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.

该副本是利用众多研究子项目之一由NIH/NCRR资助的中心赠款提供的资源。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构是对于中心，这不一定是调查员的机构。我们正在使用蛋白质组学分析来鉴定在衣原体鞭毛中动力蛋白调节复合物的先前描述和新成分。纤毛和鞭毛是广泛的细胞器，在整个进化过程中一直是高度保守的，并且在运动性，感知和发展的真核生物中起着重要作用，从生物到生物到哺乳动物[1,2]。 9+2纤毛和鞭毛的振荡击败高度协调，需要精确的调节[3]。但是，该调节的详细结构和分子基础尚待阐明。单细胞藻类衣原体是一种良好的模型生物体，具有大量可用突变体，包括影响鞭毛运动的人，这对于解决挑战性问题而言，这使该原则是无价的[4]。先前对抑制径向辐条突变体“瘫痪的鞭毛”表型的衣原体突变体的研究已将动力蛋白调节复合物（DRC）鉴定为Dynein活性调节系统的关键参与者，从而成为鞭毛运动性[5-7]。七个轴突多肽被生化鉴定为DRC成分，形成了一个大型复合物，其明显的分子量至少为500 kDa [5,6]。使用野生型和DRC突变剂的电子显微镜，将DRC结构大致映射到径向辐条rs2底部附近的新月形区域[8,9]。我们最近的冷冻电子层析成像数据已以前所未有的细节和分辨率将完整的轴突中的七个DRC组件定位在完整的轴突中，包括它们与Nexin Link（准备中的手稿）的空间关系。此外，观察到其他未表征的DRC密度。然而，迄今为止，只有在分子水平上表征了由PF2基因编码的蛋白质DRC4 [10]。为了确定其余6个描述的DRC成分和未知亚基的基因，我们开始对来自衣原体野生型和突变鞭毛的分离轴突进行全面的蛋白质组学分析。到目前为止，二维（2-D）的页面显示，野生型和DRC突变体之间有38个蛋白质斑点在统计上显着变化。 MALDI-TOF MS分析将这些斑点确定为18个单独的蛋白质。其中包括六个已知的DRC成分（DRC1至DRC6），以及不同修饰的蛋白质，其中一种野生型鞭毛中至少有6种同工型。将来，我们计划扩大研究，以确定候选刚果民主共和国的组件未通过2-D页面解决。可能的途径是采用无标签的定量蛋白质组学方法，或者是基于标签的方法（例如ITRAQ），以将野生型和突变体鞭毛蛋白质组相关联。这项研究代表了DRC复合物的首次蛋白质组学分析，并具有鉴定新型DRC成分的潜力。这些发现可能会阐明纤毛和鞭毛运动调节的分子事件，并可能有助于鉴定生物标志物和治疗靶标，以治疗人类纤毛疾病。参考： [1]波特我，销售WS。（2000）9 + 2轴突锚定了多个内臂动力蛋白以及控制运动能力的激酶和磷酸酶网络。 J Cell Biol，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。 J Cell Biol，170：103-13。 [3] Smith EF，Yang P.（2004）径向辐条和中央设备：调节鞭毛运动的机械化学传感器。细胞Motil细胞骨架，57：8-17。 [4] Harris Eh。（2001）衣原体作为模型生物。 Annu Rev Plant Physiol Plant Mol Biol，52：363-406。 [5] Huang B，Ramanis Z，Luck DJ。（1982）衣原体中的抑制突变揭示了鞭毛功能的调节机制。牢房，28：115-24。 [6] Piperno G，Mead K，Ledizet M，Moscatelli A.（1994）“动力蛋白调节络合物”中的突变改变了衣原体轴突中内臂动力蛋白的ATP不敏感结合位点。 J Cell Biol，125：1109-17。 [7] Piperno G，Mead K，Shestak W.（1992）Chlamydomonas鞭毛中内部动力蛋白臂I2与“动力蛋白调节复合物”相互作用。 J Cell Biol，118：1455-63。 [8] Mastronarde DN，O'Toole ET，McDonald KL，McIntosh JR，Porter ME。（1992）在野生型和衣原体的突变鞭毛中内部动力蛋白臂的布置。 J Cell Biol，118：1145-62。 [9] Gardner LC，O'Toole E，Perrone CA，Giddings T，Porter ME。（1994）“动力蛋白调节络合物”的组件位于径向辐条和衣原体鞭毛中的动力蛋白臂之间的连接。 J Cell Biol，127：1311-25。 [10] Rupp G，波特我。（2003）衣原体中动力蛋白调节复合物的亚基是生长逮捕特异性基因产物的同源物。 J Cell Biol，162：47-57。