A New Rapid Freezing Apparatus for Electron Microscopy

一种新型电子显微镜快速冷冻装置

基本信息

  • 批准号:
    02558026
  • 负责人:
  • 金额:
    $ 5.63万
  • 依托单位:
  • 依托单位国家:
    日本
  • 项目类别:
    Grant-in-Aid for Developmental Scientific Research (B)
  • 财政年份:
    1990
  • 资助国家:
    日本
  • 起止时间:
    1990 至 1991
  • 项目状态:
    已结题

项目摘要

The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P^3-1-(2-nitropheny1)ethy1 ester of ATP (caged ATP) was analyzed with a newly-developed freezing system using liquid helium. Actin and S1 (100muM each) formed a rope-like double helix characteristic of rigor in the presence of 5 mM caged ATP at room temperature. At 15 ms after photolysis, the rope-like double helix was partially disintegrated. The number of S1 attached to actin filaments gradually decreased up to 35 ms after photolysis, and no more changes were detected from 35 to 200 ms. After depletion of ATP. the rope-like double helix was reformed. Taking recent analyses of actomyosin kinetics into consideration, we concluded that most S1 observed on actin filaments at 25-200 ms are so called "weakly-bound S1" (S1.ATP or S1.ADP.Pi) and that the weakly-bound S1 under a rapid association-dissociation equilibrium with actin filaments can be captured by electron microscopy by means of our newly-developed freezing system.This enabled us to directly compare the conformation of weakly- and strongly-bound S1. Within the resolution of deep-etch replica technique, there were no significant conformational differences between weakly- and strongly-bound S1, and neither types of S1 showed any positive cooperativity in their binding to actin filaments. Close comparison revealed that the weakly- and strongly-bound S1 have different angles of attachment. As compared to strongly-bound S1, weakly-bound S1 showed broad distribution of attachment angle and a decreased tilt from the perpendicular to the filaments. These results discussed with special reference to the molecular mechanism of acto-myosin interaction in the presence of ATP.
采用新型液氦冷冻系统,分析了P^3-1-(2-nitropheny1)乙基ATP(笼式ATP)光解后肌凝蛋白亚片段1 (S1)与肌动蛋白丝的相互作用。肌动蛋白和S1(各100muM)在室温下,在5 mM笼状ATP存在下形成绳状双螺旋结构,具有刚性特征。光解15ms后,绳状双螺旋部分解体。在光解后35 ms内,附着在肌动蛋白丝上的S1数量逐渐减少,在35 ~ 200 ms内没有变化。在ATP耗尽之后。绳状双螺旋结构被改造。考虑到最近对肌动球蛋白动力学的分析,我们得出结论,在25-200 ms时,在肌动蛋白丝上观察到的大多数S1是所谓的“弱结合S1”(S1)。ATP或s1, adp。Pi),并且利用我们新开发的冷冻系统可以在电子显微镜下捕获与肌动蛋白丝快速结合-解离平衡的弱结合S1。这使我们能够直接比较弱键和强键S1的构象。在深蚀刻复制技术的分辨率范围内,弱结合和强结合的S1之间没有显着的构象差异,并且两种类型的S1在与肌动蛋白丝的结合中都没有表现出任何积极的协同性。通过比较发现,弱结合和强结合的S1具有不同的附着角度。与强结合的S1相比,弱结合的S1的附着角分布较宽,从垂直于细丝的角度倾斜较小。这些结果特别讨论了ATP存在下肌动蛋白-肌球蛋白相互作用的分子机制。

项目成果

期刊论文数量(9)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
Itoh,M.: "A 220-kD undercoat-constitutive protein:Its specific localization at cadherin-based cell-cell adhesion sites." Journal of Cell Biology. 115. 1449-1462 (1991)
Itoh,M.:“一种 220 kD 的底毛组成蛋白:其特定定位于基于钙粘蛋白的细胞间粘附位点。”
  • DOI:
  • 发表时间:
  • 期刊:
  • 影响因子:
    0
  • 作者:
  • 通讯作者:
Muto,E.: "Doubleーrowed organization of inner dynein arms in Chlamydomonas flagella revealed by tiltーseries thinーsection electron microscopy." J.Cell Sci.
Muto,E.:“通过倾斜系列薄切片电子显微镜揭示衣藻鞭毛内动力蛋白臂的双排组织。”
  • DOI:
  • 发表时间:
  • 期刊:
  • 影响因子:
    0
  • 作者:
  • 通讯作者:
Nagafuchi,A.: "The 102kd cadherin-associated protein: Similarity to vinculin and posttranscriptional regulation of expression." Cell. 65. 1-20 (1991)
Nagafuchi,A.:“102kd 钙粘蛋白相关蛋白:与纽蛋白和转录后表达调控的相似性。”
  • DOI:
  • 发表时间:
  • 期刊:
  • 影响因子:
    0
  • 作者:
  • 通讯作者:
Nagafuchi,A.: "The 102kd cadherin-associated protein:Similarity to vinculin and posttranscriptional regulation of expression." Cell. 65. 849-857 (1991)
Nagafuchi,A.:“102kd 钙粘蛋白相关蛋白:与纽蛋白的相似性和表达的转录后调节。”
  • DOI:
  • 发表时间:
  • 期刊:
  • 影响因子:
    0
  • 作者:
  • 通讯作者:
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TSUKITA Shoichiro其他文献

TSUKITA Shoichiro的其他文献

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{{ truncateString('TSUKITA Shoichiro', 18)}}的其他基金

Claudins in the epithelium/endothelium barrier dysfucrition
上皮/内皮屏障功能障碍中的 Claudins
  • 批准号:
    14207008
  • 财政年份:
    2002
  • 资助金额:
    $ 5.63万
  • 项目类别:
    Grant-in-Aid for Scientific Research (A)
Molecular mechanism for cell-cell adhesion in canceration and metastasis
癌变和转移中细胞粘附的分子机制
  • 批准号:
    12219210
  • 财政年份:
    2000
  • 资助金额:
    $ 5.63万
  • 项目类别:
    Grant-in-Aid for Scientific Research on Priority Areas
The claudin family : Its involvement in interecellular sealing and epithelial polarity
密蛋白家族:参与细胞间密封和上皮极性
  • 批准号:
    11307002
  • 财政年份:
    1999
  • 资助金额:
    $ 5.63万
  • 项目类别:
    Grant-in-Aid for Scientific Research (A)
CLAUDINS AND OCCLUDIN : COMPARISON WITH CONNEXIN
CLAUDINS 和 OCCLUDIN:与 CONNEXIN 的比较
  • 批准号:
    11694270
  • 财政年份:
    1999
  • 资助金额:
    $ 5.63万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B).
Development of a new drug delivery method by the use of occludin molecules
利用occludin分子开发新的药物递送方法
  • 批准号:
    10557011
  • 财政年份:
    1998
  • 资助金额:
    $ 5.63万
  • 项目类别:
    Grant-in-Aid for Scientific Research (B)
Structure and function of occludin in tight junctions : comparison with connexin gap junctions
紧密连接中occludin的结构和功能:与连接蛋白间隙连接的比较
  • 批准号:
    09044290
  • 财政年份:
    1997
  • 资助金额:
    $ 5.63万
  • 项目类别:
    Grant-in-Aid for international Scientific Research
The structure and functions of occludin
occludin的结构和功能
  • 批准号:
    08407006
  • 财政年份:
    1996
  • 资助金额:
    $ 5.63万
  • 项目类别:
    Grant-in-Aid for Scientific Research (A)
MODULATION OF BLOOD-BRAIN BARRIER
血脑屏障的调节
  • 批准号:
    06557014
  • 财政年份:
    1994
  • 资助金额:
    $ 5.63万
  • 项目类别:
    Grant-in-Aid for Developmental Scientific Research (B)
CELL ADHESION-DEPENDENT REGULATION OF CELL GROWTH AND DIFFERENTIATION
细胞生长和分化的细胞粘附依赖性调节
  • 批准号:
    06404083
  • 财政年份:
    1994
  • 资助金额:
    $ 5.63万
  • 项目类别:
    Grant-in-Aid for General Scientific Research (A)
Time-lapse Electron Microscopy with Caged Compounds
笼状化合物的延时电子显微镜
  • 批准号:
    04558034
  • 财政年份:
    1992
  • 资助金额:
    $ 5.63万
  • 项目类别:
    Grant-in-Aid for Developmental Scientific Research (B)

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Connectmics analysis of functional synapses by microwave rapid freezing method
微波快速冷冻法对功能突触的连接组学分析
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    2015
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Study of rapid freezing method of wet bio-specimens
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通过快速冷冻捕获的收缩肌肉的即时视图。
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一种简易快速冷冻方法保存农畜及小动物蛋的研究
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