IMAGE ANALYSIS OF INNER DYNEIN ARM MUTANTS IN CHLAMYDOMONAS

衣藻内动力蛋白臂突变体的图像分析

• 批准号: 8170829
• 负责人: EILEEN T O TOOLE
• 金额: $ 0.62万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170829
• 关键词: Binding; Biochemical; Chemicals; Chlamydomonas; Cilia; Complex; Computer Retrieval of Information on Scientific Projects Database; Cyclin-Dependent Kinase Inhibitor 3; Defect; Dynein ATPase; Flagella; Funding; Grant; Hydrolysis; Image; Image Analysis; Institution; Light; Lobe; Mechanics; Microscopy; Microtomy; Microtubules; Modeling; Motor; Mutation; N-terminal; Phenotype; Phosphoproteins; Phosphorylation; Radial; Regulation; Research; Research Personnel; Resources; Signal Pathway; Slide; Source; Structure; United States National Institutes of Health; arm; cell motility; digital imaging; driving force; mutant; sample fixation; stem

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. The motility of eukaryotic cilia and flagella depends on the presence of multiple dynein motors that convert the chemical energy derived from ATP binding and hydrolysis into mechanical forces that drive microtubule sliding within the axoneme. One unresolved issue is the structural arrangement of inner arm dynein subunits within the axoneme and their relationship to the regulatory machinery that coordinates their activity. The current model for the regulation of flagellar motility is that mechanical interactions between the central pair microtubules and radial spokes are converted into a biochemical signaling pathway that ultimately alters the phosphorylation state of the dynein arms within the 96 nm axoneme repeat. We have focused on the I1 inner arm dynein, which is an important target of the radial spoke-central pair complex. The I1 dynein is composed of two distinct heavy chains (1-alpha and 1-beta), three intermediate chains (IC140, IC138, IC97), and several light chains, and its activity is altered by changes in the phosphorylation state of IC138. Thus far we have characterized mutations in both DHC subunits and two IC subunits that alter the assembly of the I1 dynein. Analysis of wild-type and I1 mutant axonemes using chemical fixation and conventional thin section microscopy in combination with computer image averaging have provided a 2-D image of the I1 dynein as a tri-lobed structure located proximal to the first radial spoke within each 96 nm axoneme repeat. Transformations with dynein heavy chain constructs encoding only the N-terminal stem domain result in partial rescue of the mutant phenotypes and reassembly of I1 dynein complexes lacking either the 1-alpha or 1-beta motor domains. Image analysis of the rescued strains demonstrated that each motor domain could be correlated with one lobe of the I1 structure. These studies further suggested the IC/LC complex would be located within the third lobe of the I1 structure, in close proximity to the axonemal kinases and phosphatases associated with the radial spokes. Recent studies of a new mutant, 6F5, which assembles an I1 dynein lacking the IC138 phosphoprotein, has revealed defects within the third lobe of the I1 structure, consistent with previous predictions

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 真核生物纤毛和鞭毛的运动依赖于多个动力蛋白马达的存在，这些马达将来自ATP结合和水解的化学能转化为驱动微管在轴丝内滑动的机械力。一个悬而未决的问题是轴丝内臂动力蛋白亚基的结构安排，以及它们与协调其活动的调节机制的关系。目前的鞭毛运动调控模型是，中央微管和放射状辐条之间的机械相互作用转化为生化信号通路，最终改变96 nm轴丝重复序列中动力蛋白臂的磷酸化状态。我们重点研究了I1内臂动力蛋白，它是径向辐条-中心对复合体的一个重要靶点。I1动力蛋白由两条不同的重链(1-α和1-β)、三条中间链(IC140、IC138、IC97)和几条轻链组成，其活性随着IC138磷酸化状态的改变而改变。到目前为止，我们已经鉴定了DHC亚基和两个IC亚基的突变，这些突变改变了I1动力蛋白的组装。使用化学固定和常规切片显微镜结合计算机图像平均对野生型和I1突变型轴丝进行分析，提供了I1动力蛋白的二维图像，该图像为位于每个96 nm轴丝重复内第一径向辐条近端的三叶结构。仅编码N-末端茎结构域的动力蛋白重链结构的转化导致突变表型的部分挽救和缺少1-α或1-β运动区的I1动力蛋白复合体的重组。对挽救的菌株的图像分析表明，每个运动域可以与I1结构的一个叶相关。这些研究进一步表明，IC/LC复合体可能位于I1结构的第三叶内，非常接近与放射状辐条相关的轴丝蛋白激酶和磷酸酶。最近对一种新的突变体6F5的研究发现，I1结构的第三叶存在缺陷，这与之前的预测一致