The Dynein Family at a Glance Peter Höök and Richard B

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Cell Science at a Glance 4369

The dynein family at a functions. Although at least 14 classes of accessory subunits bind; and a ~380 kDa kinesin and 17 classes of myosin have motor domain. The motor domain glance been identiﬁed, the dyneins fall into contains six discernible AAA ATPase Peter Höök* and Richard B. only two major classes, axonemal and units, identifying the dynein HC as a Vallee cytoplasmic dyneins, based on both divergent member of the AAA+ family functional and structural criteria. of ATPases (Neuwald et al., 1999). Department of Pathology and Cell Biology, Columbia University, New York, NY 10032, USA. Axonemal dyneins are responsible for Members of the AAA+ family are *Author for correspondence (e-mail: ciliary and ﬂagellar beating; cytoplasmic involved in a very wide range of [email protected]) dyneins are involved in intracellular functions but have a common feature: the

Journal of Cell Science 119, 4369-4371 transport, mitosis, cell polarization and formation of ring-shaped oligomeric Published by The Company of Biologists 2006 directed cell movement complexes of the AAA ATPase module. doi:10.1242/jcs.03176 Within the AAA+ proteins, dynein All dynein forms that have occupies a divergent branch along with Three families of cytoskeletal motor been identiﬁed biochemically are midasin (Iyer et al., 2004). This branch protein – the myosins, kinesins and multisubunit proteins. Each has one to is characterized by the incorporation of dyneins – have evolved to mediate three heavy chains (HCs) of >500 kDa; all six AAA modules within a single transport of cells and of structures and these correspond to the number of giant polypeptide. The AAA family has materials within cells in eukaryotes. morphologically identiﬁable heads and members in prokaryotes and it seems Whereas myosin uses actin polymers to contain the motor domains of the likely, therefore, that the dyneins had carry out its tasks, kinesin and dynein are molecule. The dynein HC forms two their origin very early in evolution. microtubule-associated motors. Dyneins prominent structures: a ~160 kDa N- In dynein, energy from nucleotide use energy from ATP hydrolysis to terminal domain that forms the base of hydrolysis at the AAA units is conveyed power a wide variety of cellular the molecule, to which most of the to the base of the molecule and to the

The Dynein Family at a Glance Peter Höök and Richard B. Vallee

+ S. pombe D. discoideum C. elegans - D. melanogaster D. rerio H. sapiens R. norvegicus M. musculus U. maydis

Journal of Cell Science E. nidulans + + A. fumigatus Dynein 1 I A. oryzae - + - + N. crassa - - N. haematococca - - + - + P. tetraurelia

T. thermophila Cytoplasmic + C. albicans E. gossypii S. cerevisiae G. lamblia D. melanogaster L. mexicana T. thermophila C. elegans Dynein 2 II T. gratilla Microtubule Axoneme R. norvegicus M. musculus Dynein 1 Inner-arm dynein C. reinhardtii Outer-arm dynein III Dynein 2 Outer-arm dynein D. melanogaster D. melanogaster H. sapiens DHC9 Clamp loader A. crassispina DnaA/Cdc6/ORC T. gratilla H. sapiens DHC11 Outer-arm dynein IV Classical M. musculus DHC11 AAA D. melanogaster AAA+ D. hydei DHC7 l HsIU/ClpX/Lon/Clp C. reinhardtii jcs.biologists.org Dynein T. thermophila Helix 2 C. reinhardtii

insert M. musculus DHC5 Axonema Midasin H. sapiens DHC5 Outer-arm dynein V M. musculus DHC8 H. sapiens DHC8 D. melanogaster Inner-arm dynein C. reinhardtii DHC1 VI Stem/tail Linker AAA1 AAA2 AAA3 AAA4Stalk AAA5 AAA6 CT D. melanogaster C. reinhardtii DHC10 Inner-arm dynein H. sapiens DHC7 VII ...... H. sapiens DHC3 LIC C. reinhardtii DHC9 IC HC Conserved residues

(See poster insert) 4370 Journal of Cell Science 119 (21)

microtubule-binding stalk for force arm dyneins are two- and one-headed and below), it coexists with axonemal production (Burgess et al., 2003). The arrayed in a repeating pattern along the A- dyneins in cilia and flagella. stalk is predicted to consist of a 15-nm- microtubule. Each of the flagellar dyneins long antiparallel coiled-coil ␣-helix interacts with the B microtubule of the crowned with a globular structure for adjacent outer doublet. Seven subtypes of Evolutionary relationship among microtubule association (Gee et al., Chlamydomonas axonemal inner-arm dyneins 1997). dynein have been identified (Kagami and The diverse forms of kinesin and myosin Kamiya, 1992); one forms a HC have been judged to comprise clear A diversity of accessory subunits, heterodimer; the rest are monomeric, one- subfamilies. The definition of the referred to as intermediate, light headed structures. subcategories has been made on the basis intermediate and light chains are also of phylogenetic analysis of the motor found associated with the dyneins. Most, subunits (HCs), but also on the but not all, of these subunits are Cytoplasmic dyneins composition and organization of the associated with the cargo-binding base Two forms of cytoplasmic dynein have nonmotor domains, which are highly of the dynein molecule. Dynein-isoform- been identified: cytoplasmic dynein 1 distinctive between subfamilies. specific intermediate chains are found in and cytoplasmic dynein 2. Both Differences in physiological and some axonemal dyneins as well as in assemble as homodimers, but their mechanochemical properties have cytoplasmic dynein 1. Some light chains distributions and functions within the correlated well with family boundaries. are shared between axonemal dyneins, cell differ markedly. Cytoplasmic dynein By contrast, the organization of both the cytoplasmic dynein 1 and other proteins. 1 is far more abundant and is found motor and nonmotor portion of the Isoform-specific light intermediate in all microtubule-containing cells. It dynein HCs is remarkably well chains are associated with both associates with and transports elements conserved. Differences in physiological cytoplasmic dyneins but not with of the Golgi apparatus, lysosomes, and function and subcellular localization axonemal dyneins. late and recycling endosomes, and is have provided the clearest indication of responsible for retrograde axonal boundaries. Some differences in domain transport. It also associates with diverse organization have been discerned Axonemal dyneins protein and RNA-containing complexes, between dyneins, but these are very The axoneme is a highly specialized and which it transports or maintains at limited in scope. For example, the C- highly conserved, array of microtubules. microtubule minus ends. Cytoplasmic terminal (CT) region of the motor Motile axonemes in flagella and many dynein 1 also associates with domain is found in some, but not all, forms of cilia almost universally consist kinetochores, where it has been dynein HCs, although the functional of 20 microtubules, of which two are implicated in microtubule capture and significance of this region is centrally located and nine are fused pairs the removal of checkpoint proteins to incompletely understood (Vallee and (the outer doublet) forming a surrounding permit entry into anaphase. This form of Höök, 2006). Isoforms of the

Journal of Cell Science cylinder. Dyneins connect the outer cytoplasmic dynein also associates with intermediate chain (IC) family bind to doublets and force them to slide against the cell cortex during division and during some axonemal dyneins and cytoplasmic each other (Summers and Gibbons, 1971). directed migration, as well as residing at dynein 1 but not cytoplasmic dynein 2. Most ultrastructural and genetic evidence other sites. These latter roles have The full range of IC-binding axonemal for subspecialization of the axonemal suggested an additional feature of dynein dynein forms remains to be explored. dyneins has been obtained in studies function: it may serve not only in The light chains (LCs), which bind of the biflagellate green alga transport motor, but also in producing to dynein almost exclusively through Chlamydomonas reinhardtii and the tension along or at the ends of the ICs as scaffolds, are found in ciliate protozoan Tetrahymena pyriformis. microtubules from fixed cellular sites. cytoplasmic dynein 1 and some, but not Sea urchin sperm flagella have also been all, axonemal dyneins, as well as non- used for biochemical analysis of By contrast, cytoplasmic dynein 2 is dynein proteins. The LCs, therefore, like axonemal dyneins, but limited work has found almost exclusively within and the ICs, have not been of significant been performed in vertebrates. At least around the base of cilia and flagella, value in distinguishing dynein five biochemically defined forms of where it is engaged in retrograde subfamilies. The LICs, however, are dynein (and even more gene products) can intraflagellar transport (Porter et al., associated with cytoplasmic dynein 1 be found in a given type of cilium or 1999; Pazour et al., 1999; Mikami et al., and dynein 2, but, so far, with no known flagellum, but correlating dynein genes 2002). This form of motility is required axonemal dyneins. These subunits, with specific dynein structures in the for axonemal maintenance. In addition, therefore, serve as a further means to axoneme has been a long and challenging cytoplasmic dynein 2 is responsible for distinguish the two major dynein process. All axonemal dyneins are stably transport through modified ciliary subfamilies. Their binding site within associated with the complete microtubule structures, such as the connecting cilium their respective dynein HCs is also (the A microtubule) of the outer doublet of photoreceptors. The transported conserved (Habura et al., 1999; Mikami and aligned as rows along the outer or material is in the form of proteinaceous et al., 2002). inner side of the A microtubule. The rafts, which travel along the outer surface outer-arm dyneins have three motor of flagellar microtubules. Thus, although We have carried out a phylogenetic domains (heads) in protozoans, but only this form of dynein is cytoplasmic, as analysis of 51 full-length dynein HCs two are retained in metazoans. The inner- judged by a number of criteria (see from 24 diverse organisms by multiple Journal of Cell Science 119 (21) 4371

sequence alignment (using the Clustal W the ~600-residue portion of the stem Photosensitized cleavage of dynein heavy chains. program with default parameters) and proximal to AAA1. This region, referred Cleavage at the “V1 site” by irradiation at 365 nm in the presence of ATP and vanadate. J. Biol. Chem. 262, 2780- constructed a phylogenetic tree (using to as the ‘linker’, has been recently 2786. the Neighbor Joining method). Based on implicated in force transduction and has Habura, A., Tikhonenko, I., Chisholm, R. L. and this and existing functional analysis been proposed to generate force through Koonce, M. P. (1999). Interaction mapping of a dynein heavy chain. Identification of dimerization and we have labelled subclasses of dynein its interaction with the AAA ring intermediate-chain binding domain. J. Biol. Chem. 274, from I-VII, which is consistent with (Burgess et al., 2003). AAA1 has been 15447-15553. Höök, P., Mikami, A., Shafer, B., Chait, B. T., current kinesin and myosin family deduced to serve as the principal site for Rosenfeld, S. S. and Vallee, R. B. (2005). Long range nomenclature. The axonemal dyneins ATP hydrolysis, based on the effects of allosteric control of cytoplasmic dynein ATPase by the appear to have diverged into five vanadate-mediated UV photocleavage at stalk and C-terminal domains. J. Biol. Chem. 280, 33045- 33054 subclasses, each of which is represented this site (Gibbons et al., 1987; Gee et al., Iyer, L. M., Leipe, D. D., Koonin, E. V. and Aravind, L. by a single gene in Chlamydomonas: the 1997). More recent studies on the dynein (2004). Evolutionary history and higher order classification ␣ inner-arm dynein; the ␤ inner-arm motor domain suggest that hydrolytic of AAA+ ATPases. J. Struct. Biol. 146, 11-31. Kagami, O. and Kamiya, R. (1992). Translocation and dynein; the ␥ outer-arm dynein, which is activity in AAA1 also requires the rotation of microtubules caused by multiple species of present only in protozoans; the ␣ and ␤ structural involvement of AAA2 Chlamydomonas inner-arm dynein. J. Cell Sci. 103, 653- 664. outer-arm dyneins, which branched from (Takahashi et al., 2004; Höök et al., Kon, T., Nishiura, M., Ohkura, R., Toyoshima, Y. Y. a common trunk to form separate ␣ and 2005). Consequently, more than half of and Sutoh, K. (2004). Distinct functions of nucleotide- ␤ dyneins. The phylogenetic origin of the conserved residues are located within binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein. Biochem. 43, 11266-11274. one-headed inner-arm dyneins is poorly the boundaries of the linker and the first Mikami, A., Tynan, S. H., Hamo, T., Luby-Phelps, K., understood. We included the sequence of two AAA domains, stressing the region’s Daito, T., Crandall, J. E., Besharse, J. C. and Vallee, R. Chlamydomonas DHC9, or subspecies c, significance as the source of force B. (2002). Molecular structure of cytoplasmic dynein 2 and its distribution in neuronal and ciliated cells. J. Cell the only one-headed full-length dynein production within the dynein motor. Sci. 115, 4801-4808. sequence presently available. The Additional clusters of conserved residues Neuwald, A. F., Aravind, L., Spouge, J. L. and Koonin, E. V. (1999). AAA+: a class of chaperone-like ATPases alignment assigned subspecies c to the are found within AAA3, in particular associated with the assembly, operation and disassembly ␤ inner-arm dyneins, suggesting that among motifs that are involved in of protein complexes. Genome Res. 9, 27-43. human DHC7 and DHC3 represent nucleotide binding and hydrolysis, Pazour, G. J., Dickert, B. L. and Witman, G. B. (1999). The DHC1b (DHC2) isoform of cytoplasmic dynein is single-headed inner-arm dyneins. Five which is consistent with the role of this required for flagellar assembly. J. Cell Biol. 144, 473-481. additional one-headed dyneins have been ATPase in dynein function (Silvanovich Porter, M. E., Bower, R., Knott, J. A., Byrd, P. and identified biochemically (Kagami and et al., 2003; Kon et al., 2004; Reck- Dentler, W. (1999). Cytoplasmic dynein heavy chain 1b is required for flagellar assembly in Chlamydomonas. Mol. Kamiya, 1992). Which subclass of inner- Peterson, 2004). Biol. Cell 10, 693-712. arm dynein these represent should be Reck-Peterson, S. L. and Vale, R. D. (2004). Molecular dissection of the roles of nucleotide binding and hydrolysis determined as their sequences become Sporadic residues along the dynein in dynein’s AAA domains in Saccharomyces cerevisiae. available. polypeptide sequence, including the Proc. Natl. Acad. Sci. 101, 1491-1495. microtubule-binding stalk, have been Silvanovich, A., Li, M.-G., Serr, M., Mische, S. and Hays, T. S. (2003). The third P-loop domain in

Journal of Cell Science kept remarkably unchanged throughout cytoplasmic dynein heavy chain is essential for dynein Sequence conservation within dynein evolution. Three of the four motor function and ATP-sensitive microtubule binding. dynein HC functional domains entirely conserved residues within the Mol. Biol. Cell 14, 1355-1365. Summers, K. E. and Gibbons, I. R. (1971). Adenosine Whereas the motor domain displays a stalk are prolines, which have the unique triphosphate-induced sliding of tubules in trypsin-treated high level of sequence conservation, ability to act as coiled-coil breakers and ﬂagella of sea-urchin sperm. Proc. Natl. Acad. Sci. 68, 3092-3096. especially among motifs implicated in may in that role function as the boundary Takahashi, Y., Edamatsu, M. and Toyoshima, Y. Y. nucleotide binding and hydrolysis, the between the coiled coil and microtubule- (2004). Multiple ATP-hydrolyzing sites that potentially sequence within the nonmotor region binding elements function in cytoplasmic dynein Proc. Natl. Acad. Sci. 101, 12865-12869. (traditionally referred to as the ‘stem’ Vallee, R. B. and Höök, P. (2006). Autoinhibitory and and more recently as the ‘tail’) is more We thank Dr Toshiki Yagi for useful discussions. other autoregulatory elements within the dynein motor divergent, and only a few residues are domain. J. Struct. Biol. 156, 175-181. entirely conserved. As noted above, the References LIC binding region within cytoplasmic Burgess, S. A., Walker M. L., Sakakibara, H., Knight, Cell Science at a Glance on the Web dynein 1 and cytoplasmic dynein 2 P. J. and Oiwa, K. (2003). Dynein structure and power stroke. Nature 421, 715-718. Electronic copies of the poster insert are shows evidence of conservation, but can Gee, M. A., Heuser, J. E. and Vallee, R. B. (1997). An available in the online version of this article be identiﬁed in intraclass comparisons extended microtubule-binding structure within the dynein at jcs.biologists.org. The JPEG images can (Mikami et al., 2002). Conserved motor domain. Nature 390, 636-639. be downloaded for printing or used as Gibbons, I. R., Lee-Eiford, A., Mocz, G., Phillipson, C. slides. residues are more readily detected along A., Tang, W. J. and Gibbons, B. H. (1987)