COMPONENTS OF THE DYNEIN REGULATORY COMPLEX IN CHLAMYDOMONAS FLAGELLA

鞭毛衣藻中动力蛋白调节复合体的组成部分

基本信息

批准号：
7955977
负责人：
DANIELA NICASTRO
金额：
$ 0.47万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-01 至 2010-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7955977
关键词：
Address Adopted Affect Agrin Algae Animal Model Binding Sites Biological Markers Biology Cells Chemicals Chlamydomonas Cilia Complex Computer Retrieval of Information on Scientific Projects Database Cytoskeleton Data Development Disease Dynein ATPase Electron Microscopy Eukaryota Event Evolution Exhibits Flagella Funding Future Genes Grant Growth Homologous Gene Human Individual Institution Label Light Link Mammals Manuscripts Maps Mass Spectrum Analysis Medicine Methods Molecular Molecular Weight Mutation Organelles Paralysed Phenotype Phosphoric Monoester Hydrolases Phosphotransferases Plants Play Preparation Protein Isoforms Proteins Proteome Proteomics Radial Regulation Research Research Personnel Resolution Resources Role Route Sales Shapes Source Spottings Structure Suppressor Mutations System Transducers United States National Institutes of Health Upper arm Yang base cell motility cilium/flagellum motility density electron tomography mutant nexin novel polypeptide spatial relationship therapeutic target two-dimensional

项目摘要

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.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目及研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。我们正在使用蛋白质组分析来鉴定衣藻鞭毛中动力蛋白调节复合物的先前描述的和新的成分。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、感知和发育中发挥着重要作用[1,2]。 9+2纤毛和鞭毛的振荡跳动高度协调，需要精确调节[3]。然而，这种调节的详细结构和分子基础仍有待阐明。单细胞藻类衣藻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有价值[4]。先前对抑制径向辐条突变体“麻痹鞭毛”表型的衣藻突变体的研究已确定动力蛋白调节复合物 (DRC) 是动力蛋白活性调节系统中的关键参与者，从而影响鞭毛运动 [5-7]。七种轴丝多肽被生化鉴定为 DRC 成分，它们形成表观分子量至少 500 kDa 的大型复合物 [5,6]。使用野生型和 drc 突变体的电子显微镜，DRC 结构被粗略地映射到径向辐条 RS2 底部附近的新月形区域 [8,9]。我们最近的冷冻电子断层扫描数据以前所未有的细节和分辨率定位了完整轴丝中的七个 DRC 组件，包括它们与 nexin 链接的空间关系（手稿正在准备中）。此外，还观察到了先前未表征的其他 DRC 密度。然而，迄今为止，只有 PF2 基因编码的蛋白质 DRC4 在分子水平上得到了表征 [10]。为了鉴定其余 6 个描述的 DRC 成分和未知亚基的基因，我们开始对来自衣藻野生型和突变鞭毛的分离轴丝进行全面的蛋白质组学分析。迄今为止，二维 (2-D) PAGE 显示 38 个蛋白质点在野生型和 DRC 突变体之间表现出统计上显着的变化。 MALDI-TOF MS 分析将这些点鉴定为 18 个单独的蛋白质；其中包括六种已知的 DRC 成分（DRC1 至 DRC6），以及不同修饰的蛋白质，包括野生型鞭毛中存在至少 6 种亚型的蛋白质。将来，我们计划扩大研究范围，以确定 2-D PAGE 无法解析的候选 DRC 成分。可能的途径是采用无标记定量蛋白质组学方法或基于标记的方法（如 iTRAQ）来关联野生型和突变型鞭毛蛋白质组。这项研究代表了 DRC 复合物的首次蛋白质组学分析，并有可能鉴定新的 DRC 成分。这些发现可能揭示纤毛和鞭毛运动调节的分子事件，并可能有助于识别人类纤毛疾病的生物标志物和治疗靶点。参考： [1] 波特 ME，销售 WS。 (2000) 9 + 2 轴丝锚定多个内臂动力蛋白以及控制运动的激酶和磷酸酶网络。细胞生物学杂志，151(5)：F37-42。 [2] Pazour GJ、Agrin N、Leszyk J、Witman GB。 (2005)真核纤毛的蛋白质组学分析。细胞生物学杂志，170：103-13。 [3] 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传感器。细胞运动细胞骨架，57：8-17。 [4] 哈里斯·EH。 (2001) 衣藻作为模式生物。年鉴植物生理学植物分子生物学，52：363-406。 [5] 黄斌，Ramanis Z，Luck DJ. (1982) 衣藻的抑制突变揭示了鞭毛功能的调节机制。细胞，28：115-24。 [6] Piperno G、Mead K、LeDizet M、Moscatelli A. (1994)“动力蛋白调节复合物”中的突变改变了衣藻轴丝中内臂动力蛋白的 ATP 不敏感结合位点。细胞生物学杂志，125：1109-17。 [7] Piperno G、Mead K、Shestak W. (1992) 内动力蛋白臂 I2 与衣藻鞭毛中的“动力蛋白调节复合物”相互作用。细胞生物学杂志，118：1455-63。 [8] Mastronarde DN、O'Toole ET、McDonald KL、McIntosh JR、Porter ME。 (1992)衣藻野生型和突变型鞭毛内动力蛋白臂的排列。细胞生物学杂志，118：1145-62。 [9] 加德纳 LC、奥图尔 E、佩罗内 CA、吉丁斯 T、波特 ME。 (1994)“动力蛋白调节复合体”的组成部分位于衣藻鞭毛中径向辐条和动力蛋白臂之间的连接处。细胞生物学杂志，127：1311-25。 [10]鲁普·G，波特·ME。 (2003) 衣藻中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。细胞生物学杂志，162：47-57。