A New Rapid Freezing Apparatus for Electron Microscopy

一种新型电子显微镜快速冷冻装置

• 批准号: 02558026
• 负责人: TSUKITA Shoichiro
• 金额: $ 5.63万
• 依托单位: National Institute for Physiological Sciences
• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02558026/
• 关键词: caged compounds; rapid freezing; electron microscopy; actin; myosin; liquid helium; etching; replica; 急速凍結法; ケ-ジド化合物; 光分解; 時間分解能; ATP

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.

用一种新研制的液氦冷冻系统研究了P^3-1-（2-硝基苯）乙基ATP酯（笼状ATP）光解后肌球蛋白亚片段1（S1）与肌动蛋白丝的相互作用。肌动蛋白和S1（各100 μ M）在室温下存在5 mM笼状ATP时形成具有僵直特征的绳状双螺旋。在光解后15 ms，绳状双螺旋部分解体。附着在肌动蛋白丝的S1的数量逐渐减少到35毫秒后光解，并没有更多的变化被检测到从35至200毫秒。耗尽ATP后。重新形成绳状双螺旋。考虑到最近对肌动球蛋白动力学的分析，我们的结论是，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），并且在与肌动蛋白丝的快速缔合-解离平衡下弱结合的S1可以通过电子显微镜通过我们新的-这使我们能够直接比较弱结合和强结合S1的构象。在深蚀刻复制技术的分辨率内，弱结合和强结合的S1之间没有显著的构象差异，并且两种类型的S1在与肌动蛋白丝的结合中均未表现出任何正协同性。通过比较发现，弱结合和强结合的S1具有不同的连接角度。与强结合的S1相比，弱结合的S1显示出较宽的附着角分布，并且从垂直于细丝的方向的倾斜度减小。这些结果进行了讨论，特别是参考在ATP的存在下的肌动蛋白-肌球蛋白相互作用的分子机制。

项目成果

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。”

Tsukita, Sa., Oishi. K., Akiyama. T., Yamanashi. Y., Yamamoto. T. & Tsukita. Sh.: "Specific proto-oncogenic tyrosine kinases of src family are enriched in cell-to-cell adherens junctions where the level of tyrosine phosphorylation is elevated." J. Cell Bi

月田，佐治亚州，大石。

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。”

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。”

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。”