IMAGE ANALYSIS OF INNER DYNEIN ARM MUTANTS IN CHLAMYDOMONAS

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。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。列出的机构是 中心，不一定是研究者的机构。 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，动力蛋白马达将ATP结合和水解产生的化学能转化为驱动微管在轴丝内滑动的机械力。一个尚未解决的问题是内臂动力蛋白亚基的轴丝内的结构安排和它们的关系，协调其活动的监管机制。 目前的模型为鞭毛运动的调节是，中央对微管和径向辐条之间的机械相互作用被转换成一个生化信号通路，最终改变96 nm轴丝重复内的动力蛋白臂的磷酸化状态。我们集中在I1内臂动力蛋白，这是一个重要的目标，径向轮辐中心对复合物。 I1动力蛋白由两条不同的重链（1-α和1-β）、三条中间链（IC 140、IC 138、IC 97）和几条轻链组成，其活性因IC 138磷酸化状态的变化而改变。到目前为止，我们已经在DHC亚基和两个IC亚基的特征突变，改变装配的I1动力蛋白。野生型和I1突变体轴丝使用化学固定和传统的薄切片显微镜结合计算机图像平均分析提供了一个2-D图像的I1动力蛋白作为一个三叶结构位于近端的第一径向辐条内每个96 nm轴丝重复。用仅编码N末端茎结构域的动力蛋白重链构建体进行转化，可以部分拯救突变表型，并重新组装缺乏1-α或1-β运动结构域的I1动力蛋白复合物。图像分析的获救菌株表明，每个电机域可以与I1结构的一个叶。 这些研究进一步表明IC/LC复合物将位于I1结构的第三叶内，靠近与径向辐条相关的轴丝激酶和磷酸酶。 最近对一种新的突变体6 F5的研究表明，该突变体组装了一种缺乏IC 138磷蛋白的I1动力蛋白，该突变体在I1结构的第三叶内存在缺陷，这与先前的预测一致

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