title: Building blocks of the nexin-dynein regulatory complex in Chlamydomonas flagella.
abstract: The directional flow generated by motile cilia and flagella is critical for many processes, including human development and organ function. Normal beating requires the control and coordination of thousands of dynein motors, and the nexin-dynein regulatory complex (N-DRC) has been identified as an important regulatory node for orchestrating dynein activity. The nexin link appears to be critical for the transformation of dynein-driven, linear microtubule sliding to flagellar bending, yet the molecular composition and mechanism of the N-DRC remain largely unknown. Here, we used proteomics with special attention to protein phosphorylation to analyze the composition of the N-DRC and to determine which subunits may be important for signal transduction. Two-dimensional electrophoresis and MALDI-TOF mass spectrometry of WT and mutant flagellar axonemes from Chlamydomonas identified 12 N-DRC-associated proteins, including all seven previously observed N-DRC components. Sequence and PCR analyses identified the mutation responsible for the phenotype of the sup-pf-4 strain, and biochemical comparison with a radial spoke mutant revealed two components that may link the N-DRC and the radial spokes. Phosphoproteomics revealed eight proteins with phosphorylated isoforms for which the isoform patterns changed with the genotype as well as two components that may play pivotal roles in N-DRC function through their phosphorylation status. These data were assembled into a model of the N-DRC that explains aspects of its regulatory function.
authors.affiliation: Biology Department, Rosenstiel Center, MS029, Brandeis University, Waltham, Massachusetts 02454.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Lin
authors.affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, and.
authors.forename: Douglas
authors.initials: D
authors.lastname: Tritschler
authors.affiliation: Biology Department, Rosenstiel Center, MS029, Brandeis University, Waltham, Massachusetts 02454.
authors.forename: Kangkang
authors.initials: K
authors.lastname: Song
authors.affiliation: Biology Department, Rosenstiel Center, MS029, Brandeis University, Waltham, Massachusetts 02454.
authors.forename: Cynthia F
authors.initials: CF
authors.lastname: Barber
authors.affiliation: Chemistry Department, MS015, Brandeis University, Waltham, Massachusetts 02454.
authors.forename: Jennifer S
authors.initials: JS
authors.lastname: Cobb
authors.affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, Minnesota 55455, and.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Porter
authors.affiliation: Biology Department, Rosenstiel Center, MS029, Brandeis University, Waltham, Massachusetts 02454,. Electronic address: nicastro@brandeis.edu.
authors.forename: Daniela
authors.initials: D
authors.lastname: Nicastro
mesh_terms: D054468:Axoneme / Q000235:genetics / Q000378:metabolism*; D016825:Chlamydomonas reinhardtii / Q000235:genetics / Q000378:metabolism*; D004398:Dyneins / Q000235:genetics / Q000378:metabolism*; D005407:Flagella / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation / Q000502:physiology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods
chemical_list: D008869:Microtubule-Associated Proteins; D010940:Plant Proteins; C484652:microtubule nexin; D004398:Dyneins