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. The postdoctoral fellow has visited for instruction in MS methods. Additional samples are now being prepared. 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.
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