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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。真核生物的纤毛和鞭毛是高度保守的运动系统，它们建立在一个叫做轴丝的微管支架上。这个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以前的描述轴丝，但新的功能已经被发现。外部动力蛋白武器已被提取从断层扫描的弯曲鞭毛和分类到结构组，使用单粒子平均和特征向量-特征值分析。这种方法已经确定了三组动力蛋白结构沿着一个单一的双重微管。轴丝内这三个臂结构中的每一个的位置都与沿着鞭毛弯曲的位置相关联，这些位置对应于轴丝的直的、弯曲的和弯曲的部分。这些结果表明，通过客观方法鉴定的基团可能代表动力蛋白复合物的不同构象状态。然而，出现了两个问题：1）鞭毛嵌入其中的冰层的厚度是分辨率限制因素，因此只要鞭毛不被压缩，将其最小化就很重要。事实上，在我们早期的制备中，大多数冷冻水合的鞭毛都有些压缩，但我们通过使用在冷冻前具有改进的印迹控制的插入式冷冻机解决了这个问题。2)该缺楔是迄今为止不可避免的伪影的冷冻电子断层重建;它会导致各向异性失真的断层图像。因此，必须开发更复杂的图像处理工具，例如用于3-D颗粒对准、分类和“缺失楔形物”校正。虽然目前的数据是令人鼓舞的，但它们必须得到改进，才能真正提供动力蛋白作用机制的信息。