CRYOET OF WT & MUTANT FLAGELLA OF CHLAMYDOMONAS ALLOWS MAPPING OF IDA SUBUNITS

WT 的秘密

• 批准号: 7355011
• 负责人: DANIELA NICASTRO
• 金额: $ 0.94万
• 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/7355011
• 关键词: Chlamydomonas; arm; dynein ATPase; flagellum; mutant

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. Axonemal dyneins are large, microtubule-based motor enzymes that convert the chemical energy derived from ATP binding and hydrolysis into the mechanical forces that bend cilia and flagella. While great progress has been made in understanding some aspects of flagellar mechanisms, much remains to be learned about the function of individual dynein subunits, their structural organization within the axoneme¿s 96 nm axial repeat, and the relationship between the motors and the regulatory machinery that coordinates their pattern of beat. We are therefore studying the 3-D structure of wild-type and mutant flagella using cryo-electron tomography in combination with correlation averaging (see Subproject T0066) to improve the signal-to-noise ratio in the tomographic reconstructions, and thus to enhance their resolution. We have recorded several tilt series of demembranated and rapidly frozen axonemes isolated from wild-type Chlamydomonas and from different inner dynein arm mutant strains (pf9, 6F5, pf10). The 3-D reconstructions and volume averages of the 96nm repeat of Chlamydomonas wild type axonemes confirm the main structural features of the inner and outer dynein arm complexes. However, in comparison to the 2-D pictures provided by previous structural studies, the tomographic volumes allow a 3D analysis and reveal new structural details, such as two novel linker structures between the inner and outer dynein arm complexes. We are using 3-D difference maps of averaged wild type and mutant axonemes to determine in situ the structural alterations that result from specific polypeptide defects.

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。轴突动力蛋白是基于微管的大型运动酶，它将ATP结合和水解产生的化学能转化为弯曲纤毛和鞭毛的机械力。虽然鞭毛机制的某些方面已经取得了很大的进展，但关于单个动力蛋白亚基的功能、轴突内96 nm轴向重复的结构组织以及马达与协调其跳动模式的调节机制之间的关系，仍有许多有待了解的地方。因此，我们正在使用冷冻电子断层扫描结合相关平均（见子项目T0066）来研究野生型和突变型鞭毛的三维结构，以提高断层扫描重建中的信噪比，从而提高它们的分辨率。我们记录了从野生型衣单胞菌和不同的内动力蛋白臂突变菌株（pf9, 6F5, pf10）中分离的轴突脱膜和快速冷冻的倾斜系列。野生衣藻轴突体96nm重复序列的三维重建和体积平均值证实了内外动力蛋白臂复合物的主要结构特征。然而，与之前的结构研究提供的二维图像相比，断层扫描可以进行三维分析并揭示新的结构细节，例如内外动力蛋白臂复合物之间的两个新的连接结构。我们正在使用平均野生型和突变轴突体的三维差异图来原位确定由特定多肽缺陷引起的结构改变。