficiency of
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mitochondrial Ca2+ uptake in response to IP3 R-driven stimuli. In addition, coexpression of Sigma-1R with Sigma-1SR blocked its action on mitochondrial Ca2+ mobilization. Most importantly, Sigma-1R overexpression suppressed hydrogen peroxide-induced ATP depletion and cell apoptosis, whereas Sigma-1SR overexpression had opposite effects in Neuro-2a cells. Taken together, Sigma-1SR dimerizes with Sigma-1R and inhibits its function, thereby promoting apoptosis in neurons. Research fund: KAKENHI23110501. doi:10.1016/j.neures.2011.07.1393
P4-b08 Dimension of GluAs reconstituted in lipid bilayer Nahoko Kasai 1 , Chandra S. Ramanujan 2 , Jelena Barnovic 2 , John F. Ryan 2 , Koji Sumitomo 1 , Keiichi Torimitsu 1 1
NTT Basic Research Labs 2 University of Oxford
As one of ligand-gated ion channel glutamate receptors, ␣-amino-3hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) are present throughout the vertebrate central nervous system, located mainly at postsynaptic membranes where they act as the main mediators of fast excitatory neurotransmission. AMPAR is a tetrameric which consists of four subunits (GluA1–GluA4). AMPAR density is reported ∼1000/m2 in a synapse of Purkinje cells in rat cerebellum, and in dentate gyrus in hippocampus the density becomes 5–10 times less in extra-synaptic membrane than in synaptic membrane. This density is also revealed to relate to short-term memory formation. We have focused on imaging single, full-length AMPARs, which were purified and reconstituted into artificial lipid membrane, using atomic force microscopy (AFM) in near physiological condition. In the previous study we reported that the single molecule measurements of the reconstituted homomeric GluA3 revealed the AMPAR extracellular domain observed in AFM was clearly tetrameric, but it appeared to be somewhat smaller in height and more extended laterally in the membrane than expected on the basis of cryo-EM and X-ray measurements. In this study, we examined the dimension of the reconstituted AMPARs in different receptor density in lipid bilayer. Full-length GluA3 homomer were overexpressed in High Five cells, purified, and reconstituted into lipid bilayer consisting phosphatidylcholine from egg yolk and phosphatidylserine from porcine brain (3:1). AFM (Dimension 3100TM , Digital Instruments Veeco Metrology, Woodbury, USA) measurements were made in tapping mode in buffer solution (30 mM HEPES (pH7.4), 260 mM KCl and 40 mM NaCl). We observed the smaller height under our low receptor density conditions, and larger height under the higher receptor density. This result implied the different receptor interaction in different density, suggesting that different receptor function at different density. Research fund: This research was supported in part by KAKENHI (B20360014). doi:10.1016/j.neures.2011.07.1394
P4-b09 Quantitative imaging of synaptic receptor dynamics using a fluorescence activation-coupled protein labeling (FAPL) method Yuichiro Ishii 1 , Hiroyuki Okuno 1 , Toru Nagano 2 , Yasuteru Urano 3 , Haruhiko Bito 1,4
Komatsu 2 , Tetsuo
1
Dep. of Neurochemistry, Grad. Sch. of Med, Univ. of Tokyo, Tokyo, Japan Lab. of Chemistry and Biology, Grad. Sch. of Pharmaceutical Sci., Univ. of Tokyo, Tokyo, Japan 3 Lab. of Chemical Biology and Molecular Imaging, Grad. Sch. of Med, Univ. of Tokyo, Tokyo, Japan 4 CREST-JST 2
Long-term potentiation (LTP) of glutamatergic synaptic transmission represents an elementary adaptive response that is essential for learning and memory. One class of glutamate-gated ion channels, the AMPA-type glutamate receptors (AMPARs), plays a central role in this process, and mechanisms that allow AMPARs to enter synapses, such as vesicular trafficking, exocytosis and lateral diffusion, are critical in controlling and altering the strength of synaptic transmission. Multiple methods have been developed to measure AMPARs trafficking in real-time, but it still remains a challenge to dissect the contribution of distinct mechanisms of AMPAR synaptic delivery. In particular, it has been difficult to segregate new AMPAR insertion by exocytosis from lateral diffusion of preexisting AMPARs into the synaptic membranes. To begin to address this issue, we here designed a novel strategy to measure AMPARs dynamics with single synapse resolution. We tagged GluA1, an AMPAR subunit, by inserting into its extracellular N-terminus an enzymatic tag SNAP, which covalently binds benzylguanine-substituted organic-dyes. We then used a novel membrane-impermeable SNAP ligand with improved fluorescence properties (Komatsu et al. JACS, 2011), and
characterizations of this non-neuronal tissue may pave the way for novel perspectives on the central nervous system physiology and pathology. We have demonstrated previously that choroid plexus epithelial cells (CPECs) express multiple primary cilia, and that the cilia modulate the cellular transport activity across the epithelial layer. Since the mode of ciliary expression is highly unusual, we performed a proteomic analysis of CPEC cilia to elucidate the molecular basis of extraordinariness. CPEC cilia were isolated and purified from swine choroid plexus tissue. The subsequent proteomic analysis identified ∼1,100 proteins. Among them, we found several molecules implicated in ciliary motility. Real-time PCR also validated the expression of these molecules in CPECs. Immunostaining for one of them, radial spoke head 9 homolog (Rsph9), demonstrated that the molecule localized to some, but not all, swine CPEC cilia. Surprisingly, live imaging of mouse choroid plexus demonstrated that CPEC cilia could beat at the neonatal stage. The beating pattern appeared to be different from that of nodal cilia. By contrast, motile cilia were not found in adult choroid plexus. In conclusion, our CPEC ciliome revealed a novel heterogeneous nature of CPECs in terms of ciliary motility and the subsequent analyses demonstrated striking postnatal transition of this property. These results also suggested that CPEC cilia could be classified as either hybrid or mosaic type. We propose to rearrange the concept of ciliary subtype classifications. Research fund: KAKENHI 22770190. doi:10.1016/j.neures.2011.07.1391
P4-b06 Putative function of TRPV4 on the choroid plexus primary cilia Shohei Sasamoto , Keishi Narita, Sen Takeda Department of Anatomy and Cell Biology, Faculty of Medicine, University of Yamanashi Primary cilia are eukaryotic cell organelle projecting from the cell surface. It has been shown that primary cilia, in general, function as a biosensor to sense diverse mechanical and chemical stimuli from extracellular environment. However, the detailed molecular mechanisms of cilia-mediated sensing as well as its diversity among different cell types have not been fully characterized. Recently, we performed a proteomic analysis of primary cilia isolated from choroid plexus epithelial cells (CPECs), which produce the cerebrospinal fluid (CSF) of the ventricular system in vertebrate brain, and identified the transient receptor potential channel vanilloid superfamily type 4 (TRPV4), a divalent cation channel activated by various mechanical and chemical stresses. In the present study, we hypothesized that TRPV4 in CPEC primary cilia may play a role in regulation of CSF production. We demonstrated that TRPV4 was localized in primary cilia of CPECs by immunocytochemistry. TRPV4 was present in 75% of total CPEC primary cilia. The intracellular Ca2+ levels in CPECs were elevated by GSK1016790A, a TRPV4 agonist, with the EC50 value of 34 nM, validating the function of TRPV4 in CPECs. A 24-hr treatment with 4 mM chloral hydrate, a potent deciliation reagent, reduced the number of cilia-positive cells as well as the TRPV4 protein levels in cultured CPECs. In addition, we found that GSK1016790A increased the basal-to-apical fluid transcytosis, which reflects CSF production activities in vitro. In conclusion, our study revealed that functional TRPV4 is expressed in CPECs, and that its activity is abolished by deciliation. Our study also suggested that TRPV4 in CPEC primary cilia may play an important role in the regulation of CSF homeostasis. doi:10.1016/j.neures.2011.07.1392
P4-b07 Identification and characterization of a novel splicing variant, short form sigma-1 receptor Norifumi Shioda , Kiyoshi Ishikawa, Kohji Fukunaga Dept. Pharmacol., Tohoku University Grad. Sch. Pharm. Sci., Sendai, Japan Sigma-1 receptor (Sigma-1R) is characterized a endoplasmic reticulum (ER)associated chaperone protein, regulating ER-mitochondrial interorganellar Ca2+ mobilization through type-3 inositol 1,4,5-trisphosphate receptors (IP3 R3) and promoting cell survival. We identified a novel splice variant of Sigma-1R, termed short form Sigma-1R (Sigma-1SR) from mouse brain cDNA libraries. Sigma-1SR lacks 50 ribonucleotides that encode the second transmembrane domain, resulting in a frame shift and giving rise to a novel 4 amino acid sequence in the C-terminus of the protein that not seen in Sigma-1R reported previously. The Sigma-1SR protein expression level is 30–40% of Sigma-1R in mouse cortex, hippocampus and striatum. When Sigma-1SR was expressed in neuroblastoma Neuro-2a cells, it localized on ER and golgi apparatus, and interacted with Sigma-1R, but not with IP3 R3. The stable expression of Sigma-1SR reduced the efficiency of
e319
mitochondrial Ca2+ uptake in response to IP3 R-driven stimuli. In addition, coexpression of Sigma-1R with Sigma-1SR blocked its action on mitochondrial Ca2+ mobilization. Most importantly, Sigma-1R overexpression suppressed hydrogen peroxide-induced ATP depletion and cell apoptosis, whereas Sigma-1SR overexpression had opposite effects in Neuro-2a cells. Taken together, Sigma-1SR dimerizes with Sigma-1R and inhibits its function, thereby promoting apoptosis in neurons. Research fund: KAKENHI23110501. doi:10.1016/j.neures.2011.07.1393
P4-b08 Dimension of GluAs reconstituted in lipid bilayer Nahoko Kasai 1 , Chandra S. Ramanujan 2 , Jelena Barnovic 2 , John F. Ryan 2 , Koji Sumitomo 1 , Keiichi Torimitsu 1 1
NTT Basic Research Labs 2 University of Oxford
As one of ligand-gated ion channel glutamate receptors, ␣-amino-3hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) are present throughout the vertebrate central nervous system, located mainly at postsynaptic membranes where they act as the main mediators of fast excitatory neurotransmission. AMPAR is a tetrameric which consists of four subunits (GluA1–GluA4). AMPAR density is reported ∼1000/m2 in a synapse of Purkinje cells in rat cerebellum, and in dentate gyrus in hippocampus the density becomes 5–10 times less in extra-synaptic membrane than in synaptic membrane. This density is also revealed to relate to short-term memory formation. We have focused on imaging single, full-length AMPARs, which were purified and reconstituted into artificial lipid membrane, using atomic force microscopy (AFM) in near physiological condition. In the previous study we reported that the single molecule measurements of the reconstituted homomeric GluA3 revealed the AMPAR extracellular domain observed in AFM was clearly tetrameric, but it appeared to be somewhat smaller in height and more extended laterally in the membrane than expected on the basis of cryo-EM and X-ray measurements. In this study, we examined the dimension of the reconstituted AMPARs in different receptor density in lipid bilayer. Full-length GluA3 homomer were overexpressed in High Five cells, purified, and reconstituted into lipid bilayer consisting phosphatidylcholine from egg yolk and phosphatidylserine from porcine brain (3:1). AFM (Dimension 3100TM , Digital Instruments Veeco Metrology, Woodbury, USA) measurements were made in tapping mode in buffer solution (30 mM HEPES (pH7.4), 260 mM KCl and 40 mM NaCl). We observed the smaller height under our low receptor density conditions, and larger height under the higher receptor density. This result implied the different receptor interaction in different density, suggesting that different receptor function at different density. Research fund: This research was supported in part by KAKENHI (B20360014). doi:10.1016/j.neures.2011.07.1394
P4-b09 Quantitative imaging of synaptic receptor dynamics using a fluorescence activation-coupled protein labeling (FAPL) method Yuichiro Ishii 1 , Hiroyuki Okuno 1 , Toru Nagano 2 , Yasuteru Urano 3 , Haruhiko Bito 1,4
Komatsu 2 , Tetsuo
1
Dep. of Neurochemistry, Grad. Sch. of Med, Univ. of Tokyo, Tokyo, Japan Lab. of Chemistry and Biology, Grad. Sch. of Pharmaceutical Sci., Univ. of Tokyo, Tokyo, Japan 3 Lab. of Chemical Biology and Molecular Imaging, Grad. Sch. of Med, Univ. of Tokyo, Tokyo, Japan 4 CREST-JST 2
Long-term potentiation (LTP) of glutamatergic synaptic transmission represents an elementary adaptive response that is essential for learning and memory. One class of glutamate-gated ion channels, the AMPA-type glutamate receptors (AMPARs), plays a central role in this process, and mechanisms that allow AMPARs to enter synapses, such as vesicular trafficking, exocytosis and lateral diffusion, are critical in controlling and altering the strength of synaptic transmission. Multiple methods have been developed to measure AMPARs trafficking in real-time, but it still remains a challenge to dissect the contribution of distinct mechanisms of AMPAR synaptic delivery. In particular, it has been difficult to segregate new AMPAR insertion by exocytosis from lateral diffusion of preexisting AMPARs into the synaptic membranes. To begin to address this issue, we here designed a novel strategy to measure AMPARs dynamics with single synapse resolution. We tagged GluA1, an AMPAR subunit, by inserting into its extracellular N-terminus an enzymatic tag SNAP, which covalently binds benzylguanine-substituted organic-dyes. We then used a novel membrane-impermeable SNAP ligand with improved fluorescence properties (Komatsu et al. JACS, 2011), and