Time-lapse Electron Microscopy with Caged Compounds

笼状化合物的延时电子显微镜

• 批准号: 04558034
• 负责人: TSUKITA Shoichiro
• 金额: $ 4.99万
• 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: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-04558034/
• 关键词: muscle; caged compounds; rapid freezing; liquid helium; platelet; electron microscopy; IP3; temporal resolution; caged化合物; ケージドATP; 急速凍結法; アクチン; ミオシン; 紫外線; ミリ秒

The interaction between myosin subfragment 1 (S1) and actin filaments after the photolysis of P^3-1-(2-nitrophenyl)ethyl 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 analyzes 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 significatn 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 are 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-硝基苯基）乙基ATP（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。附着在肌动蛋白丝上的S1的数量在光解后35 ms逐渐减少，在35 ~ 200 ms之间没有检测到更多的变化。ATP耗尽后，绳状双螺旋被重新形成。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范围内，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显示出任何正的协同性。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，特别提到的ATP的存在下，acto-myosin相互作用的分子机制。

项目成果

Furuse,M.: "Occludin:A novel integral membrane protein localizing at tight junctions." J.Cell Biol.123. 1777-1788 (1993)

Furuse,M.：“Occludin：一种位于紧密连接处的新型整合膜蛋白。”

Shimoyama,Y.: "Cadherin dysfunction in a human cancer cell line:Possible involvement of loss of alpha-catenin expression in reduced cell-cell adhesive-ness." Cancer Research. 52. 5770-5774 (1992)

Shimoyama, Y.：“人类癌细胞系中的钙粘蛋白功能障碍：可能与 α-连环蛋白表达缺失导致细胞间粘附性降低有关。”

Matsuyoshi,N.: "Cadherin-medicated cell-cell adhesion is perturbed by v-src tyrosine phosphorylation in metastatic fibroblasts." Journal of Cell Biology. 118. 703-714 (1992)

Matsuyoshi,N.：“钙粘蛋白介导的细胞间粘附受到转移性成纤维细胞中 v-src 酪氨酸磷酸化的干扰。”

Oda, H., Uemura, T., Shiomi, K., Nigafuchi, A., Tsukita, S., and Takeichi, M.: "Identification of a Drosophila homologue of alpha-catenin and its association with the armadillo protein." J.Cell Biol.121. 1133-1140 (1993)

Oda, H.、Uemura, T.、Shiomi, K.、Nigafuchi, A.、Tsukita, S. 和 Takeichi, M.：“果蝇 α-连环蛋白同源物的鉴定及其与犰狳蛋白的关联。”

Takeuchi,K.: "Perturbation of cell adhesion and microvilli formation by antisense oligonucleotides to ERM family members." J.Cell Biol.(in press). (1994)

Takeuchi,K.：“ERM 家族成员的反义寡核苷酸对细胞粘附和微绒毛形成的干扰。”