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。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是针对该中心的，这不是调查人员的机构。真核生物纤毛和鞭毛是建立在基于微管的支架上的高度保守的通气系统。这种细胞器的复杂性以及其主要运动蛋白动力蛋白的大小使得很难理解鞭毛搏动的分子机制。我们正在使用冷冻水鞭毛的电子断层扫描来解决此问题。 3-D重建使我们能够在轴突中识别和定位单个大分子。我们的许多结果证实了先前对轴突的描述，但已经揭示了新的特征。使用单个粒子平均和特征向量 - 元素值分析，已经从Ben Flagelum的断层图中提取了外动臂，并将其分类为结构组。这种方法已经沿单个双重微管确定了三组动力蛋白结构。这三个臂结构中每个臂结构中每个位置的位置与沿鞭毛弯曲的位置相关，与轴突的直，弯曲和弯曲部分相对应。这些结果表明，通过客观方法识别的组可能代表动力蛋白复合物的不同会议状态。但是，出现了两个问题：1）嵌入鞭毛的冰层厚度是一个分辨率限制因素，因此，只要鞭毛不被压缩，将其最小化。的确，在我们的早期制剂中，大多数冷冻水合的鞭毛受到了压缩，但是我们通过在冻结之前使用猛烈的冰柜来改善对印迹的控制，解决了这一问题。 2）缺少楔形是迄今为止不可避免的冷冻电子层析成像重建的伪像；它在断层图中引起各向异性畸变。这是更复杂的图像处理工具，例如对于3-D粒子的比对，必须开发分类和缺失的纽维奇校正。尽管当前的数据令人鼓舞，但必须对它们进行改进，以便对Dynein的作用机理真正提供信息。