COMPONENTS OF THE DYNEIN REGULATORY COMPLEX IN CHLAMYDOMONAS FLAGELLA

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。 我们正在使用蛋白质组学分析来鉴定之前描述的鞭毛衣藻动力蛋白调节复合体的新成分。 纤毛和鞭毛是广泛存在的细胞器，在整个进化过程中高度保守，在从原生动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]。纤毛和鞭毛的振荡跳动高度协调，需要精确的调节[3]。然而，这一调控的详细结构和分子基础仍有待阐明。单细胞藻衣藻属是一种成熟的模式生物，拥有大量可用的突变体，包括那些影响鞭毛运动性的突变体，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]。先前对衣藻突变体抑制放射状辐条突变体的“瘫痪鞭毛”表型的研究发现，动力蛋白调节复合体(DRC)是动力蛋白活性调节系统中的关键角色，因此鞭毛运动[5-7]。七个轴丝多肽被生化鉴定为DRC组分，它们形成了一个表观分子量至少为500 kDa的大型复合体[5，6]。利用野生型和DRC突变体的电子显微镜，DRC结构被粗略地映射到放射状RS2[8，9]底部附近的新月形区域。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成分，包括它们与Nexin链接(手稿正在准备中)的空间关系。此外，还观察到了以前没有表征的其他DRC密度。 然而，到目前为止，只有由PF2基因编码的蛋白质DRC4在分子水平上被表征。为了确定其余6个DRC组分和未知亚基的基因，我们对衣藻野生型和突变型鞭毛中分离的轴丝进行了全面的蛋白质组学分析。到目前为止，二维(2D)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的显著变化。MALDI-TOF MS分析确定这些斑点为18个单独的蛋白质；其中包括6个已知的DRC组分(DRC1至DRC6)，以及不同修饰的蛋白质，包括在野生型鞭毛中存在的至少6个异构体的蛋白质。 在未来，我们计划扩大我们的研究范围，以确定2D PAGE未解析的候选DRC组件。可能的途径是采用无标记的定量蛋白质组方法，或者采用基于标记的方法，如iTRAQ，来关联野生型和突变型鞭毛蛋白质组。这项研究是首次对DRC复合体进行蛋白质组学分析，并有可能识别新的DRC组分。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]搬运工我，销售WS。(2000)的92轴丝锚定多个内臂动力蛋白和一个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，Agin N，Leszyk J，Witman GB。(2005)真核纤毛的蛋白质组学分析。J细胞生物学，170：103-13。 [3]史密斯·艾夫，杨鹏(2004)径向辐条与中枢器官：调节鞭毛运动的机械力化学换能器。细胞运动细胞骨架，57：8-17。 [4]Harris EH.(2001)衣藻作为模式生物。植物生理学年鉴，52：363-406. [5]黄斌，拉曼尼斯·Z，幸运DJ。(1982)衣藻中的抑制子突变揭示了鞭毛功能的调节机制。电话号码：28：115-24。 [6]Piperno G，Mead K，LeDizet M，Moscatelli A(1994)在“动力蛋白调节复合体”中的突变改变了轴突衣藻内臂动力蛋白的ATP不敏感结合部位。J细胞生物学，125：1109-17。 [7]Piperno G，Mead K，Shestak W.(1992)鞭毛衣藻内动力蛋白臂I2与动力蛋白调控复合体相互作用。J细胞生物学，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。J细胞生物学，118：1145-62。 [9]Gardner LC，O‘Toole E，Perrone CA，Gidding T，Porter ME。(1994)“动力蛋白调控复合体”的组件位于衣藻鞭毛的径向辐条和动力蛋白臂之间的连接处。J细胞生物学，127：1311-25。 [10]Rupp G，Porter ME。(2003)衣藻中动力蛋白调节复合体的一个亚单位是生长停滞特异性基因产物的同源物。J细胞生物学，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.