New mutations in flagellar motors identified by whole genome sequencing in Chlamydomonas

Cilia

Susan K Dutcher¹  Silas Hsu¹  Alison J Albee¹  Nicholas P Nauman¹  Huawen Lin¹ 

[1] Department of Genetics, Washington University, 660 South Euclid Avenue, St Louis, MO 63110, USA

关键词: Whole genome sequencing;    IFT recycling;    Ciliary assembly;    IFT81;    Cytoplasmic dynein;    Kinesin-2;   

Others  :  1092717
DOI  :  10.1186/2046-2530-2-14

 received in 2013-04-04, accepted in 2013-10-02,  发布年份 2013

【 摘 要 】

Background

The building of a cilium or flagellum requires molecular motors and associated proteins that allow the relocation of proteins from the cell body to the distal end and the return of proteins to the cell body in a process termed intraflagellar transport (IFT). IFT trains are carried out by kinesin and back to the cell body by dynein.

Methods

We used whole genome sequencing to identify the causative mutations for two temperature-sensitive flagellar assembly mutants in Chlamydomonas and validated the changes using reversion analysis. We examined the effect of these mutations on the localization of IFT81, an IFT complex B protein, the cytoplasmic dynein heavy chain (DHC1b), and the dynein light intermediate chain (D1bLIC).

Results

The strains, fla18 and fla24, have mutations in kinesin-2 and cytoplasmic dynein, respectively. The fla18 mutation alters the same glutamic acid (E₂₄G) mutated in the fla10-14 allele (E₂₄K). The fla18 strain loses flagella at 32?C more rapidly than the E₂₄K allele but less rapidly than the fla10-1 allele. The fla18 mutant loses its flagella by detachment rather than by shortening. The fla24 mutation falls in cytoplasmic dynein and changes a completely conserved amino acid (L₃₂₄₃P) in an alpha helix in the AAA5 domain. The fla24 mutant loses its flagella by shortening within 6 hours at 32?C. DHC1b protein is reduced by 18-fold and D1bLIC is reduced by 16-fold at 21?C compared to wild-type cells. We identified two pseudorevertants (L₃₂₄₃S and L₃₂₄₃R), which remain flagellated at 32?C. Although fla24 cells assemble full-length flagella at 21?C, IFT81 protein localization is dramatically altered. Instead of localizing at the basal body and along the flagella, IFT81 is concentrated at the proximal end of the flagella. The pseudorevertants show wild-type IFT81 localization at 21?C, but proximal end localization of IFT81 at 32?C.

Conclusions

The change in the AAA5 domain of the cytoplasmic dynein in fla24 may block the recycling of IFT trains after retrograde transport. It is clear that different alleles in the flagellar motors reveal different functions and roles. Multiple alleles will be important for understanding structure-function relationships.

【 授权许可】

   
2013 Lin et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Kozminski KG, Johnson KA, Forscher P, Rosenbaum JL: A motility in the eukaryotic flagellum unrelated to flagellar beating. Proc Natl Acad Sci U S A 1993, 90:5519-5523.
• [2]Cole DG, Chinn SW, Wedaman KP, Hall K, Vuong T, Scholey JM: Novel heterotrimeric kinesin-related protein purified from sea urchin eggs. Nature 1993, 366:268-270.
• [3]Walther Z, Vashishtha M, Hall JL: The Chlamydomonas FLA10 gene encodes a novel kinesin-homologous protein. J Cell Biol 1994, 126:175-188.
• [4]Kozminski KG, Beech PL, Rosenbaum JL: The Chlamydomonas kinesin-like protein FLA10 is involved in motility associated with the flagellar membrane. J Cell Biol 1995, 131:1517-1527.
• [5]Cole DG, Diener DR, Himelblau AL, Beech PL, Fuster JC, Rosenbaum JL: Chlamydomonas kinesin-II-dependent intraflagellar transport (IFT): IFT particles contain proteins required for ciliary assembly in Caenorhabditis elegans sensory neurons. J Cell Biol 1998, 141:993-1008.
• [6]Piperno G, Mead K: Transport of a novel complex in the cytoplasmic matrix of Chlamydomonas flagella. Proc Natl Acad Sci U S A 1997, 94:4457-4462.
• [7]Mueller J, Perrone CA, Bower R, Cole DG, Porter ME: The FLA3 KAP subunit is required for localization of kinesin-2 to the site of flagellar assembly and processive anterograde intraflagellar transport. Mol Biol Cell 2005, 16:1341-1354.
• [8]Miller MS, Esparza JM, Lippa AM, Lux FG 3rd, Cole DG, Dutcher SK: Mutant kinesin-2 motor subunits increase chromosome loss. Mol Biol Cell 2005, 16:3810-3820.
• [9]Porter ME, Bower R, Knott JA, Byrd P, Dentler W: Cytoplasmic dynein heavy chain 1b is required for flagellar assembly in Chlamydomonas. Mol Biol Cell 1999, 10:693-712.
• [10]Pazour GJ, Dickert BL, Witman GB: The DHC1b (DHC2) isoform of cytoplasmic dynein is required for flagellar assembly. J Cell Biol 1999, 144:473-481.
• [11]Iomini C, Babaev-Khaimov V, Sassaroli M, Piperno G: Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases. J Cell Biol 2001, 153:13-24.
• [12]Pigino G, Geimer S, Lanzavecchia S, Paccagnini E, Cantele F, Diener DR, Rosenbaum JL, Lupetti P: Electron-tomographic analysis of intraflagellar transport particle trains in situ. J Cell Biol 2009, 187:135-148.
• [13]Wei Q, Zhang Y, Li Y, Zhang Q, Ling K, Hu J: The BBSome controls IFT assembly and turnaround in cilia. Nat Cell Biol 2012, 14:950-957.
• [14]Nachury MV, Loktev AV, Zhang Q, Westlake CJ, Peranen J, Merdes A, Slusarski DC, Scheller RH, Bazan JF, Sheffield VC, Jackson PK: A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 2007, 129:1201-1213.
• [15]Berbari NF, Johnson AD, Lewis JS, Askwith CC, Mykytyn K: Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors. Mol Biol Cell 2008, 19:1540-1547.
• [16]Berbari NF, Lewis JS, Bishop GA, Askwith CC, Mykytyn K: Bardet-Biedl syndrome proteins are required for the localization of G protein-coupled receptors to primary cilia. Proc Natl Acad Sci U S A 2008, 105:4242-4246.
• [17]Lechtreck KF, Brown JM, Sampaio JL, Craft JM, Shevchenko A, Evans JE, Witman GB: Cycling of the signaling protein phospholipase D through cilia requires the BBSome only for the export phase. J Cell Biol 2013, 201:249-261.
• [18]Ahmed NT, Gao C, Lucker BF, Cole DG, Mitchell DR: ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery. J Cell Biol 2008, 183:313-322.
• [19]Eggenschwiler JT, Anderson KV: Cilia and developmental signaling. Annu Rev Cell Dev Bio 2007, 23:345-373.
• [20]Schmidts M, Arts HH, Bongers EM, Yap Z, Oud MM, Antony D, Duijkers L, Emes RD, Stalker J, Yntema JB, Plagnol V, Hoischen A, Gilissen C, Forsythe E, Lausch E, Veltman JA, Roeleveld N, Superti-Furga A, Kutkowska-Kazmierczak A, Kamsteeg EJ, El?io?lu N, van Maarle MC, Graul-Neumann LM, Devriendt K, Smithson SF, Wellesley D, Verbeek NE, Hennekam RC, Kayserili H, Scambler PJ: Exome sequencing identifies DYNC2H1 mutations as a common cause of asphyxiating thoracic dystrophy (Jeune syndrome) without major polydactyly, renal or retinal involvement. J Med Genet 2013, 50:309-323.
• [21]Beales PL, Bland E, Tobin JL, Bacchelli C, Tuysuz B, Hill J, Rix S, Pearson CG, Kai M, Hartley J, Johnson C, Irving M, Elcioglu N, Winey M, Tada M, Scambler PJ: IFT80, which encodes a conserved intraflagellar transport protein, is mutated in Jeune asphyxiating thoracic dystrophy. Nature Genet 2007, 39:727-729.
• [22]Bredrup C, Saunier S, Oud MM, Fiskerstrand T, Hoischen A, Brackman D, Leh SM, Midtbo M, Filhol E, Bole-Feysot C, Nitschk? P, Gilissen C, Haugen OH, Sanders JS, Stolte-Dijkstra I, Mans DA, Steenbergen EJ, Hamel BC, Matignon M, Pfundt R, Jeanpierre C, Boman H, R?dahl E, Veltman JA, Knappskog PM, Knoers NV, Roepman R, Arts HH: Ciliopathies with skeletal anomalies and renal insufficiency due to mutations in the IFT-A gene WDR19. Am J Hum Genet 2011, 89:634-643.
• [23]Perrault I, Saunier S, Hanein S, Filhol E, Bizet AA, Collins F, Salih MA, Gerber S, Delphin N, Bigot K, Orssaud C, Silva E, Baudouin V, Oud MM, Shannon N, Le Merrer M, Roche O, Pietrement C, Goumid J, Baumann C, Bole-Feysot C, Nitschke P, Zahrate M, Beales P, Arts HH, Munnich A, Kaplan J, Antignac C, Cormier-Daire V, Rozet JM: Mainzer-Saldino syndrome is a ciliopathy caused by IFT140 mutations. Am J Hum Genet 2012, 90:864-870.
• [24]Huang B, Rifkin MR, Luck DJ: Temperature-sensitive mutations affecting flagellar assembly and function in Chlamydomonas reinhardtii. J Cell Biol 1977, 72:67-85.
• [25]Adams GM, Huang B, Luck DJ: Temperature-sensitive, assembly-defective flagella mutants of Chlamydomonas reinhardtii. Genetics 1982, 100:579-586.
• [26]Pedersen LB, Miller MS, Geimer S, Leitch JM, Rosenbaum JL, Cole DG: Chlamydomonas IFT172 is encoded by FLA11, interacts with CrEB1, and regulates IFT at the flagellar tip. Curr Biol 2005, 15:262-266.
• [27]Engel BD, Ishikawa H, Wemmer KA, Geimer S, Wakabayashi K, Hirono M, Craige B, Pazour GJ, Witman GB, Kamiya R, Marshall WF: The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function. J Cell Biol 2012, 199:151-167.
• [28]Parker JD, Quarmby LM: Chlamydomonas fla mutants reveal a link between deflagellation and intraflagellar transport. BMC Cell Biol 2003, 4:11. BioMed Central Full Text
• [29]Ramanis Z, Luck DJ: Loci affecting flagellar assembly and function map to an unusual linkage group in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 1986, 83:423-426.
• [30]Dutcher SK, Li L, Lin H, Meyer L, Giddings TH Jr, Kwan AL, Lewis BL: Whole-genome sequencing to identify mutants and polymorphisms in Chlamydomonas reinhardtii. 2012, G3 2:15-22.
• [31]Iomini C, Li L, Esparza JM, Dutcher SK: Retrograde intraflagellar transport mutants identify complex a proteins with multiple genetic interactions in Chlamydomonas reinhardtii. Genetics 2009, 183:885-896.
• [32]Witman GB: Dynein and Intraflagellar Transport. In Dyneins: Structure, Biology and Disease. Edited by King SM. London: Elsevier; 2012.
• [33]Matsuura K, Lefebvre PA, Kamiya R, Hirono M: Kinesin-II is not essential for mitosis and cell growth in Chlamydomonas. Cell Motil Cytoskeleton 2002, 52:195-201.
• [34]Pazour GJ, Wilkerson CG, Witman GB: A dynein light chain is essential for the retrograde particle movement of intraflagellar transport (IFT). J Cell Biol 1998, 141:979-992.
• [35]Hou Y, Qin H, Follit JA, Pazour GJ, Rosenbaum JL, Witman GB: Functional analysis of an individual IFT protein: IFT46 is required for transport of outer dynein arms into flagella. J Cell Biol 2007, 176:653-665.
• [36]Lucker BF, Miller MS, Dziedzic SA, Blackmarr PT, Cole DG: Direct interactions of intraflagellar transport complex B proteins IFT88, IFT52, and IFT46. J Biol Chem 2010, 285:21508-21518.
• [37]Brazelton WJ, Amundsen CD, Silflow CD, Lefebvre PA: The bld1 mutation identifies the Chlamydomonas osm-6 homolog as a gene required for flagellar assembly. Curr Biol 2001, 11:1591-1594.
• [38]Deane JA, Cole DG, Seeley ES, Diener DR, Rosenbaum JL: Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles. Curr Biol 2001, 11:1586-1590.
• [39]Lucker BF, Behal RH, Qin H, Siron LC, Taggart WD, Rosenbaum JL, Cole DG: Characterization of the intraflagellar transport complex B core: direct interaction of the IFT81 and IFT74/72 subunits. J Biol Chem 2005, 280:27688-27696.
• [40]Pazour GJ, Baker SA, Deane JA, Cole DG, Dickert BL, Rosenbaum JL, Witman GB, Besharse JC: The intraflagellar transport protein, IFT88, is essential for vertebrate photoreceptor assembly and maintenance. J Cell Biol 2002, 157:103-113.
• [41]Behal RH, Miller MS, Qin H, Lucker BF, Jones A, Cole DG: Subunit interactions and organization of the Chlamydomonas reinhardtii intraflagellar transport complex A proteins. J Biol Chem 2012, 287:11689-11703.
• [42]Hou Y, Pazour GJ, Witman GB: A dynein light intermediate chain, D1bLIC, is required for retrograde intraflagellar transport. Mol Biol Cell 2004, 15:4382-4394.
• [43]Qin H, Wang Z, Diener D, Rosenbaum J: Intraflagellar transport protein 27 is a small G protein involved in cell-cycle control. Curr Biol 2007, 17:193-202.
• [44]Fan ZC, Behal RH, Geimer S, Wang Z, Williamson SM, Zhang H, Cole DG, Qin H: Chlamydomonas IFT70/CrDYF-1 is a core component of IFT particle complex B and is required for flagellar assembly. Mol Biol Cell 2010, 21:2696-2706.
• [45]Mitchell DR, Kang Y: Identification of oda6 as a Chlamydomonas dynein mutant by rescue with the wild-type gene. J Cell Biol 1991, 113:835-842.
• [46]LeDizet M, Piperno G: Ida, 4?1, ida4?2, and ida4?3 are intron splicing mutations affecting the locus encoding p28, a light chain of Chlamydomonas axonemal inner dynein arms. Mol Biol Cell 1995, 6:713-723.
• [47]Bhogaraju S, Cajanek L, Fort C, Blisnick T, Weber K, Taschner M, Mizuno N, Lamla S, Bastin P, Nigg EA, Lorentzen E: Molecular basis of tubulin transport within the cilium by IFT74 and IFT81. Science 2013, 341:1009-1012.
• [48]Neff MM, Turk E, Kalishman M: Web-based primer design for single nucleotide polymorphism analysis. Trends Genet 2002, 18:613-615.
• [49]Piperno G, Huang B, Luck DJ: Two-dimensional analysis of flagellar proteins from wild-type and paralyzed mutants of Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A 1977, 74:1600-1604.
• [50]Rupp G, O?Toole E, Gardner LC, Mitchell BF, Porter ME: The sup-pf-2 mutations of Chlamydomonas alter the activity of the outer dynein arms by modification of the gamma-dynein heavy chain. J Cell Biol 1996, 135:1853-1865.
• [51]Dutcher SK, Morrissette NS, Preble AM, Rackley C, Stanga J: Epsilon-tubulin is an essential component of the centriole. Mol Biol Cell 2002, 13:3859-3869.
• [52]Dutcher SK, Huang B, Luck DJ: Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii. J Cell Biol 1984, 98:229-236.
• [53]Lin H, Miller M, Granas D, Dutcher S: Whole genome sequencing reveals distinct functions of a protein phosphatase 2A in Chlamydomonas mating. PLoS Genet 2013, 9:e1003841.
• [54]Albee AJ, Kwan AL, Lin H, Granas D, Stormo GD, Dutcher SK: Identification of cilia genes that affect cell-cycle progression using whole-genome transcriptome analysis in Chlamydomonas reinhardtti. 2013, G3 3:979-991.
• [55]Firestone AJ, Weinger JS, Maldonado M, Barlan K, Langston LD, O?Donnell M, Gelfand VI, Kapoor TM, Chen JK: Small-molecule inhibitors of the AAA+ ATPase motor cytoplasmic dynein. Nature 2012, 484:125-129.
• [56]Shih SM, Engel BD, Kocabas F, Bilyard T, Gennerich A, Marshall WF, Yildiz A: Intraflagellar transport drives flagellar surface motility. Elife 2013, 2:e00744.
• [57]Kon T, Oyama T, Shimo-Kon R, Imamula K, Shima T, Sutoh K, Kurisu G: The 2.8 A crystal structure of the dynein motor domain. Nature 2012, 484:345-350.
• [58]Schmidt H, Gleave ES, Carter AP: Insights into dynein motor domain function from a 3.3-A crystal structure. Nat Struct Mol Biol 2012, 19:492-497.
• [59]Crooks GE, Hon G, Chandonia JM, Brenner SE: WebLogo: a sequence logo generator. Genome Res 2004, 14:1188-1190.
• [60]Sivagurunathan S, Schnittker RR, Razafsky DS, Nandini S, Plamann MD, King SJ: Analyses of dynein heavy chain mutations reveal complex interactions between dynein motor domains and cellular dynein functions. Genetics 2012, 191:1157-1179.