- Email: [email protected]
P-F2-08 GDP inverts the polarity of microtubule polymerization
F2 The lower cytoskeleton P-F24
P-F246
ITOH TJ’. WADA Y2., HISANAGA S2,
MORPHQLW#XL C-W Q@? LWOSOMES CAUWiB BY AQIW4 THE EFFECT OF a-ACl’INIlW AND #%4MIN ’ TAKlGUCHl K. HONDA M. HOTANI H. Dept. of Mol. BioL, School of&i., Nagoya Univ. (JPN)
ISHIGURO KS, KISHIMOTO T2. & HOTANI H’ ‘Nagoya Univ., Dept. Mol. Biol., (IPN), 2Tokyo Inst. Tech., Dent. Biosci.. (JPN). %itsubishi Kasei Life Sci. Inst.. (lP@ Purpose: To know the regulation of tau within ceils, tau was phosphorylated by cdk5 kinase with or without microtubules (MT) in vitro. Method: Phosphorylation sites in tau were determined by western blotting. Effects of phosphoryiations on MT dynamics were examined by dark-field microscopy and light scattering measurement. Resultst Phosphoryi pups were incorporated into both Ser-235 and Ser-404 independent of the presence of MT, while both Ser-202 and Thr-205 were phosphorylated only in the presence of MT. Dark-field microscopic observation demonstrated that MT-stabilizing activity of tau decreased independent of the presence of MT. However, its MT-nucleating activity was strongly inhibited only when phosphoryiated in the presence of MT. Conclusion: Tau can be phosphoryiated at Ser202 and Thr-205 only when it bound to MT. Phosphoryiation of these sites is important to inhibit the MT-nucleating activity.
Purpose: Living ceils and cell organeiies are ail compartmentalized by biomembrane, and their morphoiogies are thought to be determined and maintained by cytoskeletai networks. We focused on the role of microfilaments on their morphogenesis, and developed a model system using the iiposomes containing actin and actinbinding proteins. Methods: In the liposomes, the encapsulated monomeric actin and its bindi,ng protein were polymerized to actin filaments by raising temperature. We observed the subsequent morphological changes of the liposomes by high intensity dark-field video micrascopy. Results: The liposome containing actin alone transformed into a disk or dumbbell shape. When actin polymerized together with a-actinin, the l&some transformed into a disk shape possessing long tubular projections. When actin polymerized together with filamin, the iiposome became very rigid and took a stable shape. Conclusions: The difference in morphology between the actin containing liposomes depended on the type of co-encapsulated actin crosslinking protein, indicates that it can determine liposome shape by organizing actin networks.
ON MICROTUBULE
DYNAWrtCS
P-F2-07 Liposome shape is sta&itized by MAPS mediated interaction between microtubules and membrane.
P-F2-08 GDP INVERTS ‘iX@ PQ~RK4.Y OR
Kaneko T, ltoh TJ, Hotani H. Dept. of Mol. Biol., School of Sci.. Nagoya Univ. (JPN)
MICROTUBULE POLYM#D8?iATION TANAKA Y, IT0 TJ, HOTANI H. Dept. of Mol. Biol., School of Sci.. Nagoya Univ. (JPN)
Purpose: To understand the role of cytoskeletons on the morphogenesis of ceils, we examined morphological changesof liposome caused by the polymerization of encapsulated tubulin. Methods: We studied effects of MAPSon the microtubule (MT)-dependent morphological changes of liposomes by dark-field microscopy. To examine the interaction of MAPS with liposomes. we made the cosedimentation assay with sucrose cushion. Results: Upon MT polymerization, a spherical liposome transformed into a “dipolar” liposome that has a central sphere and two straight projections aligned on a line. Without MAPS. the “dipolar” liposome subsequently transformed to “monopolar” one by moving the central sphere without change in a total projection length. The number ratio of “dipoiar” to “monopolar” shape increased with MAPS concentrations. MT cosedimented with liposomes in the presence of MAPS. while MAPS-free MT did not. Particularly MAPI and MAP2 bound to liposome. Conclusions: These results suggest that MAPS medi;lte the binding bGtween microtubules and membrane, hence s&i#ze Eiposome shape.
Purpose: Guanine-nucieotide on tubulin molecules and its hydrolysis should play an important role not only in the frequency but also in the polarity of microtubule (MT) polymerization-depolymerization. We examined the effect of GDP on the polarity of MT elongation. Methods: MTs were polymerized from Tetruh.~rtma axonemes in the presence of only GTP or both of GTP and GDP. We determined that MTs elongated from which end bf “oneended axonemes,” the axonthat MTs elongated from their only one end, using NEMtubulin. MTs were observed by dark-field microscopy. Resulti 85% of one-ended axonemes observed at low tubulin concentration in the presence of only GTP, elongated MTs from their plus ends as predicted by the difference of critical concentrations of both ends. In the copresence of GDP, however, 94% of one-ended axonemes elongated MTs from their minus ends despite the higher tybulin concentratiqns. Conclusions: These results suggest that GDP can invcfi the polnity,of MT &ng@tion. 176
P-F246
ITOH TJ’. WADA Y2., HISANAGA S2,
MORPHQLW#XL C-W Q@? LWOSOMES CAUWiB BY AQIW4 THE EFFECT OF a-ACl’INIlW AND #%4MIN ’ TAKlGUCHl K. HONDA M. HOTANI H. Dept. of Mol. BioL, School of&i., Nagoya Univ. (JPN)
ISHIGURO KS, KISHIMOTO T2. & HOTANI H’ ‘Nagoya Univ., Dept. Mol. Biol., (IPN), 2Tokyo Inst. Tech., Dent. Biosci.. (JPN). %itsubishi Kasei Life Sci. Inst.. (lP@ Purpose: To know the regulation of tau within ceils, tau was phosphorylated by cdk5 kinase with or without microtubules (MT) in vitro. Method: Phosphorylation sites in tau were determined by western blotting. Effects of phosphoryiations on MT dynamics were examined by dark-field microscopy and light scattering measurement. Resultst Phosphoryi pups were incorporated into both Ser-235 and Ser-404 independent of the presence of MT, while both Ser-202 and Thr-205 were phosphorylated only in the presence of MT. Dark-field microscopic observation demonstrated that MT-stabilizing activity of tau decreased independent of the presence of MT. However, its MT-nucleating activity was strongly inhibited only when phosphoryiated in the presence of MT. Conclusion: Tau can be phosphoryiated at Ser202 and Thr-205 only when it bound to MT. Phosphoryiation of these sites is important to inhibit the MT-nucleating activity.
Purpose: Living ceils and cell organeiies are ail compartmentalized by biomembrane, and their morphoiogies are thought to be determined and maintained by cytoskeletai networks. We focused on the role of microfilaments on their morphogenesis, and developed a model system using the iiposomes containing actin and actinbinding proteins. Methods: In the liposomes, the encapsulated monomeric actin and its bindi,ng protein were polymerized to actin filaments by raising temperature. We observed the subsequent morphological changes of the liposomes by high intensity dark-field video micrascopy. Results: The liposome containing actin alone transformed into a disk or dumbbell shape. When actin polymerized together with a-actinin, the l&some transformed into a disk shape possessing long tubular projections. When actin polymerized together with filamin, the iiposome became very rigid and took a stable shape. Conclusions: The difference in morphology between the actin containing liposomes depended on the type of co-encapsulated actin crosslinking protein, indicates that it can determine liposome shape by organizing actin networks.
ON MICROTUBULE
DYNAWrtCS
P-F2-07 Liposome shape is sta&itized by MAPS mediated interaction between microtubules and membrane.
P-F2-08 GDP INVERTS ‘iX@ PQ~RK4.Y OR
Kaneko T, ltoh TJ, Hotani H. Dept. of Mol. Biol., School of Sci.. Nagoya Univ. (JPN)
MICROTUBULE POLYM#D8?iATION TANAKA Y, IT0 TJ, HOTANI H. Dept. of Mol. Biol., School of Sci.. Nagoya Univ. (JPN)
Purpose: To understand the role of cytoskeletons on the morphogenesis of ceils, we examined morphological changesof liposome caused by the polymerization of encapsulated tubulin. Methods: We studied effects of MAPSon the microtubule (MT)-dependent morphological changes of liposomes by dark-field microscopy. To examine the interaction of MAPS with liposomes. we made the cosedimentation assay with sucrose cushion. Results: Upon MT polymerization, a spherical liposome transformed into a “dipolar” liposome that has a central sphere and two straight projections aligned on a line. Without MAPS. the “dipolar” liposome subsequently transformed to “monopolar” one by moving the central sphere without change in a total projection length. The number ratio of “dipoiar” to “monopolar” shape increased with MAPS concentrations. MT cosedimented with liposomes in the presence of MAPS. while MAPS-free MT did not. Particularly MAPI and MAP2 bound to liposome. Conclusions: These results suggest that MAPS medi;lte the binding bGtween microtubules and membrane, hence s&i#ze Eiposome shape.
Purpose: Guanine-nucieotide on tubulin molecules and its hydrolysis should play an important role not only in the frequency but also in the polarity of microtubule (MT) polymerization-depolymerization. We examined the effect of GDP on the polarity of MT elongation. Methods: MTs were polymerized from Tetruh.~rtma axonemes in the presence of only GTP or both of GTP and GDP. We determined that MTs elongated from which end bf “oneended axonemes,” the axonthat MTs elongated from their only one end, using NEMtubulin. MTs were observed by dark-field microscopy. Resulti 85% of one-ended axonemes observed at low tubulin concentration in the presence of only GTP, elongated MTs from their plus ends as predicted by the difference of critical concentrations of both ends. In the copresence of GDP, however, 94% of one-ended axonemes elongated MTs from their minus ends despite the higher tybulin concentratiqns. Conclusions: These results suggest that GDP can invcfi the polnity,of MT &ng@tion. 176