David Rees Mitchell

Outer dynein arms (ODAs) are multiprotein complexes that drive flagellar beating. Based on genetic and biochemical analyses, ODAs preassemble in the cell body and then move into the flagellum by intraflagellar transport (IFT). To study ODA transport in vivo, we expressed the essential intermediate chain 2 tagged with mNeonGreen (IC2-NG) to rescue the corresponding Chlamydomonas reinhardtii mutant oda6. IC2-NG moved by IFT; the transport was of low processivity and increased in frequency during flagellar growth. As expected, IFT of IC2-NG was diminished in oda16, lacking an ODA specific IFT adapter, and in ift46 IFT46ΔN lacking the ODA16-interacting portion of IFT46. IFT loading appears to involve ODA16-dependent recruitment of ODAs to basal bodies followed by hand-over to IFT. Upon unloading from IFT, ODAs rapidly docked to the axoneme. Transient docking still occurred in the docking complex mutant oda3 indicating that the docking complex stabilizes rather than initiates ODA-microtubule interactions. In full-length flagella, ODAs continued to enter and move inside cilia by short-term bidirectional IFT and diffusion and the newly imported complexes frequently replaced axoneme-bound ODAs. We propose that the low processivity of ODA IFT contributes to flagellar maintenance by ensuring the availability of replacement ODAs along the length of flagella. (199 max. 200) Video S1: IFT of IC2‐NG in oda3 flagella. TIRF video and kymogram showing IFT transport and diffusion of IC2‐NG in oda3 flagella. Images were acquired at 10 fps and the timer counts seconds. The movie corresponds to Fig. 2A. Download Original Video (.2 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-05-0291/20180820/media/mc-e18-05-0291-s05.,150,.mp4.m3u8?b92b4ad1b4f274c70877518613abb28b424615f7925cf55ec9c2cb79b9a9dfcd69a8987b50485f1f25a8d2d0ec021e2ce182d7453bfa175ee96febde725867105baee910659abb2d71f22960d149172236fdfd7ea23cc81a6ba5fbcc3ee332424e4077244437ace454a3c3c597992ee191b30ac0e8eb71197b8dc5e5515ce1b1bf9c1531c9698fb8881fe9a325b0249b29b36531ad043de4d02d923bab Video S2: IC2‐NG co‐migrates with IFT20‐mC. Two‐color TIRF video showing IC2‐NG and IFT20‐mC in the corresponding oda6 ift20‐1 double mutant background. Images were acquired at 10 fps and timer counts seconds. Download Original Video (1.4 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-05-0291/20180820/media/mc-e18-05-0291-s06.,150,.mp4.m3u8?b92b4ad1b4f274c70877518613abb28b424615f7925cf55ec9c2cb79b9a9dfcd69a8987b50485f1f25a8d2d0ec021e2ce182d7453bfa175ee96febde725867105baee910659abb2d71f22960d149172236fdfd7ea23cc81a6ba5fbcc3ee332424e4077244437ace454a3c3c597992ee191b30ac0e8eb7119788dc5e5515ce1b1a17b41fb88a8179c59f08e4a081d70e24bacfc3fff8b313255fe95c2b0 Video S3: IFT trains carrying two IC2‐NG particles. TIRF video and kymogram showing transport and unloading of IC2‐NG from IFT. One of the two particles was photobleached during the recording. Images were acquired at 10 fps and the timer counts seconds. The movie corresponds to Fig. S3 C. Download Original Video (.3 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-05-0291/20180820/media/mc-e18-05-0291-s07.,150,.mp4.m3u8?b92b4ad1b4f274c70877518613abb28b424615f7925cf55ec9c2cb79b9a9dfcd69a8987b50485f1f25a8d2d0ec021e2ce182d7453bfa175ee96febde725867105baee910659abb2d71f22960d149172236fdfd7ea23cc81a6ba5fbcc3ee332424e4077244437ace454a3c3c597992ee191b30ac0e8eb7119798dc5e5515ce1b11ab157e04f638bbf8562adfb7837f619b75ffb708d84cc82db67b2d0cd Video S4: Docking of IC2‐NG particles in oda6 flagella. TIRF video showing transport, unloading and rapid immobilization of IC2‐NG in an oda6‐derived flagellum of an oda6 x oda6 IC2‐NG zygote. The arrows mark the positions of the immobilized IC2‐NG particles. Images were acquired at 10 fps and the timer counts seconds. The movie corresponds to Fig. 3C top panel. Download Original Video (.2 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-05-0291/20180820/media/mc-e18-05-0291-s08.,150,.mp4.m3u8?b92b4ad1b4f274c70877518613abb28b424615f7925cf55ec9c2cb79b9a9dfcd69a8987b50485f1f25a8d2d0ec021e2ce182d7453bfa175ee96febde725867105baee910659abb2d71f22960d149172236fdfd7ea23cc81a6ba5fbcc3ee332424e4077244437ace454a3c3c597992ee191b30ac0e8eb7119768dc5e5515ce1b1c07e815b3e54c18c3e866737df6761dd799422058c7f2d3aba771a2eb8 Video S5: Diffusion and IFT of IC2‐NG in zygotic oda8 flagella. TIRF video showing IC2‐NG in two flagella of an oda8 oda6 x oda8 oda6 IC2‐NG zygote. Note long‐lasting diffusion of IC2‐NG particles compared to Video S6 showing zygotic oda3 flagella. Images were acquired at 10 fps and the timer counts ss:mm. The movie corresponds to Fig. 5B. Download Original Video (1.9 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-05-0291/20180820/media/mc-e18-05-0291-s09.,150,.mp4.m3u8?b92b4ad1b4f274c70877518613abb28b424615f7925cf55ec9c2cb79b9a9dfcd69a8987b50485f1f25a8d2d0ec021e2ce182d7453bfa175ee96febde725867105baee910659abb2d71f22960d149172236fdfd7ea23cc81a6ba5fbcc3ee332424e4077244437ace454a3c3c597992ee191b30ac0e8eb7119778dc5e5515ce1b11a5d0434e398c860b72a71c743750c78d0bbd24a0a36a046371982548c Video S6: Transport of IC2‐NG in flagella of an oda3 oda6 x oda3 oda6 IC2‐NG zygote. TIRF video showing IC2‐NG behavior in the absence of the DC. Note particles cycling between IFT, diffusion, and transient docking. Images were acquired at 10 fps and the timer counts seconds. A corresponding kymogram is shown in Fig. S3B top row, right. Download Original Video (.9 MB) https://ascb-prod-streaming.literatumonline.com/journals/content/mboc/0/mboc.ahead-of-print/mbc.e18-05-0291/20180820/media/mc-e18-05-0291-s10.,150,.mp4.m3u8?b92b4ad1b4f274c70877518613abb28b424615f7925cf55ec9c2cb79b9a9dfcd69a8987b50485f1f25a8d2d0ec021e2ce182d7453bfa175ee96febde725867105baee910659abb2d71f22960d149172236fdfd7ea23cc81a6ba5fbcc3ee332424e4077244437ace454a3c3c597992ee191b30ac0e8eb7118ba53acecc03e8eab24b18bda3abae6842f01f170f95a921048c4d0c255633092840a720217

Anton van Leeuwenhoek’s startling microscopic observations in the 1600s first stimulated fascination with the way that cells use cilia to generate currents and to swim in a fluid environment. Research in recent decades has yielded deep knowledge about the mechanical and biochemical nature of these organelles but only opened a greater fascination about how such beautifully intricate and multifunctional structures arose during evolution. Answers to this evolutionary puzzle are not only sought to satisfy basic curiosity, but also, as stated so eloquently by Dobzhansky (Am Zool 4: 443 [1964]), because “nothing in biology makes sense except in the light of evolution.” Here I attempt to summarize current knowledge of what ciliary organelles of the last eukaryotic common ancestor (LECA) were like, explore the ways in which cilia have evolved since that time, and speculate on the selective processes that might have generated these organelles during early eukaryotic evolution.

Axonemal dyneins are multi-subunit enzymes that must be pre-assembled in the cytoplasm, transported into cilia by intraflagellar transport, and bound to specific sites on doublet microtubules where their activity facilitates microtubule sliding-based motility. Outer dynein arms (ODAs) require assembly factors to assist their preassembly, transport and attachment to cargo (specific doublet A-tubule sites). In Chlamydomonas, three assembly factors, ODA5, ODA8 and ODA10, show genetic interactions and have been proposed to interact in a complex, but we recently showed that flagellar ODA8 does not copurify with ODA5 or ODA10 (Desai et al., 2015). Here we show that ODA5 and ODA10 depend on each other for stability and coexist in a complex in both cytoplasmic and flagellar extracts. Immunofluorescence and immunoelectron microscopy reveal that ODA10 in flagella localizes strictly to a proximal region of doublet number one (DM1), which completely lacks ODAs in Chlamydomonas. Studies of the in vitro binding of ODAs to axonemal doublets reveal a role for the ODA5/ODA10 assembly complex in cytoplasmic maturation of ODAs into a form that can bind to doublet microtubules.

The Chlamydomonas reinhardtii oda8 mutation blocks assembly of flagellar outer dynein arms (ODAs), and interacts genetically with ODA5 and ODA10, which encode axonemal proteins thought to aid dynein binding onto axonemal docking sites. We positionally cloned ODA8 and identified the gene product as the algal homolog of vertebrate LRRC56. Its flagellar localization depends on ODA5 and ODA10, consistent with genetic interaction studies, but phylogenomics suggests that LRRC56 homologs play a role in intraflagellar transport (IFT)‐dependent assembly of outer row dynein arms, not axonemal docking. ODA8 distribution between cytoplasm and flagella is similar to that of IFT proteins and about half of flagellar ODA8 is in the soluble matrix fraction. Dynein extracted in vitro from wild type axonemes will rebind efficiently to oda8 mutant axonemes, without re‐binding of ODA8, further supporting a role in dynein assembly or transport, not axonemal binding. Assays comparing preassembled ODA complexes from the cytoplasm of wild type and mutant strains show that dynein in oda8 mutant cytoplasm has not properly preassembled and cannot bind normally onto oda axonemes. We conclude that ODA8 plays an important role in formation and transport of mature dynein complexes during flagellar assembly. © 2014 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.

Assembly of outer dynein arms (ODAs) requires multiple steps and involves multiple proteins in addition to dynein subunits. The Chlamydomonas ODA10, ODA5, and ODA8 loci genetically interact and are hypothesized to function as an axonemal accessory complex, but only ODA5p was previously characterized. We positionally cloned ODA10 and identified the gene by rescuing an oda10 mutant with a hemagglutinin-tagged cDNA. ODA10 sequence predicts a conserved coiled-coil protein homologous to mouse ccdc151. ODA10p is present in cytoplasm and flagella, remains axonemal after detergent treatment, and is extracted with 0.6 M NaCl. Both outer arm dynein and ODA10p rebound to the axonemes when desalted extracts are mixed with oda10-mutant axonemes. Sucrose gradient separation of these extracts shows that ODA10p sediments near the top of the gradient, not with 23S outer dynein arm proteins. Unexpectedly, dynein and ODA10p fractions are able to bind individually to oda10 axonemes. ODA10p is present on oda8-mutant flagella at wild-type levels. However, ODA10p does not assemble into oda5 flagella and is absent from oda5 cytoplasm, suggesting a necessity of ODA5p for stability of ODA10p in vivo. The results suggest that ODA10p does not function as a part of a traditionally defined docking complex.

Cilia and flagella are conserved hair‐like appendages of eukaryotic cells that function as sensing and motility generating organelles. Motility is driven by thousands of axonemal dyneins that require precise regulation. One essential motility regulator is the central pair complex (CPC) and many CPC defects cause paralysis of cilia/flagella. Several human diseases, such as immotile cilia syndrome, show CPC abnormalities, but little is known about the detailed three‐dimensional (3D) structure and function of the CPC. The CPC is located in the center of typical [9+2] cilia/flagella and is composed of two singlet microtubules (MTs), each with a set of associated projections that extend toward the surrounding nine doublet MTs. Using cryo‐electron tomography coupled with subtomogram averaging, we visualized and compared the 3D structures of the CPC in both the green alga Chlamydomonas and the sea urchin Strongylocentrotus at the highest resolution published to date. Despite the evolutionary distance between these species, their CPCs exhibit remarkable structural conservation. We identified several new projections, including those that form the elusive sheath, and show that the bridge has a more complex architecture than previously thought. Organism‐specific differences include the presence of MT inner proteins in Chlamydomonas, but not Strongylocentrotus, and different overall outlines of the highly connected projection network, which forms a round‐shaped cylinder in algae, but is more oval in sea urchin. These differences could be adaptations to the mechanical requirements of the rotating CPC in Chlamydomonas, compared to the Strongylocentrotus CPC which has a fixed orientation. © 2012 Wiley Periodicals, Inc

David Mitchell, Hannah Mitchison and colleagues identify a new Chlamydomonas protein required for the preassembly of axonemal dyneins before their transport into cilia. They further show that mutations in the homologous gene in humans result in primary ciliary dyskinesia accompanied by defects in the assembly of inner and outer dynein arms. Primary ciliary dyskinesia most often arises from loss of the dynein motors that power ciliary beating. Here we show that DNAAF3 (also known as PF22), a previously uncharacterized protein, is essential for the preassembly of dyneins into complexes before their transport into cilia. We identified loss-of-function mutations in the human DNAAF3 gene in individuals from families with situs inversus and defects in the assembly of inner and outer dynein arms. Knockdown of dnaaf3 in zebrafish likewise disrupts dynein arm assembly and ciliary motility, causing primary ciliary dyskinesia phenotypes that include hydrocephalus and laterality malformations. Chlamydomonas reinhardtii PF22 is exclusively cytoplasmic, and a PF22-null mutant cannot assemble any outer and some inner dynein arms. Altered abundance of dynein subunits in mutant cytoplasm suggests that DNAAF3 (PF22) acts at a similar stage as other preassembly proteins, for example, DNAAF2 (also known as PF13 or KTU) and DNAAF1 (also known as ODA7 or LRRC50), in the dynein preassembly pathway. These results support the existence of a conserved, multistep pathway for the cytoplasmic formation of assembly competent ciliary dynein complexes.

The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, 54 proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologs of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles, and orthologs has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans.

In the alga Chlamydomonas reinhardtii, Oda16 functions during ciliary assembly as an adaptor for intraflagellar transport of outer arm dynein. Oda16 orthologs only occur in genomes of organisms that use motile cilia; however, such cilia play multiple roles during vertebrate development and the contribution of Oda16 to their assembly remains unexplored. We demonstrate that the zebrafish Oda16 ortholog (Wdr69) is expressed in organs with motile cilia and retains a role in dynein assembly. Antisense morpholino knockdown of Wdr69 disrupts ciliary motility and results in multiple phenotypes associated with vertebrate ciliopathies. Affected cilia included those in Kupffer's vesicle, where Wdr69 plays a role in generation of asymmetric fluid flow and establishment of organ laterality, and otic vesicles, where Wdr69 is needed to develop normal numbers of otoliths. Analysis of cilium ultrastructure revealed loss of outer dynein arms in morphant embryos. These results support a remarkable level of functional conservation for Oda16/Wdr69. Developmental Dynamics 239:2190–2197, 2010.