title: Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella.
abstract: Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo-electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named "radial spoke 3 stand-in," which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.
authors.affiliation: Biology Department, Rosenstiel Center, Brandeis University, Waltham, MA 02454, USA.
authors.forename: Cynthia F
authors.initials: CF
authors.lastname: Barber
authors.forename: Thomas
authors.initials: T
authors.lastname: Heuser
authors.forename: Blanca I
authors.initials: BI
authors.lastname: Carbajal-González
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Botchkarev
authors.forename: Daniela
authors.initials: D
authors.lastname: Nicastro
mesh_terms: D054468:Axoneme / Q000648:ultrastructure; D016825:Chlamydomonas reinhardtii / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D004398:Dyneins / Q000378:metabolism*; D055032:Electron Microscope Tomography; D005407:Flagella / Q000378:metabolism / Q000648:ultrastructure*; D008958:Models, Molecular; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding; D020836:Protein Structure, Quaternary
chemical_list: D010940:Plant Proteins; D004398:Dyneins
references.citation: Cell Motil Cytoskeleton. 2004 Jan;57(1):8-17
references.pmid: 14648553
references.citation: J Cell Biol. 2009 Dec 14;187(6):921-33
references.pmid: 20008568
references.citation: J Cell Biol. 2007 Nov 5;179(3):515-26
references.pmid: 17967944
references.citation: Proc Natl Acad Sci U S A. 1977 Aug;74(8):3456-60
references.pmid: 269405
references.citation: J Cell Biol. 1981 Jan;88(1):73-9
references.pmid: 6451632
references.citation: J Biol Chem. 2011 Aug 19;286(33):29175-29191
references.pmid: 21700706
references.citation: J Cell Biol. 1985 Jun;100(6):2008-18
references.pmid: 2860115
references.citation: Proc Natl Acad Sci U S A. 1971 Dec;68(12):3092-6
references.pmid: 5289252
references.citation: Science. 1992 Sep 11;257(5076):1557-9
references.pmid: 1387971
references.citation: J Cell Biol. 1992 Sep;118(5):1163-76
references.pmid: 1387404
references.citation: Nat Rev Mol Cell Biol. 2007 Nov;8(11):880-93
references.pmid: 17955020
references.citation: J Cell Biol. 1993 Oct;123(1):183-90
references.pmid: 8408197
references.citation: Methods Cell Biol. 2009;91:1-39
references.pmid: 20409778
references.citation: Exp Cell Res. 1972 Oct;74(2):471-9
references.pmid: 4628123
references.citation: J Cell Biol. 1978 Mar;76(3):729-47
references.pmid: 632325
references.citation: Proc Natl Acad Sci U S A. 2011 Jul 5;108(27):11105-8
references.pmid: 21690369
references.citation: N Engl J Med. 1979 Jan 11;300(2):53-6
references.pmid: 152870
references.citation: J Pathol. 2004 Nov;204(4):470-7
references.pmid: 15495266
references.citation: Mol Biol Cell. 2011 Jul 15;22(14):2520-31
references.pmid: 21613541
references.citation: J Cell Biol. 1968 Oct;39(1):77-94
references.pmid: 5678451
references.citation: J Comput Chem. 2004 Oct;25(13):1605-12
references.pmid: 15264254
references.citation: J Cell Sci. 2000 Jan;113 ( Pt 1):91-102
references.pmid: 10591628
references.citation: Proc Natl Acad Sci U S A. 1977 Apr;74(4):1600-4
references.pmid: 266200
references.citation: Proc Natl Acad Sci U S A. 1965 Dec;54(6):1665-9
references.pmid: 4379719
references.citation: J Cell Biol. 2001 Jun 11;153(6):1315-26
references.pmid: 11402073
references.citation: J Cell Biol. 1981 Jan;88(1):80-8
references.pmid: 7204490
references.citation: J Cell Biol. 1970 Oct;47(1):159-82
references.pmid: 4935335
references.citation: Cytoskeleton (Hoboken). 2011 Jul;68(7):389-400
references.pmid: 21692193
references.citation: J Cell Biol. 1997 Jan 13;136(1):167-76
references.pmid: 9008711
references.citation: Cell. 2009 Feb 6;136(3):485-95
references.pmid: 19203583
references.citation: J Cell Sci. 2006 Mar 15;119(Pt 6):1165-74
references.pmid: 16507594
references.citation: Cell Motil Cytoskeleton. 2007 Jul;64(7):489-95
references.pmid: 17366641
references.citation: J Morphol. 1977 Jul;153(1):143-51
references.pmid: 408499
references.citation: Cell Motil Cytoskeleton. 1993;24(4):224-32
references.pmid: 8477455
references.citation: J Ultrastruct Res. 1977 Oct;61(1):21-43
references.pmid: 915974
references.citation: J Cell Biol. 2003 Jul 7;162(1):47-57
references.pmid: 12847082
references.citation: Int Rev Cytol. 2002;219:115-55
references.pmid: 12211628
references.citation: J Biol Chem. 2000 Jun 23;275(25):18905-12
references.pmid: 10858448
references.citation: Science. 2006 Aug 18;313(5789):944-8
references.pmid: 16917055
references.citation: Acta Crystallogr D Biol Crystallogr. 2000 Jul;56(Pt 7):791-4
references.pmid: 10930825
references.citation: J Cell Biol. 2008 Dec 1;183(5):923-32
references.pmid: 19029338
references.citation: J Cell Biol. 1974 Oct;63(1):35-63
references.pmid: 4424314
references.citation: J Struct Biol. 2005 Oct;152(1):36-51
references.pmid: 16182563
references.citation: Exp Cell Res. 1974 Mar 15;84(1):374-80
references.pmid: 4594106
references.citation: J Cell Biol. 2000 Nov 27;151(5):F37-42
references.pmid: 11086017
references.citation: J Cell Biol. 1998 May 18;141(4):979-92
references.pmid: 9585416
references.citation: Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):15889-94
references.pmid: 16246999
references.citation: Cell. 1982 Jan;28(1):115-24
references.pmid: 6461414
references.citation: Proc Natl Acad Sci U S A. 1977 May;74(5):2045-9
references.pmid: 266725
references.citation: Cytoskeleton (Hoboken). 2011 Apr;68(4):237-46
references.pmid: 21391306
references.citation: J Cell Biol. 1972 Sep;54(3):507-39
references.pmid: 4558009
references.citation: J Muscle Res Cell Motil. 1981 Mar;2(1):83-130
references.pmid: 7240408
references.citation: Mol Biol Cell. 2004 Aug;15(8):3891-902
references.pmid: 15194815
references.citation: J Cell Biol. 1994 Dec;127(6 Pt 1):1683-92
references.pmid: 7798320
references.citation: Cytoskeleton (Hoboken). 2011 Jul;68(7):363-72
references.pmid: 21692192
references.citation: J Struct Biol. 1996 Jan-Feb;116(1):71-6
references.pmid: 8742726