COMPONENTS OF THE DYNEIN REGULATORY COMPLEX IN CHLAMYDOMONAS FLAGELLA

这个子项目是许多研究子项目中的一个 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，不一定是研究者的机构。 我们正在使用蛋白质组学分析，以确定以前描述的和新的组件的动力蛋白调节复合体在衣原体鞭毛。 纤毛和鞭毛是广泛分布的细胞器，在整个进化过程中高度保守，在从原生生物到哺乳动物的真核生物的运动、传感和发育中发挥重要作用[1，2]。9+2纤毛和鞭毛的振荡跳动是高度协调的，需要精确的调节[3]。然而，这种调节的详细结构和分子基础仍有待阐明。单细胞藻类衣原体是一种成熟的模式生物，具有大量可用的突变体，包括影响鞭毛运动的突变体，这使得这种原生生物对于解决具有挑战性的问题非常宝贵[4]。先前对抑制辐射状辐条突变体的“瘫痪鞭毛”表型的衣原体突变体的研究已经将动力蛋白调节复合物（DRC）鉴定为动力蛋白活性的调节系统中的关键参与者，从而鉴定为鞭毛运动性[5-7]。七种轴丝多肽被生化鉴定为DRC组分，其形成具有至少500 kDa的表观分子量的大复合物[5，6]。使用野生型和DRC突变体的电子显微镜，DRC结构大致映射到靠近径向辐条RS 2基部的新月形区域[8，9]。我们最近的冷冻电子断层扫描数据已本地化的七个DRC组件在完整的轴丝在前所未有的细节和分辨率，包括它们的空间关系的连接蛋白链接（手稿准备）。此外，观察到先前未表征的额外DRC密度。 然而，到目前为止，只有DRC 4，由PF 2基因编码的蛋白质，已在分子水平上进行了表征[10]。为了确定剩余的6个描述的DRC组件和未知的亚基的基因，我们已经开始了一个全面的蛋白质组学分析分离的轴丝衣原体野生型和突变体鞭毛。双向（2-D）聚丙烯酰胺凝胶电泳显示，迄今为止，38个蛋白质点表现出统计上显着的变化之间的野生型和DRC突变体。MALDI-TOF MS分析将这些点鉴定为18种单独的蛋白质;其中包括六种已知的DRC组分（DRC 1至DRC 6），以及不同修饰的蛋白质，包括一种具有至少6种野生型鞭毛中存在的同种型的蛋白质。 在未来，我们计划扩大我们的研究，以确定候选DRC组件没有解决的2-D PAGE。可能的途径是采用无标记的定量蛋白质组学方法，或者基于标记的方法，如iTRAQ，将野生型和突变体鞭毛蛋白质组相关联。这项研究代表了第一个蛋白质组学分析的刚果民主共和国复杂，并有可能确定新的刚果民主共和国的组成部分。这些发现可能揭示了纤毛和鞭毛运动调节的分子事件，并可能有助于识别用于治疗人类睫状体疾病的生物标志物和治疗靶点。 参考资料： [1]第一章 Porter ME，销售WS.（2000）9 + 2轴丝锚定多个内臂动力蛋白和控制运动的激酶和磷酸酶网络。J Cell Biol，151（5）：F37-42. [二]《中国日报》 Pazour GJ，Agrin N，Leszyk J，Witman GB.（2005）真核纤毛的蛋白质组学分析。J Cell Biol，170：103-13. [3]第一章 Smith EF，Yang P.（2004）径向辐条和中央装置：调节鞭毛运动的机械化学换能器。Cell Motil Cytoskeleton，57：8-17. [4]美国 哈里斯2001年，以衣原体为模式生物。Annu Rev Plant Physiol Plant Mol Biol，52：363-406. [五]《中国日报》 黄B，拉马尼斯Z，幸运DJ。（1982）衣原体中的抑制突变揭示了鞭毛功能的调节机制。Cell，28：115-24. [6]美国 杨伟杰，王伟杰，王伟杰.（1994）“动力蛋白调节复合物”中的突变改变衣原体轴丝中内臂动力蛋白的ATP不敏感结合位点。J Cell Biol，125：1109-17. [七]《中国日报》 李文龙，李文龙，李文龙.（1992）内动力蛋白臂I2与衣原体鞭毛中的“动力蛋白调节复合物”相互作用。J Cell Biol，118：1455-63. [八]《中国日报》 Mastronarde DN，O 'Toole ET，McDonald KL，McIntosh JR，Porter ME（1992）衣原体野生型和突变型鞭毛内动力蛋白臂的排列。J Cell Biol，118：1145-62. [9]第一章 Gardner LC，O 'Toole E，Perrone CA，Giddings T，Porter ME.（1994）“动力蛋白调节复合物”的组分位于衣原体鞭毛中径向辐条和动力蛋白臂之间的连接处。J Cell Biol，127：1311-25. [10个国家] Rupp G，Porter ME.（2003）衣原体动力蛋白调节复合物的亚基是生长停滞特异性基因产物的同源物。J Cell Biol，162：47-57.

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. We are using proteomic analysis for identification of previously described and novel Components of the dynein regulatory complex in Chlamydomonas flagella. Cilia and flagella are widespread organelles that have been highly conserved throughout evolution and play important roles in motility, sensing and development of eukaryotes ranging from protists to mammals [1,2]. The oscillatory beating of 9+2 cilia and flagella is highly coordinated and requires precise regulation [3]. However, the detailed structural and molecular basis of this regulation remains to be elucidated. The unicellular algae Chlamydomonas is a well-established model organism with a large arsenal of available mutants, including those affecting flagellar motility, which have made this protist invaluable for addressing challenging questions [4]. Previous studies on Chlamydomonas mutants that suppressed the "paralyzed flagella" phenotype of radial spoke mutants have identified the dynein regulatory complex (DRC) as a key player in the regulation system of dynein's activity and thus flagellar motility [5-7]. Seven axonemal polypeptides were biochemically identified as DRC components that form a large complex with an apparent molecular weight of at least 500 kDa [5,6]. Using electron microscopy of wild type and drc-mutants the DRC structure was roughly mapped to a crescent shaped region near the base of radial spoke RS2 [8,9]. Our recent cryo-electron tomography data have localized the seven DRC components in intact axonemes in unprecedented detail and resolution, including their spatial relationship to the nexin link (manuscript in preparation). Moreover, additional DRC densities not previously characterized were observed. However, to date, only DRC4, the protein encoded by the PF2 gene, has been characterized at the molecular level [10]. To identify the genes of the remaining 6 described DRC components and the unknown subunits, we have begun a comprehensive proteomic analysis of isolated axonemes from Chlamydomonas wild type and mutant flagella. Two-dimensional (2-D) PAGE so far revealed that 38 protein spots exhibited statistically significantly changes among the wild type and DRC mutants. MALDI-TOF MS analysis identified these spots as 18 individual proteins; among those were six of the known DRC components (DRC1 to DRC6), as well as differently modified proteins, including one with at least 6 isoforms present in wild type flagella. In the future we plan to expand our study to identify the candidate DRC components not resolved by 2-D PAGE. Possible routes would be to adopt label-free quantitative proteomic approaches or alternatively a label-based method, like iTRAQ, to correlate the wild type and mutant flagellar proteome. This study represents the first proteomic analysis of the DRC complex and has the potential to identify novel DRC components. These findings may shed light on the molecular events underlying cilia and flagella motility regulation and may aid in the identification of biomarkers and therapeutic targets for the treatment of human ciliary diseases. References: [1] Porter ME, Sale WS. (2000) The 9 + 2 axoneme anchors multiple inner arm dyneins and a network of kinases and phosphatases that control motility. J Cell Biol, 151(5):F37-42. [2] Pazour GJ, Agrin N, Leszyk J, Witman GB. (2005) Proteomic analysis of a eukaryotic cilium. J Cell Biol, 170:103-13. [3] Smith EF, Yang P. (2004) The radial spokes and central apparatus: mechano-chemical transducers that regulate flagellar motility. Cell Motil Cytoskeleton, 57:8-17. [4] Harris EH. (2001) Chlamydomonas as a model organism. Annu Rev Plant Physiol Plant Mol Biol, 52:363-406. [5] Huang B, Ramanis Z, Luck DJ. (1982) Suppressor mutations in Chlamydomonas reveal a regulatory mechanism for flagellar function. Cell, 28:115-24. [6] Piperno G, Mead K, LeDizet M, Moscatelli A. (1994) Mutations in the "dynein regulatory complex" alter the ATP-insensitive binding sites for inner arm dyneins in Chlamydomonas axonemes. J Cell Biol, 125:1109-17. [7] Piperno G, Mead K, Shestak W. (1992) The inner dynein arms I2 interact with a "dynein regulatory complex" in Chlamydomonas flagella. J Cell Biol, 118:1455-63. [8] Mastronarde DN, O'Toole ET, McDonald KL, McIntosh JR, Porter ME. (1992) Arrangement of inner dynein arms in wild-type and mutant flagella of Chlamydomonas. J Cell Biol, 118:1145-62. [9] Gardner LC, O'Toole E, Perrone CA, Giddings T, Porter ME. (1994) Components of a "dynein regulatory complex" are located at the junction between the radial spokes and the dynein arms in Chlamydomonas flagella. J Cell Biol,127:1311-25. [10] Rupp G, Porter ME. (2003) A subunit of the dynein regulatory complex in Chlamydomonas is a homologue of a growth arrest-specific gene product. J Cell Biol, 162:47-57.