Published by the International Society of Protistologists
Journal of Eukaryotic Microbiology ISSN 1066-5234
SHORT COMMUNICATION
Evolution of the Dynein Heavy Chain Family in Ciliates Vidyalakshmi Rajagopalan & David E. Wilkes Department of Biological Sciences, Indiana University South Bend, South Bend, Indiana 46634
Keywords Ichthyophthirius; Oxytricha; Paramecium; Tetrahymena. Correspondence D. Wilkes, Department of Biological Sciences, Indiana University South Bend, 1700 Mishawaka Avenue, P.O. Box 7111, South Bend, Indiana 46634, USA Telephone number: +1 574-520-4411; FAX number: +1 574-520-4482; e-mail: [email protected]
ABSTRACT Dynein heavy chains are motor proteins that comprise a large gene family found across eukaryotes. We have investigated this gene family in four ciliate species: Ichthyophthirius, Oxytricha, Paramecium, and Tetrahymena. Ciliates appear to encode more dynein heavy chain genes than most eukaryotes. Phylogenetic comparisons demonstrated that the last common ancestor of the ciliates that were examined expressed at least 14 types of dynein heavy chains with most of the expansion coming from the single-headed inner arm dyneins. Each of the dyneins most likely performed different functions within the cell.
Received: 21 January 2015; revised 11 June 2015; accepted June 11, 2015. doi:10.1111/jeu.12245
DYNEINS are microtubule-based motor complexes that perform a variety of functions in eukaryotic cells (Porter 1996; Vale 2003). They comprise a family well conserved across eukaryotic species. The family can be divided into two broad classes: (1) axonemal dyneins and (2) nonaxonemal, or cytoplasmic, dyneins. Axonemal dyneins are the molecular motors that power the movement of cilia and eukaryotic flagella and they can be further divided into outer arms and inner arms. Identical outer arms are found every 24 nm along the length of an axoneme and are required for establishing the high beat frequencies of flagella (Brokaw and Kamiya 1987; Gibbons and Gibbons 1973, 1976). Inner arm dyneins exist in many forms as opposed to the single type of outer arm dynein. Along the length of an axoneme one- and two-headed inner arm dyneins are organized in 96 nm repeating units in which each unit contains one each of an I1, I2, and I3 sub-region (Dutcher 1995). The I1 region contains a two-headed dynein while the I2 and I3 regions each have two singleheaded dyneins. The inner arms are involved in regulating the amplitude of axonemal bending (Brokaw and Kamiya 1987). Two groups of nonaxonemal or cytoplasmic dyneins exist. Cytoplasmic dynein-1 is found in all eukaryotes except higher plants and is the only dynein present in species without cilia or flagella. It is important for mitotic movements and for the organization and trafficking of
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intracellular membranes (Pfister et al. 2006; Vale 2003). Cytoplasmic dynein-2 is found only in organisms that have cilia or eukaryotic flagella and is the motor for retrograde intraflagellar transport (Blacque et al. 2008; Rosenbaum and Witman 2002; Scholey 2003). Each of the dynein complexes is made of a core of one or more heavy chains (DYHs) associated with a set of smaller subunits. The DYHs form the heads of the dynein complexes and contain both the microtubule-binding site 2004; and the site of ATP hydrolysis (Koonce and Samso Mocz and Gibbons 2001; Neuwald et al. 1999). Organisms with motile cilia or flagella express multiple DYH genes (Andrews et al. 1996; Asai et al. 1994; Gibbons et al. 1994; Porter et al. 1996; Rasmussen et al. 1994; Tanaka et al. 1995). Comparisons of the DYH motor domain sequences have shown that the sequences correspond to functional classes (Asai 1995; Gibbons 1995). The family evolved early with at least nine different DYHs present in the last eukaryotic common ancestor (Wickstead and Gull 2007; Wilkes et al. 2008). The cytoplasmic dynein 1 and 2 heavy chains form homodimers of their respective complexes. The outer arm a and b/c heavy chains assemble into the heterodimeric or heterotrimeric axonemal outer arm dynein complex. The inner arm 1a and 1b heavy chains form the heterodimeric I1 inner arm dynein. The other three groups of heavy chains are thought to
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2016, 63, 138–141
Dynein Evolution in Ciliates
Rajagopalan & Wilkes
comprise the single-headed inner arm dyneins (sh-IAD). Many eukaryotic genomes contain more than one DYH gene per ancestral group and thus have more than nine total DYH genes. The Chlamydomonas, sea urchin, and human genomes have 14, 15, and 16 DYH genes, respectively. Previous examination of the genomes of two ciliates found more DYH genes than typical. The Tetrahymena and Ichthyophthirius genomes contain 25 and 19 DYH genes, respectively (Coyne et al. 2011; Wilkes et al. 2008). We have extended the analysis of ciliate DYH genes to the recently available Paramecium and Oxytricha genomes. Our results support the idea that ciliates do have more DYHs than most organisms and that some of the expansion occurred before speciation of the ciliates. Of particular interest, the last common ancestor of the examined ciliates appears to have had seven distinct DYH subgroups of the single-headed inner arms as opposed to only three for the last eukaryotic common ancestor. MATERIALS AND METHODS Gene sequences The dynein heavy chain sequences from Tetrahymena thermophila and Ichthyophthirius multifiliis have been published previously (Coyne et al. 2011; Wilkes et al. 2008). Paramecium sequences were obtained by searching the ParameciumDB (http://paramecium.cgm.cnrs-gif.fr/; Arnaiz and Sperling 2011; Arnaiz et al. 2007) and Oxytricha sequences were obtained from the OxyDB (http://oxy.ciliate.org/index.php/home/welcome/). The conserved region of Tetrahymena DYH4 motor domain from the first P loop to the sequence coding for the conserved amino acid sequence PATVSR (residues 2307-2312 of DYH4) was used as the query sequence to BLAST (Altschul et al. 1997) each genome. Phylogenetic analyses The DYH motor domain sequences of the four ciliate species were compared by Clustal Omega alignment (www.ebi.ac.uk/Tools/msa/clustalo/). The phylogenetic analysis was conducted on 84 amino acid sequences. All positions containing gaps and missing data were eliminated, and a total of 169 positions were retained in the final dataset. The phylogeny was inferred using the Maximum Likelihood method implemented in MEGA5 (Tamura et al. 2011). The JTT matrix (Jones et al. 1992) was used, with evolutionary rate differences among sites modelled using a discrete approximation of a Gamma distribution ([+G], 5 categories; alpha parameter = 0.6668) plus an invariable sites category ([+I], 5.0365% of sites). Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was < 100 or less than one-fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. A 500-pseudoreplicate bootstrap analysis was performed to assess robustness.
TetDYH8 Ich2313.m000021 ParaGSPATP00025924001 96 ParaGSPATP00033121001 Ich1999.m000059 Ich2733.m000052 IAD group 3A TetDYH11 ParaGSPATP00019919001 ParaGSPATP00001961001 91 94 ParaGSPATP00020187001 82 ParaGSPATP00005122001 ParaGSPATP00001980001 OxyContig17454 0 g50 OxyContig9415 0 1 g18 ParaGSPATP00004069001 99 IAD group 3B TetDYH17 93 TetDYH14 86 Ich2913.m000242 99 TetDYH12 Ich2300.m000062 IAD group 3C 81 ParaGSPATP00012521001 OxyContig10418 0 g55 94 TetDYH10 ParaGSPATP00007189001 94 IAD group 3D TetDYH25 TetDYH13 Ich2443.m000020 Ich2883.m000117 OxyContig16305 0 g10 OxyContig15709 0 g85 85 ParaGSPATP00008239001 ParaPTETP6000001001 ParaGSPATP00008111001 TetDYH9 IAD group 4 94 Ich2895.m000270 TetDYH19 TetDYH20 83 TetDYH16 Ich2630.m000021 TetDYH21 Ich2678.m000182 TetDYH15 79 IAD group 5A OxyContig14020 0 g49 ParaPTETP1000023001 Ich2625.m000030 95 Ich2484.m000119 TetDYH24 ParaPTETP8700005001 75 ParaGSPATP00009095001 99 TetDYH18 OxyContig2564 0 g105 IAD group 5B OxyContig21237 0 g67 92 TetDYH23 ParaGSPATP00018717001 TetDYH22 Ich2507.m000068 OxyContig16067 0 g66 OxyContig335 1 g130 92 TetDYH7 98 IAD 1beta Ich2751.m000110 ParaGSPATP00038674001 TetDYH5 92 Ich2880.m000134 OAD gamma 100 ParaGSPATP00038423001 87 OxyContig19406 0 0 g75 TetDYH4 OAD beta ParaPTETP5700005001 97 OxyContig44 1 g4 TetDYH3 Ich2946.m000074 100 OAD alpha ParaPTETP300004001 OxyContig1360 1 g26 94 TetDYH6 78 Ich1778.m000066 IAD 1alpha 87 ParaGSPATP00015638001 OxyContig19661 0 g109 TetDYH1 94 Ich1655.m000083 Cyto 1 99 ParaGSPATP00002017001 OxyContig11184 0 g31 OxyContig11800 0 g24 95 TetDYH2 100 Cyto 2 Ich1642.m000052 77
83 76
ParaPTETP9700007001
0.2
Figure 1 Phylogenetic analysis of the ca. 400 residues conserved catalytic domain of the dynein heavy chains revealed that ciliate genomes examined contain at least 14 distinct types of dynein heavy chains. The original maximum likelihood tree is shown with bootstrap values > 75% (500 iterations) at branch points. There were a total of 169 positions in the final dataset. Cyto 1 = cytoplasmic dynein 1; Cyto 2 = cytoplasmic dynein 2; OAD = axonemal outer arm dynein; IAD = axonemal inner arm dynein. Ich = Ichthyophthirius; Oxy = Oxytricha; Para = Paramecium; Tet = Tetrahymena. Scale = 0.2 substitutions/site.
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2016, 63, 138–141
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CONCLUSIONS
RESULTS AND DISCUSSION Ciliate genomes have larger dynein heavy chain families than most eukaryotes Previously, we have shown that the ciliates, Tetrahymena and Ichthyophthirius, have 25 and 19 DYH genes, respectively (Coyne et al. 2011; Wilkes et al. 2008). Our searches found 50 DYH genes in Paramecium and at least 15 in Oxytricha. Oxytricha may encode as many as 25 DYH genes however many are unclear due to the quality and incomplete annotation of the genome. For example, Oxytricha Contig14590.0.g69 may be the IAD 1b that appears to be missing in our analysis. The number of Paramecium DYH genes is much higher than typical because of a recent whole genome duplication (Aury et al. 2006). Even without the genome duplication Paramecium has 25 DYH genes.
Dyneins are an ancient family of microtubule-based motors. The last eukaryotic common ancestor encoded at least nine distinct types of dynein heavy chains that probably had different functions within the cell (Wickstead and Gull 2007; Wilkes et al. 2008). Our analyses of ciliate genomes show that the nine DYH groups had expanded to at least 14 before the speciation of the ciliates examined. Most of this expansion was with the sh-IAD heavy chains. Further analyses of other taxa should help define the evolution of this large, functionally important, and widespread family. ACKNOWLEDGMENTS This study was supported by NSF grant 1026159 to DEW. LITERATURE CITED
At least seven sh-IAD heavy chains were present in the last common ancestor of the ciliate species examined The sh-IAD heavy chains across eukaryotes can be divided into three groups, IAD groups 3–5 (Morris et al. 2006; Wilkes et al. 2008). Our phylogenetic analysis of the ciliate DYHs found that the ciliate IAD group 3 could be divided into four subgroups and IAD group 5 into two subgroups (Fig. 1). In addition to these subgroups, the ciliate IAD group 5B may actually be two subgroups although these branches are not well-defined. Therefore, the last common ancestor of the four ciliate species that we examined expressed at least seven sh-IAD heavy chains. Later duplication of these sh-IADs resulted in even more species specific DYHs. For example, Tetrahymena expresses 18 sh-IAD DYH genes in the seven ancestral groups. The large number of sh-IADs in ciliates may be a result of the different types of cilia found associated with individual ciliates. For example, Tetrahymena cells are covered with body and oral apparatus cilia and the anterior and posterior body cilia show different characteristics (Frankel 2000). Specific sh-IADs may only be present within a single type of cilium. Alternatively, the large number of sh-IADs in ciliates may be especially important for ciliates that need to generate a large number of cilia rapidly. The last common ancestor of the ciliates examined possessed an outer arm dynein containing three heavy chains The ancestral outer arm dynein of the last eukaryotic common ancestor appears to have been a heterodimer of a and b subunits. There was a duplication of the OAD b isoform into the OAD c isoform before the speciation of the various ciliates examined (Fig. 1). The OAD a, b, and c appear to form a three-headed outer arm in ciliates as opposed to a two-headed outer arm.
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Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Res., 25:3389–3402. Andrews, K. K., Nettesheim, P., Asai, D. J. & Ostrowski, L. E. 1996. Identification of seven rat axonemal dynein heavy chain genes: expression during ciliated cell differentiation. Mol. Biol. Cell, 7:71–79. Arnaiz, O., Cain, S., Cohen, J. & Sperling, L. 2007. ParameciumDB: a community resource that integrates the Paramecium tetraurelia genome sequence with genetic data. Nucleic Acids Res., 35:D439–444. Arnaiz, O. & Sperling, L. 2011. ParameciumDB in 2011: new tools and new data for functional and comparative genomics of the model ciliate Paramecium tetraurelia. Nucleic Acids Res., 39: D632–636. Asai, D. J. 1995. Multi-dynein hypothesis. Cell Motil. Cytoskeleton, 32:129–132. Asai, D. J., Beckwith, S. M., Kandl, K. A., Keating, H. H., Tjandra, H. & Forney, J. D. 1994. The dynein genes of Paramecium tetraurelia: sequences adjacent to the catalytic P-loop identify cytoplasmic and axonemal heavy chain isoforms. J. Cell Sci., 107:839–847. Aury, J. M., Jaillon, O., Duret, L., Noel, B., Jubin, C., Porcel, B. gurens, B., Daubin, V., Anthouard, V., Aiach, N., Arnaiz, M., Se O., Billaut, A., Beisson, J., Blanc, I., Bouhouche, K., C^ amara, F., Duharcourt, S., Guigo, R., Gogendeau, D., Katinka, M., Keller, €l, A., Lepe re, A. M., Kissmehl, R., Klotz, C., Koll, F., Le Moue G., Malinsky, S., Nowacki, M., Nowak, J. K., Plattner, H., Pouterlain, J., Ruiz, F., Serrano, V., Zagulski, M., Dessen, P., Be mier, M., Weissenbach, J., Scarpelli, C., Sch€ achter, V., Sperling, L., Meyer, E., Cohen, J. & Wincker, P. 2006. Global trends of whole-genome duplications revealed by the ciliate Paramecium tetraurelia. Nature, 444:171–178. Blacque, O. E., Cevik, S. & Kaplan, O. I. 2008. Intraflagellar transport: from molecular characterisation to mechanism. Front Biosci., 13:2633–2652. Brokaw, C. J. & Kamiya, R. 1987. Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function. Cell Motil. Cytoskeleton, 8:68–75. Coyne, R. S., Hannick, L., Shanmugam, D., Hostetler, J. B., Brami, D., Joardar, V. S., Johnson, J., Radune, D., Singh, I., Badger,
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2016, 63, 138–141
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Rajagopalan & Wilkes
J. H., Kumar, U., Saier, M., Wang, Y., Cai, H., Gu, J., Mather, M. W., Vaidya, A. B., Wilkes, D. E., Rajagopalan, V., Asai, D. J., Pearson, C. G., Findly, R. C., Dickerson, H. W., Wu, M., Martens, C., Van de Peer, Y., Roos, D. S., Cassidy-Hanley, D. M. & Clark, T. G. 2011. Comparative genomics of the pathogenic ciliate Ichthyophthirius multifiliis, its free-living relatives and a host species provide insights into adoption of a parasitic lifestyle and prospects for disease control. Genome Biol., 12:R100. Dutcher, S. K. 1995. Flagellar assembly in two hundred and fifty easy-to-follow steps. Trends Genet., 11:398–404. Frankel, J. 2000. Cell biology of Tetrahymena thermophila. Methods Cell Biol., 62:27–125. Gibbons, I. R. 1995. Dynein family of motor proteins: present status and future questions. Cell Motil. Cytoskeleton, 32:136– 144. Gibbons, B. H., Asai, D. J., Tang, W. J. Y., Hays, T. S. & Gibbons, I. R. 1994. Phylogeny and expression of axonemal and cytoplasmic dynein genes in sea urchins. Mol. Biol. Cell, 5:57–70. Gibbons, B. H. & Gibbons, I. R. 1973. The effect of partial extraction of dynein arms on the movement of reactivated sea-urchin sperm. J. Cell Sci., 13:337–357. Gibbons, B. H. & Gibbons, I. R. 1976. Functional recombination of dynein 1 with demembranated sea urchin sperm partially extracted with KC1. Biochem. Biophys. Res. Com., 73:1–6. Jones, D. T., Taylor, W. R. & Thornton, J. M. 1992. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci., 8:275–282. , M. 2004. Of rings and levers: the dynein Koonce, M. P. & Samso motor comes of age. Trends Cell Biol., 14:612–619. Mocz, G. & Gibbons, I. R. 2001. Model for the motor component of dynein heavy chain based on homology to the AAA family of oligomeric ATPases. Structure, 9:93–103. Morris, R. L., Hoffman, M. P., Obar, R. A., McCafferty, S. S., Gibbons, I. R., Leone, A. D., Cool, J., Allgood, E. L., Musante, A. M., Judkins, K. M., Rossetti, B. J., Rawson, A. P. & Burgess,
D. R. 2006. Analysis of cytoskeletal and motility proteins in the sea urchin genome assembly. Dev. Biol., 300:219–237. Neuwald, A. F., Aravind, L., Spouge, J. L. & Koonin, E. V. 1999. AAA+: a class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes. Genome Res., 9:27–43. Pfister, K. K., Shah, P. R., Hummerich, H., Russ, A., Cotton, J., Annuar, A. A., King, S. M. & Fisher, E. M. C. 2006. Genetic analysis of the cytoplasmic dynein subunit families. PLoS Genet., 2:e1. Porter, M. E. 1996. Axonemal dyneins: assembly, organization, and regulation. Curr. Opin. Cell Biol., 8:10–17. Porter, M. E., Knott, J. A., Myster, S. H. & Farlow, S. J. 1996. The dynein gene family in Chlamydomonas reinhardtii. Genetics, 14:569–585. Rasmussen, K., Serr, M., Gepner, J., Gibbons, I. & Hays, T. S. 1994. A family of dynein genes in Drosophila melanogaster. Mol. Biol. Cell, 5:45–55. Rosenbaum, J. L. & Witman, G. B. 2002. Intraflagellar transport. Nat. Rev. Mol. Cell Biol., 3:813–825. Scholey, J. M. 2003. Intraflagellar transport. Annu. Rev. Cell Dev. Biol., 19:423–443. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol., 28:2731–2739. Tanaka, Y., Zhang, Z. & Hirokawa, N. 1995. Identification and molecular evolution of new dynein-like protein sequences in rat brain. J. Cell Sci., 108:1883–1893. Vale, R. D. 2003. The molecular motor toolbox for intracellular transport. Cell, 112:467–480. Wickstead, B. & Gull, K. 2007. Dyneins across eukaryotes: a comparative genomic analysis. Traffic, 8:1708–1721. Wilkes, D. E., Watson, H. E., Mitchell, D. R. & Asai, D. J. 2008. Twenty-five dyneins in Tetrahymena: a re-examination of the multidynein hypothesis. Cell Motil. Cytoskeleton, 65:342–351.
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Journal of Eukaryotic Microbiology ISSN 1066-5234
SHORT COMMUNICATION
Evolution of the Dynein Heavy Chain Family in Ciliates Vidyalakshmi Rajagopalan & David E. Wilkes Department of Biological Sciences, Indiana University South Bend, South Bend, Indiana 46634
Keywords Ichthyophthirius; Oxytricha; Paramecium; Tetrahymena. Correspondence D. Wilkes, Department of Biological Sciences, Indiana University South Bend, 1700 Mishawaka Avenue, P.O. Box 7111, South Bend, Indiana 46634, USA Telephone number: +1 574-520-4411; FAX number: +1 574-520-4482; e-mail: [email protected]
ABSTRACT Dynein heavy chains are motor proteins that comprise a large gene family found across eukaryotes. We have investigated this gene family in four ciliate species: Ichthyophthirius, Oxytricha, Paramecium, and Tetrahymena. Ciliates appear to encode more dynein heavy chain genes than most eukaryotes. Phylogenetic comparisons demonstrated that the last common ancestor of the ciliates that were examined expressed at least 14 types of dynein heavy chains with most of the expansion coming from the single-headed inner arm dyneins. Each of the dyneins most likely performed different functions within the cell.
Received: 21 January 2015; revised 11 June 2015; accepted June 11, 2015. doi:10.1111/jeu.12245
DYNEINS are microtubule-based motor complexes that perform a variety of functions in eukaryotic cells (Porter 1996; Vale 2003). They comprise a family well conserved across eukaryotic species. The family can be divided into two broad classes: (1) axonemal dyneins and (2) nonaxonemal, or cytoplasmic, dyneins. Axonemal dyneins are the molecular motors that power the movement of cilia and eukaryotic flagella and they can be further divided into outer arms and inner arms. Identical outer arms are found every 24 nm along the length of an axoneme and are required for establishing the high beat frequencies of flagella (Brokaw and Kamiya 1987; Gibbons and Gibbons 1973, 1976). Inner arm dyneins exist in many forms as opposed to the single type of outer arm dynein. Along the length of an axoneme one- and two-headed inner arm dyneins are organized in 96 nm repeating units in which each unit contains one each of an I1, I2, and I3 sub-region (Dutcher 1995). The I1 region contains a two-headed dynein while the I2 and I3 regions each have two singleheaded dyneins. The inner arms are involved in regulating the amplitude of axonemal bending (Brokaw and Kamiya 1987). Two groups of nonaxonemal or cytoplasmic dyneins exist. Cytoplasmic dynein-1 is found in all eukaryotes except higher plants and is the only dynein present in species without cilia or flagella. It is important for mitotic movements and for the organization and trafficking of
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intracellular membranes (Pfister et al. 2006; Vale 2003). Cytoplasmic dynein-2 is found only in organisms that have cilia or eukaryotic flagella and is the motor for retrograde intraflagellar transport (Blacque et al. 2008; Rosenbaum and Witman 2002; Scholey 2003). Each of the dynein complexes is made of a core of one or more heavy chains (DYHs) associated with a set of smaller subunits. The DYHs form the heads of the dynein complexes and contain both the microtubule-binding site 2004; and the site of ATP hydrolysis (Koonce and Samso Mocz and Gibbons 2001; Neuwald et al. 1999). Organisms with motile cilia or flagella express multiple DYH genes (Andrews et al. 1996; Asai et al. 1994; Gibbons et al. 1994; Porter et al. 1996; Rasmussen et al. 1994; Tanaka et al. 1995). Comparisons of the DYH motor domain sequences have shown that the sequences correspond to functional classes (Asai 1995; Gibbons 1995). The family evolved early with at least nine different DYHs present in the last eukaryotic common ancestor (Wickstead and Gull 2007; Wilkes et al. 2008). The cytoplasmic dynein 1 and 2 heavy chains form homodimers of their respective complexes. The outer arm a and b/c heavy chains assemble into the heterodimeric or heterotrimeric axonemal outer arm dynein complex. The inner arm 1a and 1b heavy chains form the heterodimeric I1 inner arm dynein. The other three groups of heavy chains are thought to
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2016, 63, 138–141
Dynein Evolution in Ciliates
Rajagopalan & Wilkes
comprise the single-headed inner arm dyneins (sh-IAD). Many eukaryotic genomes contain more than one DYH gene per ancestral group and thus have more than nine total DYH genes. The Chlamydomonas, sea urchin, and human genomes have 14, 15, and 16 DYH genes, respectively. Previous examination of the genomes of two ciliates found more DYH genes than typical. The Tetrahymena and Ichthyophthirius genomes contain 25 and 19 DYH genes, respectively (Coyne et al. 2011; Wilkes et al. 2008). We have extended the analysis of ciliate DYH genes to the recently available Paramecium and Oxytricha genomes. Our results support the idea that ciliates do have more DYHs than most organisms and that some of the expansion occurred before speciation of the ciliates. Of particular interest, the last common ancestor of the examined ciliates appears to have had seven distinct DYH subgroups of the single-headed inner arms as opposed to only three for the last eukaryotic common ancestor. MATERIALS AND METHODS Gene sequences The dynein heavy chain sequences from Tetrahymena thermophila and Ichthyophthirius multifiliis have been published previously (Coyne et al. 2011; Wilkes et al. 2008). Paramecium sequences were obtained by searching the ParameciumDB (http://paramecium.cgm.cnrs-gif.fr/; Arnaiz and Sperling 2011; Arnaiz et al. 2007) and Oxytricha sequences were obtained from the OxyDB (http://oxy.ciliate.org/index.php/home/welcome/). The conserved region of Tetrahymena DYH4 motor domain from the first P loop to the sequence coding for the conserved amino acid sequence PATVSR (residues 2307-2312 of DYH4) was used as the query sequence to BLAST (Altschul et al. 1997) each genome. Phylogenetic analyses The DYH motor domain sequences of the four ciliate species were compared by Clustal Omega alignment (www.ebi.ac.uk/Tools/msa/clustalo/). The phylogenetic analysis was conducted on 84 amino acid sequences. All positions containing gaps and missing data were eliminated, and a total of 169 positions were retained in the final dataset. The phylogeny was inferred using the Maximum Likelihood method implemented in MEGA5 (Tamura et al. 2011). The JTT matrix (Jones et al. 1992) was used, with evolutionary rate differences among sites modelled using a discrete approximation of a Gamma distribution ([+G], 5 categories; alpha parameter = 0.6668) plus an invariable sites category ([+I], 5.0365% of sites). Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was < 100 or less than one-fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. A 500-pseudoreplicate bootstrap analysis was performed to assess robustness.
TetDYH8 Ich2313.m000021 ParaGSPATP00025924001 96 ParaGSPATP00033121001 Ich1999.m000059 Ich2733.m000052 IAD group 3A TetDYH11 ParaGSPATP00019919001 ParaGSPATP00001961001 91 94 ParaGSPATP00020187001 82 ParaGSPATP00005122001 ParaGSPATP00001980001 OxyContig17454 0 g50 OxyContig9415 0 1 g18 ParaGSPATP00004069001 99 IAD group 3B TetDYH17 93 TetDYH14 86 Ich2913.m000242 99 TetDYH12 Ich2300.m000062 IAD group 3C 81 ParaGSPATP00012521001 OxyContig10418 0 g55 94 TetDYH10 ParaGSPATP00007189001 94 IAD group 3D TetDYH25 TetDYH13 Ich2443.m000020 Ich2883.m000117 OxyContig16305 0 g10 OxyContig15709 0 g85 85 ParaGSPATP00008239001 ParaPTETP6000001001 ParaGSPATP00008111001 TetDYH9 IAD group 4 94 Ich2895.m000270 TetDYH19 TetDYH20 83 TetDYH16 Ich2630.m000021 TetDYH21 Ich2678.m000182 TetDYH15 79 IAD group 5A OxyContig14020 0 g49 ParaPTETP1000023001 Ich2625.m000030 95 Ich2484.m000119 TetDYH24 ParaPTETP8700005001 75 ParaGSPATP00009095001 99 TetDYH18 OxyContig2564 0 g105 IAD group 5B OxyContig21237 0 g67 92 TetDYH23 ParaGSPATP00018717001 TetDYH22 Ich2507.m000068 OxyContig16067 0 g66 OxyContig335 1 g130 92 TetDYH7 98 IAD 1beta Ich2751.m000110 ParaGSPATP00038674001 TetDYH5 92 Ich2880.m000134 OAD gamma 100 ParaGSPATP00038423001 87 OxyContig19406 0 0 g75 TetDYH4 OAD beta ParaPTETP5700005001 97 OxyContig44 1 g4 TetDYH3 Ich2946.m000074 100 OAD alpha ParaPTETP300004001 OxyContig1360 1 g26 94 TetDYH6 78 Ich1778.m000066 IAD 1alpha 87 ParaGSPATP00015638001 OxyContig19661 0 g109 TetDYH1 94 Ich1655.m000083 Cyto 1 99 ParaGSPATP00002017001 OxyContig11184 0 g31 OxyContig11800 0 g24 95 TetDYH2 100 Cyto 2 Ich1642.m000052 77
83 76
ParaPTETP9700007001
0.2
Figure 1 Phylogenetic analysis of the ca. 400 residues conserved catalytic domain of the dynein heavy chains revealed that ciliate genomes examined contain at least 14 distinct types of dynein heavy chains. The original maximum likelihood tree is shown with bootstrap values > 75% (500 iterations) at branch points. There were a total of 169 positions in the final dataset. Cyto 1 = cytoplasmic dynein 1; Cyto 2 = cytoplasmic dynein 2; OAD = axonemal outer arm dynein; IAD = axonemal inner arm dynein. Ich = Ichthyophthirius; Oxy = Oxytricha; Para = Paramecium; Tet = Tetrahymena. Scale = 0.2 substitutions/site.
© 2015 The Author(s) Journal of Eukaryotic Microbiology © 2015 International Society of Protistologists Journal of Eukaryotic Microbiology 2016, 63, 138–141
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CONCLUSIONS
RESULTS AND DISCUSSION Ciliate genomes have larger dynein heavy chain families than most eukaryotes Previously, we have shown that the ciliates, Tetrahymena and Ichthyophthirius, have 25 and 19 DYH genes, respectively (Coyne et al. 2011; Wilkes et al. 2008). Our searches found 50 DYH genes in Paramecium and at least 15 in Oxytricha. Oxytricha may encode as many as 25 DYH genes however many are unclear due to the quality and incomplete annotation of the genome. For example, Oxytricha Contig14590.0.g69 may be the IAD 1b that appears to be missing in our analysis. The number of Paramecium DYH genes is much higher than typical because of a recent whole genome duplication (Aury et al. 2006). Even without the genome duplication Paramecium has 25 DYH genes.
Dyneins are an ancient family of microtubule-based motors. The last eukaryotic common ancestor encoded at least nine distinct types of dynein heavy chains that probably had different functions within the cell (Wickstead and Gull 2007; Wilkes et al. 2008). Our analyses of ciliate genomes show that the nine DYH groups had expanded to at least 14 before the speciation of the ciliates examined. Most of this expansion was with the sh-IAD heavy chains. Further analyses of other taxa should help define the evolution of this large, functionally important, and widespread family. ACKNOWLEDGMENTS This study was supported by NSF grant 1026159 to DEW. LITERATURE CITED
At least seven sh-IAD heavy chains were present in the last common ancestor of the ciliate species examined The sh-IAD heavy chains across eukaryotes can be divided into three groups, IAD groups 3–5 (Morris et al. 2006; Wilkes et al. 2008). Our phylogenetic analysis of the ciliate DYHs found that the ciliate IAD group 3 could be divided into four subgroups and IAD group 5 into two subgroups (Fig. 1). In addition to these subgroups, the ciliate IAD group 5B may actually be two subgroups although these branches are not well-defined. Therefore, the last common ancestor of the four ciliate species that we examined expressed at least seven sh-IAD heavy chains. Later duplication of these sh-IADs resulted in even more species specific DYHs. For example, Tetrahymena expresses 18 sh-IAD DYH genes in the seven ancestral groups. The large number of sh-IADs in ciliates may be a result of the different types of cilia found associated with individual ciliates. For example, Tetrahymena cells are covered with body and oral apparatus cilia and the anterior and posterior body cilia show different characteristics (Frankel 2000). Specific sh-IADs may only be present within a single type of cilium. Alternatively, the large number of sh-IADs in ciliates may be especially important for ciliates that need to generate a large number of cilia rapidly. The last common ancestor of the ciliates examined possessed an outer arm dynein containing three heavy chains The ancestral outer arm dynein of the last eukaryotic common ancestor appears to have been a heterodimer of a and b subunits. There was a duplication of the OAD b isoform into the OAD c isoform before the speciation of the various ciliates examined (Fig. 1). The OAD a, b, and c appear to form a three-headed outer arm in ciliates as opposed to a two-headed outer arm.
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