3D STRUCTURE OF ACTIVATED AXONEMES STUDIED BY CRYO-ELECTRON TOMOGRAPHY

通过低温电子断层扫描研究激活轴丝的 3D 结构

• 批准号: 7354993
• 负责人: DANIELA NICASTRO
• 金额: $ 1.87万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-26 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7354993
• 关键词: arm; dynein ATPase; dysbarism; flagellum; microtubules; particle; problem solving; tomography

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Eukaryotic cilia and flagella are highly conserved motile systems built on a microtubule-based scaffold called the axoneme. The complexity of this organelle and size of its major motor protein, dynein, have made it difficult to understand the molecular mechanisms that underlie flagellar beating. We are using electron tomography of frozen-hydrated flagella to solve this problem. 3-D reconstructions have enabled us to identify and localize single macromolecules in axonemes. Many of our results confirm previous descriptions of axonemes, but novel features have already been revealed. Outer dynein arms have been extracted from tomograms of a bent flagellum and classified into structural groups, using single particle averaging and Eigenvector-Eigenvalue analysis. This approach has identified three groups of dynein structures along a single doublet microtubule. The sites within the axoneme from which each of these three arm structures have come correlate with positions along the flagellar bend that correspond to straight, curving, and curved parts of the axoneme. These results indicate that the groups identified by objective methods might represent different conformational states of the dynein complex. However, two problems have emerged: 1) the thickness of the ice-layer in which the flagella are embedded is a resolution-limiting factor, so it is important to minimize it, so long as the flagella are not compressed. Indeed, the majority of frozen-hydrated flagella in our early preparations were somewhat compressed, but we have solved this problem by using a plunge-freezer with improved control of blotting prior to freezing. 2) The ¿missing wedge¿ is to date an inevitable artifact of cryo-electron tomographic reconstructions; it causes anisotropic distortions in the tomograms. Thus, more sophisticated image processing tools, e.g. for 3-D particle alignment, classification and ¿missing-wedge¿ correction must be developed. Though current data are encouraging, they must be improved to be truly informative about dynein¿s mechanism of action.

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。真核生物的纤毛和鞭毛是高度保守的运动系统，建立在以微管为基础的支架上，称为轴丝。这种细胞器的复杂性及其主要运动蛋白动力蛋白的大小，使得人们很难理解鞭毛跳动背后的分子机制。我们正在使用冷冻水化鞭毛的电子断层扫描来解决这个问题。三维重建使我们能够识别和定位轴丝中的单个大分子。我们的许多结果证实了之前对轴丝的描述，但新的特征已经被揭示。从弯曲鞭毛的断层图像中提取了动力蛋白外臂，并利用单粒子平均和特征向量-特征值分析将其分类为结构群。这种方法已经确定了沿着单个双线微管的三组动力蛋白结构。这三个臂结构中的每一个都来自轴丝内的位置，这些位置与沿着鞭毛弯曲的位置相关，这些位置对应于轴丝的直、弯和弯部分。这些结果表明，客观方法鉴定的基团可能代表动力蛋白复合体的不同构象状态。然而，出现了两个问题：1)嵌入鞭毛的冰层的厚度是分辨率的限制因素，所以只要鞭毛不被压缩，将其最小化是很重要的。事实上，在我们的早期制剂中，大多数冷冻水合的鞭毛都有一定程度的压缩，但我们已经解决了这个问题，使用了立式冷冻机，改进了冷冻前对印迹的控制。2)楔形缺失是冷冻电子层析重建中不可避免的伪影，它会造成层析图像的各向异性失真。因此，必须开发更复杂的图像处理工具，例如用于3-D粒子对准、分类和缺失-楔形校正。尽管目前的数据令人鼓舞，但它们必须得到改进，才能真正提供有关动力蛋白S作用机制的信息。