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1224 A t-Snare is involved in axonal growth: Botulinum neurotoxin C1 induces growth cone collapse
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1224
A t-SNARE IS INVOLVED IN AXONAL GROWTE: DOTULINUY NEUROTOXIN Cl INDUCES GROWTECONE COLLAPSE. MICBI~IRO ICARASBI~. SUSUMUTERAKAWA’. CBIZUKAIDE3. YOSIIIAKIKOWIYA’. ‘Dept. Bolec. cell. Neurobiol.. Gunma University Sch. Med.. Yaebashi. Curma;‘Photon Med. Shizuoka: 3Dept. Anat., Univ. Kyoto. Kyoto. Clr., Uaaaaatsu Med. Sch., Wamamatsu.
The most likely cause of the membrane expansion for axonal growth is that the vesicles in the central domainof the growth cone are addedto the growth cone plasma membrane tbrough cxocytosis. A molecular model for vesicular targeting in exocytosis has recently been proposed. referred as the SNARE hypothesis. To test the hypothesis is applicable to membrane expansion lor axonal growth. Weobserved the effect of ncurotoxin Cl of Clostridium botulinurn (gift of S. Kozaki. Univ. Osaka Pref.). cleaving syntaxin. which is essential to targetin of synaptic vcsiclcs as a L-SNARE.to the growth cone. Neurotoxin Cl causedgrowth cone collapse and inhibition of axonal growth. Video-enhanced microscopic study showed (a) that neurotoxin Cl sclcctivcly blocked the central domain at the initial stage, but not the lamellipodia: and (b) that large vacuole formation occurred probably through fusion of smaller vesicles from the
central domainto the most distal segmentsof the normal axonal growth. Our results demonstrate that syntaxin is involved in axonal growth. indicating that syntaxin may participate directly in n cmbranc expansion in the central domain of the growth cone. in a SNARE-like way.
1225 6B4 PROTEOGLYCAN/PHOSPHACAN, A EXTRACELLULAR VARIANT OF PROTEIN TYROSINE PHOSPHATASEC, IS A REPULSIVE SUBSTRATUM BUT PROMOTES MORPHOLOGICAL DIFFERENTIATION OF CORTICAL NEURONS. NOBUAKI MAEDA. MASAHARU National Institute for BasicBioloev. Okazaki 444. Jaoan.
NODA.
Division of Molecular Neurobiolo&
6B4 proteoglycan (6B4PC) is a major solublechondroitin sulfateproteoglycanin the brain, which correspondsto the extracellular region of a receptor-like protein tyrosine phosphatase,PTPC. The influence of 6B4PG, adsorbedonto the substratum,on cell adhesionand neurite outgrowth was studiedusingdissociatedneuronsfrom the rat cerebral cortex and thalamus. When cortical and thalamicneuronswere seededon the patternedsubstrataconsistingof a grid-like structureof alternatingpoly-L-lysine (PLL) and6B4PG-coatedPLL domains,they were concentratedon the PLL domains,indicatingthat 6B4PG is a repulsivesbstratum. However, 6B4PG did not retard the differentiation of neurons,but rather promotedneurite outgrowth anddevelopmentof the dendritesof coitical neuronswhenneuronswereseededon the PLL-conditionedcoverslips continuously coated with 6B4 PG. These results suggestthat 6B4PG modulatesmorphogenesisand differentiation of neuronsdependingon its spatiotemporalexpressionin the brain.
1226 SAITQH Niigata
NERVE GROWTH CONE PROTEINS ISOLATED FROM MOUSE BRAIN : IDENTIFICATION AND CHARACTERIZATION WITH ANTIBODIES. TAKAKQ K-.-ABE, ~HIRQSHI TANAKA,~HIRQSHI and RYQZQ KUWANO, Res. Lab. for MQ~. &net,, Niigata~Univ,,~Asahimaahi-1, 951, Japan
The nerve growth cone plays an important role in pathfinding and recognition of the axon for neural network formation. Therefore, analyses of proteins on the growth cone are needed to elucidate molecular mechanisms of synapse formation. The growth cone can be isolated from neuronal cells as growth cone particles by sucrose density gradient centrifugation. We have prepared growth cone particle-enriched(fraction A, the interface between 0.32 M and 0.75 M) and -non-enriched(fraction C, the interface between 1.0 M and 2.66 M) fractions from early postnatal mouse brain. Membrane proteins of the fractions A and C were analyzed by lectin-affinity chromatography, ion exchange chromatography, 2-dimensional gel electrophoresis and SDS-PAGE. Partial amino acid sequences of several proteins concentrated in the fraction A were obtained and compared with those in the protein and nucleotide sequence databases. We have found that proteins previously reported‘ to present on the growth cone(GAP-43, NAP-22, MARCKS and M6) and others includina with uniaue.J 9P93, < those sequences were contain-e-d in the fraction A. we have raised antibodies Furthermore, against some of these proteins, and are currently analyzing their intracellular localizations in differentiated murine neural precursor cells that have been isolated from 10.5 days fetus head and cultured for several days.
1224
A t-SNARE IS INVOLVED IN AXONAL GROWTE: DOTULINUY NEUROTOXIN Cl INDUCES GROWTECONE COLLAPSE. MICBI~IRO ICARASBI~. SUSUMUTERAKAWA’. CBIZUKAIDE3. YOSIIIAKIKOWIYA’. ‘Dept. Bolec. cell. Neurobiol.. Gunma University Sch. Med.. Yaebashi. Curma;‘Photon Med. Shizuoka: 3Dept. Anat., Univ. Kyoto. Kyoto. Clr., Uaaaaatsu Med. Sch., Wamamatsu.
The most likely cause of the membrane expansion for axonal growth is that the vesicles in the central domainof the growth cone are addedto the growth cone plasma membrane tbrough cxocytosis. A molecular model for vesicular targeting in exocytosis has recently been proposed. referred as the SNARE hypothesis. To test the hypothesis is applicable to membrane expansion lor axonal growth. Weobserved the effect of ncurotoxin Cl of Clostridium botulinurn (gift of S. Kozaki. Univ. Osaka Pref.). cleaving syntaxin. which is essential to targetin of synaptic vcsiclcs as a L-SNARE.to the growth cone. Neurotoxin Cl causedgrowth cone collapse and inhibition of axonal growth. Video-enhanced microscopic study showed (a) that neurotoxin Cl sclcctivcly blocked the central domain at the initial stage, but not the lamellipodia: and (b) that large vacuole formation occurred probably through fusion of smaller vesicles from the
central domainto the most distal segmentsof the normal axonal growth. Our results demonstrate that syntaxin is involved in axonal growth. indicating that syntaxin may participate directly in n cmbranc expansion in the central domain of the growth cone. in a SNARE-like way.
1225 6B4 PROTEOGLYCAN/PHOSPHACAN, A EXTRACELLULAR VARIANT OF PROTEIN TYROSINE PHOSPHATASEC, IS A REPULSIVE SUBSTRATUM BUT PROMOTES MORPHOLOGICAL DIFFERENTIATION OF CORTICAL NEURONS. NOBUAKI MAEDA. MASAHARU National Institute for BasicBioloev. Okazaki 444. Jaoan.
NODA.
Division of Molecular Neurobiolo&
6B4 proteoglycan (6B4PC) is a major solublechondroitin sulfateproteoglycanin the brain, which correspondsto the extracellular region of a receptor-like protein tyrosine phosphatase,PTPC. The influence of 6B4PG, adsorbedonto the substratum,on cell adhesionand neurite outgrowth was studiedusingdissociatedneuronsfrom the rat cerebral cortex and thalamus. When cortical and thalamicneuronswere seededon the patternedsubstrataconsistingof a grid-like structureof alternatingpoly-L-lysine (PLL) and6B4PG-coatedPLL domains,they were concentratedon the PLL domains,indicatingthat 6B4PG is a repulsivesbstratum. However, 6B4PG did not retard the differentiation of neurons,but rather promotedneurite outgrowth anddevelopmentof the dendritesof coitical neuronswhenneuronswereseededon the PLL-conditionedcoverslips continuously coated with 6B4 PG. These results suggestthat 6B4PG modulatesmorphogenesisand differentiation of neuronsdependingon its spatiotemporalexpressionin the brain.
1226 SAITQH Niigata
NERVE GROWTH CONE PROTEINS ISOLATED FROM MOUSE BRAIN : IDENTIFICATION AND CHARACTERIZATION WITH ANTIBODIES. TAKAKQ K-.-ABE, ~HIRQSHI TANAKA,~HIRQSHI and RYQZQ KUWANO, Res. Lab. for MQ~. &net,, Niigata~Univ,,~Asahimaahi-1, 951, Japan
The nerve growth cone plays an important role in pathfinding and recognition of the axon for neural network formation. Therefore, analyses of proteins on the growth cone are needed to elucidate molecular mechanisms of synapse formation. The growth cone can be isolated from neuronal cells as growth cone particles by sucrose density gradient centrifugation. We have prepared growth cone particle-enriched(fraction A, the interface between 0.32 M and 0.75 M) and -non-enriched(fraction C, the interface between 1.0 M and 2.66 M) fractions from early postnatal mouse brain. Membrane proteins of the fractions A and C were analyzed by lectin-affinity chromatography, ion exchange chromatography, 2-dimensional gel electrophoresis and SDS-PAGE. Partial amino acid sequences of several proteins concentrated in the fraction A were obtained and compared with those in the protein and nucleotide sequence databases. We have found that proteins previously reported‘ to present on the growth cone(GAP-43, NAP-22, MARCKS and M6) and others includina with uniaue.J 9P93, < those sequences were contain-e-d in the fraction A. we have raised antibodies Furthermore, against some of these proteins, and are currently analyzing their intracellular localizations in differentiated murine neural precursor cells that have been isolated from 10.5 days fetus head and cultured for several days.