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1-1-2016 Radial Spokes-A Snapshot of the Motility Regulation, Assembly, and Evolution of Cilia and Flagella Xiaoyan Zhu Marquette University

Yi Liu Marquette University

Pinfen Yang Marquette University, [email protected]

Published version. Cold Harbor Perspectives in Biology (2016). DOI. © 2016 Cold Spring Harbor Laboratory Press. Used with permission. Downloaded from http://cshperspectives.cshlp.org/ at MARQUETTE UNIV on January 24, 2017 - Published by Cold Spring Harbor Laboratory Press

Radial Spokes—A Snapshot of the Motility Regulation, Assembly, and Evolution of Cilia and Flagella

Xiaoyan Zhu, Yi Liu, and Pinfen Yang

The Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53201 Correspondence: [email protected]

Propulsive forces generated by cilia and flagella are used in events that are critical for the thriving of diverse eukaryotic organisms in their environments. Despite distinctive strokes and regulations, the majority of them adopt the 9þ2 that is believed to exist in the last eukaryotic common ancestor. Only a few outliers have opted for a simpler format that forsakes the signature radial spokes and the central pair apparatus, although both are unnec- essary for force generation or rhythmicity. Extensive evidence has shown that they operate as an integral system for motility control. Recent studies have made remarkable progress on the . This review will trace how the new structural, compositional, and evolutional insights pose significant implications on flagella biology and, conversely, .

he intricate radial spokes (RSs) and central Witman 1981) has especially fueled much imag- Tpair apparatus (CP) in the 9þ2 axoneme ination. In contrast, the RS is relatively simple. have attracted the interest of many. By all ac- Nonetheless, the simplicity presented a distinc- counts, flagellar motility is contingent on their tive opportunity for asking questions funda- direct interactions, and thus they are often men- mental to flagella biology. Vigorous efforts in tioned in the same context. Although motile combination with brilliant ideas and new tech- cilia and flagella in some types or some nologies in recent years have shed considerable organisms naturally lack both RSs and the CP, insight on RSs from diverse organisms, includ- for the majority that possess the 9þ2 axoneme, ing humans. Although many earlier RS studies they prove to be critical for motility. Genetic used Chlamydomonas, emerging evidence defects afflicting either structure will result in showed species-specific differences in RSs and a spectrum of dyskinesia, ranging from paralysis experimental advantages. This review will nar- to jerking to a mixed population of motile and rate the converging discoveries that unveil RSs’ dysmotile cilia. structures, molecular interactions, functions As mentioned in Loreng and Smith (2016), and divergence among different organisms, the CP is substantially more complex than the and the implications. The in-depth discussion RS. The synchronized spinning of the CP with of motility control mechanism, radial spoke rhythmic beating in certain cilia (Omoto and (RSPs) and RS mutants in Chlamydo-

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X. Zhu et al.

monas could be found in a previous review spoke head proteins (Lin et al. 2014). The bio- (Yang and Smith 2008). chemical bases underlying the distinctive RS3 morphology are unknown. The molecular context at the base of each RS MORPHOLOGY AND PERIODICITY is also distinct, suggesting that the task of the OF RADIAL SPOKES three RSs are not identical. RS1 base is adjacent Physical attributes of axonemal components are to the base of the two-headed inner arm integral to the mechanics of motile cilia and I1, whose base module contacts the MIA com- flagella. RSs were named after their radial pat- plex named after the deficiencies in modifier of tern in the axoneme cross sections (Ishikawa inner arm (mia) mutants (King and Dutcher 2016). Individual RSs rendered by conventional 1997; Yamamoto et al. 2013). mia mutants, al- electron microscopy (EM) often were hard to beit containing all I1 components, are slow discern or varied substantially. Recent cryo- swimmers deficient in as I1 mutants electron tomography (ET) that bypassed fixa- (see the section Chemical Signaling for a detailed tions and metal decoration finally resolves the discussion). RS2 and RS3/RS3S are, respective- discrepancies. Interestingly, RSs from diverse ly, adjacent to the front and back of the – organisms actually bear striking resemblances dynein regulatory complex (N-DRC) and per- but indeed with distinctions. A side view of haps the calmodulin- and spoke-associated the Trypanosome axoneme (Koyfman et al. complex (CSC) (Gardner et al. 1994; Dymek 2011) in which the CP is extruded (Fig. 1A) and Smith 2007; Heuser et al. 2009, 2012; Ur- reveals the typical radial pattern of RSs, with banska et al. 2015). This arrangement is consis- the thinner spoke stalk docking to each outer tent with distinct inner dynein anomalies in mia doublet and the enlarged head projecting to- mutants (King and Dutcher 1997) and N-DRC ward the CP.In a lateral view of Chlamydomonas mutants (Piperno et al. 1992). These precise axoneme (Fig. 1B), there are two RSs—RS1 and structural relationships are predetermined by a RS2—in each 96-nm repeat, contrary to three complex coined as a molecular ruler that is, sur- RSs for most organisms. The missing third RS— prisingly, comprised of only two coiled-coil pa- RS3—is replaced by a stand-in stub, RS3S (Pi- ralogous proteins (FAP59 and FAP172) (Oda gino et al. 2011; Barber et al. 2012; Lin et al et al. 2014a). Defects in their encoding 2014). The distances among the three RSs ad- in Chlamydomonas mutants ( pf7 and pf8) result here to a multiplication of 8 nm—the length of in diminished inner dynein arms and N-DRC. ab-tubulins—namely 32, 24, and 40 nm. Interestingly, the 32- to 64-nm RS periodicity is As revealed by negative-stained splayed ax- replaced by a 32-nm only periodicity. Length- oneme (Witman et al. 1978), RS1 and RS2 ap- ening these ruler proteins also alters the period- pear as a T shape, which, as the view rotates icity of RSs or their adjacent ensemble. It is in- outward, turns into a Y shape with an enlarged terpreted that RS periodicity is founded on the head connected to a neck region by two arms molecular ruler that recruits N-DRC and inner (Fig. 1C). The top view shows spoke heads com- dynein arms, which in turn inhibits the inherent prised of two asymmetric modules arranged in a ability of RSs in binding to outer doublets. twofold rotational symmetry (Fig. 1D), pre- sumably each connected to the neck by one RADIAL SPOKE PROTEINS arm. At higher resolutions, the spoke head in IN CHLAMYDOMONAS humans (Fig. 1E) or sea urchins appeared small- er and more compact, lacking the tendril-like The repertoire of RS mutants (Huang et al. extensions in protist spoke heads. Notably, 1981), the RSP 2D map (Piperno et al. 1981), RS3 has a rather different morphology, with nuclear transformation (Diener et al. 1990), and an asymmetric head (Fig. 1E) and a kinked stalk. extraction of RS particles (Yang et al. 2001) set Furthermore, RS3 seems unaffected in primary the stage for the comprehensive identifications cilia dyskinesia (PCD) patients with defective of RSPs in Chlamydomonas, Ciona (Satouh et al.

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The Basic and Add-Ons of Radial Spokes

A B C – Head Dpy-30 – Neck RIIa

RS1RS2RS3S RS3S RS2 RS1 3 2 NDK5 RSP2 NDK5

100 nm

D F G

6/4 9 10/1

40 nm E

Figure 1. Structures of radial spokes. (A–E) Cryo-electron tomogram renditions of . (A) Cross section from Trypanosome brucei in which the central pair apparatus was extruded offered an unobstructed view of the entire radial spoke. The central pair apparatus is absent in this sample (electron microscopy [EM] Data Bank [EMDB] ID 5302; Koyfman et al. 2011). (B,C) Lateral view of the T-shaped twin radial spokes, RS1 and RS2, in one 96-nm repeat of Chlamydomonas flagella and a counterclockwise rotation view to reveal the bifurcation of the neck region (EMDB ID 1941; Pigino et al. 2011). The bottom panels depict predicted positions of the helices from RSP2, RSP3, and NDK5 (pink, green, and blue lines) that form the two arms at the neck and tether to two head modules. Spoke HSP40 at this position is not illustrated for clarity. (D,E) Top view of a 96-nm repeat in a Chlamydomonas flagellum (EMDB ID 5845; Oda et al. 2014b) and human respiratory (EMDB ID 5950; Lin et al. 2014). Each spoke head has two modules arranged in rotary symmetry. Numbers in D indicate predicted locations of RSPs. Note humans have triplet RSs, with RS3 strikingly different from RS1 and RS2. The images were generated using the EM Navigator at the Data Bank Japan (PDBj) (pdbj.org). (F–G) Crystallographic structures predicting the assembly process of the spokeneck region with Dpy-30 domains from RSP2 (pink ribbons) and NDK5 (blue ribbons) and amphipathic helices from a RSP3 dimer (green ribbons). (F) Crystallography of tetramers of the Dpy-30 domain that is comprised of two X helical bundles. The domain exists as a homodimer in solution. In crystals, staggering of the two dimers partially exposes the binding cleft for an amphipathic helix in their interacting partners. The polypeptides are from Dpy-30 protein. The illustration is generated from PDB ID 3G36 (Wanget al. 2009). This may account for the direct association of RSP2 and NDK5 human orthologs. (G) The face-to-face orientation of the two dimeric Dpy-30 domains binding to an amphi- pathic helix from Ash2 in the Set1-like histone methyltransferase complex. The illustration is generated from PDB ID 4RIQ (Tremblay et al. 2014). This may represent the conformational change as the RSP2/NDK5 complex associates with RSP3’s amphipathic helix.

2005; Satouh and Inaba 2009), and other organ- tides. Two are ubiquitous—calmodulin and isms. RSPs were first defined based on the miss- LC8, which are present in multiple protein com- ing 17 proteins in the spokeless axonemes of plexes—and therefore are not absent in pf14. Chlamydomonas mutant, pf14 (Huang et al. Together, these 19 proteins reside in the Y- 1981). Extracted RS particles contain these 17 shaped RS complex. The rest are likely tethered RSPs, tubulins, and at least six more polypep- to the RS base and coextracted when microtu-

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X. Zhu et al.

bules are disrupted by the KI buffer. All Chla- which otherwise bears no other PKA signature mydomonas RSPs have been identified except features (Fig. 1B). This led to a prediction that RSP13 (Yanget al. 2006). RSP13 may be orthol- some “AKAPs”—including RSP3 in Chlamydo- ogous to CMUB116 in Ciona RSs, a conserved monas—anchors proteins with a RIIa domain protein with both a calmodulin-binding do- that tethers functional modules irrelevant to main and a ubiquitin domain. There is no evi- PKA, and the term of “AKAPs” should be re- dence yet that the content in RS1 and RS2 dif- served for those that anchor PKA holoenzyme fers. Only those that were studied further will be in vivo (see detailed discussion in the section discussed below. Evolution of Radial Spokes). In addition, RSP3 harbors another amphipathic helix that binds Dpy-30 domains in RSP2 and NDK5. Chemical ORGANIZATION OF RSPs cross-linking suggests further association of Based on partial deficiencies in a few Chlamydo- NDK5 with RSP1, and RSP2 with RSP4/RSP6 monas RS mutants, the 17 RSPs were assigned to (Kohno et al. 2011). separate regions—five (RSP 4, 6, 1, 10, and 9) in Taken together, these observations support the head, three (RSP2, HSP40, and RSP23) at the a model that one RSP3 dimer serves as a scaffold neck, and the rest at the stalk (Huang et al. 1981; to anchor the rest of the RSPs. The conserved Patel-King et al. 2004; Yanget al. 2006) (Fig. 1B– region of RSP3 likely defines the RS length that E). The comprehensive data showed that the is strictly conserved (Gaillard et al. 2001). Its largely symmetric RS1 and RS2 contained amino-terminal region docks RSs to the outer many homodimers and paired paralogs with doublets, whereas its carboxyl terminus directs an identical domain. For example, in the spoke toward the spoke head (Sivadas et al. 2012; Oda head, RSP4 and 6 are paralogs expressed from et al. 2014b). This orientation positions the duplicated genes (Curry et al. 1992), whereas RIIa-containing RSP7 and RSP11 toward the RSP1 and 10 are homologs with membrane oc- outer doublets and the two Dpy-30-containing cupation and recognition nexus (MORN) re- RSP2 and NDK5 toward the spoke head (Fig. peats. Analysis of recombinant proteins and tag- 1B). As such, the carboxy-terminal helices in ging indicate that each spoke head contains two RSP2, RSP3, and NDK5 may form the arms copies of each protein that is inherently dimeric that bifurcate at the neck and secure two spoke (Kohno et al. 2011; Oda et al. 2014b). Further- head modules directionally (Fig. 1B,C, bottom more, the RIIa domains in RSP11 and RSP7, and panel). For the spoke head, we designate that the Dpy-30 domains in RSP2 and RSP23 RSP6 is peripheral to RSP4, and RSP1 is periph- (NDK5) in the stalk are dimerization and dock- eral to RSP10 (Fig. 1D), because both RSP6 and ing (DD) domains of striking similar tertiary RSP1 are not required for spoke head assembly structures (Sivadas et al. 2012). Finally, RSP3 in Chlamydomonas (Wei et al. 2010; X Zhu, un- that docks the RS to outer doublets also exists publ.); and RSP10 could be chemically cross- as a homodimer (Diener et al. 1993; Wirschell linked into a homodimer (Kohno et al. 2011). et al. 2008). RSP9 is required for spoke head assembly but RIIa-containing RSPs showed the physio- there is insufficient data to predict its location. logical relevance of the binding of RSP3 to the RIla-containing RII in the cAMP-dependent EVOLUTION OF RADIAL SPOKES protein kinase (PKA) (Gaillard et al. 2001) in an in vitro assay credited for the discoveries of In spite of the similar Y-shaped morphology, many PKA-anchoring proteins (AKAPs). As RSs diverged evolutionarily—toward simplic- such, RSP3 is often referred to as an AKAP.De- ity—perhaps accelerated by duplication, letion mutagenesis (Sivadas et al. 2012) and redundancy, and the versatility of DD domains. structural tagging (Oda et al. 2014b) confirmed Consistent with partial redundancy of spoke that an RII-binding amphipathic helix in RSP3 head proteins, the Basic Local Alignment Search binds to the RIIa domain in RSP7 and RSP11, Tool (BLAST) search indicates that RSP1 and

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The Basic and Add-Ons of Radial Spokes

its predicted partner, NDK5, are absent in Tet- terparts. For instance, contrary to Chlamydo- rahymena and other ciliates, whereas Ciona in- monas RSP2’s 738 aa residues, human RSP2 testinalis as well as sea urchins only have one counterparts—DYDC1 and DYDC2—are only gene for RSP4/6 (Table 1) (P Yang, unpubl.). 170-aa long, encompassing only the region This is substantiated by the presence of only one required for rhythmic beating, including the RSP4/6 and one MORN protein in purified Dpy-30 domain for docking to RSP3, and the Ciona RSs (Satouh and Inaba 2009). This may flanking helices perhaps for forming the bifur- be also true for humans, although humans have cated arms that interact with RSP4/6 (Fig. genes for RSP4, 6, 1, and 10. The abundance 1B,C) (Gopal et al. 2012). As the extensions of the transcripts (Kott et al. 2013) or express are predicted to be near the spoke head, absence sequence tags (ESTs) for RSP1 (RSPH1) and of the extension is consistent with metazoans’ RSPH10 are opposite in airway and testis. Sim- smaller, compact spoke head. Although these ilarly, RSPH4 transcripts are far more abundant carboxy-terminal tails are dispensable, RSP2s than RSPH6 transcripts. This raises the pos- and NDK5s have calmodulin-binding motifs. sibility that the spoke head in human cilia may Chlamydomonas cells lacking RSP2’s tail cannot contain homodimers of RSPH4 and RSPH1, steer properly under bright light (Gopal et al. forsaking RSPH6 and RSPH10, whereas 2012). This suggests that the calmodulin-bind- RSPH1 is absent in sperm flagella. As such, the ing extension acts on the spoke head region notion that certain defective RS genes will not (Fig. 1B,C) to coordinate flagellar motility as affect fertility and thus will be more prevalent this photosynthetic organism navigates illumi- (Onoufriadis et al. 2014) should be entertained. nated environments. Such measures apparently Simplification also reflects in the sizes of are unnecessary for most organisms. RSP1, 2, 3, and NDK5 in the head/neck region The RS epitomizes the versatility of DD do- (Table1). These proteins from Tetrahymena and mains and their amphipathic helix partners Chlamydomonas have a long carboxy-terminal (Gopal et al. 2012; Sivadas et al. 2012). The extension that is absent in their metazoan coun- DD domains bring to molecular complexes

Table 1. Divergence of radial spoke proteins (RSPs) in the head and neck region of four representative species, as shown by their sizes and selective absence Species

RSP C. r. T. t. C. i. H. s. RSP4 465 463 527 717 486 RSP6 459 493 N/D 612 RSP1 814 N/D 300 309 RSP10 216 227 N/D 870 221 RSP9 269 296 276 276 463 RSP2 (DYDC1,2) 738 628 169 177 RSP23 (NDK5) 586 N/D 257 212 RSP3 516 691 336 380 (N’160) 950 764 RSPs in the head region are shaded in gray. C.r., Chlamydomonas reinhardtii; T. t., Tetrahymena thermophile; C. i., Ciona intestinalis; H. s., Homo sapiens. The polypeptides were identified based on a BLAST search against Chlamydomonas RSPs. Those from C. r. and C. i. were also confirmed by protein biochemistry of isolated RS particles (Yang et al. 2006; Satouh and Inaba 2009). Note that some RSPs in the protists are far larger, while human RSP3 has an additional 160-aa fragment at the amino terminus (N’160) (Jivan et al. 2009). N/D, Not detectable in the genome or RS proteome.

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their tethered effector moieties—for cNMP sig- that they assemble first, perhaps mediated by naling in the case of RII in PKA, for calcium- the Dpy-30 domain that was crystallized as two sensing such as RSP7, for nucleoside metabo- partially staggered dimeric dimers (Fig. 1F). In- lism such as NDK5, and for assembly such as teractions with amphipathic helices in the RSP3 DYDC2 and RSP11. The tethered moieties dimer during the assembly of the 12S RS pre- could further duplicate or combine, as in Chla- cursors might reposition the two sets of dimers mydomonas’ RSP2 and NDK5. Mammals par- into a face-to face orientation (Fig. 1G). This ticularly have multiple genes that encode RIIa- structure-based interpretation is concordant containing proteins, most of which are enriched with the detection of RS subparticles in the in testis. But they share little semblance to each RSP3 mutant (Diener et al. 2011). other, RSP7, or RSP11 except the RIIa domain The distinct sedimentation coefficients of (e.g., Newell et al. 2008). The divergence of DD 12S and 20S RS particles are more likely caused domain proteins may allow airway and testis to by disparateness in shapes than masses. While build slightly different RSs and perhaps CPs that the Y-shaped mature RSs are about 32 nm by also contains an RIIa-binding protein (Gaillard 42 nm, the 12S RS is a D-shape of 20 nm by et al. 2001; Rao et al. 2016). Some of them bind 28 nm in negative-stained EM (Diener et al. to “AKAPs” in the fibrous sheath of sperm fla- 2011). The precursors lack the two strictly con- gella (e.g., Fiedler et al. 2012). As such, bona fide served small dimeric proteins—HSP40 and RIIa partners of RSP3 and any AKAPs may dif- LC8—that are involved in the conformational fer in each cell type, and should be determined change during RS assembly. Most organisms empirically. Although RSP3 does not bind have a multitude of HSP40 genes. Typically, PKA’s RII in Chlamydomonas, it potentially HSP40s cooperate with other chaperones to could in animals’ motile cilia or flagella as a mediate protein folding, whereas some may bona fide AKAP. Different RIIa partners may act solitarily. Flagella that lack HSP40 jerk in- explain opposite effects of PKA on Chlamydo- cessantly although the other RSPs appear nor- monas flagella and other cilia and flagella (Ha- mal (Yanget al. 2008). EM revealed irregular tilt segawa et al. 1987). It is worthwhile to point out or a lower bifurcated neck of RSs. These obser- that RSP3 indeed possesses a key feature of vations support a model that the V-shaped di- AKAPs, the scaffold of molecular complexes, meric HSP40, transported differently from 12S to integrate proteins of diverse functions for precursors, bind to the polypeptides at the neck flagellar motility control. Although Chlamydo- region at the flagellar tip where final assembly monas RSP3 is not a typical PKA-anchoring occurs (Johnson and Rosenbaum 1992). LC8 AKAP, it still anchors calcium-sensing mole- dimer, present in numerous protein complexes cules perhaps for calcium-related motility con- including multiheaded and RSs, typi- trol and structural proteins for transducing me- cally brings two polypeptides in a complex to- chanical feedback. gether. But in RSs, a stack of LC8 dimers bring together the amino terminus of an RSP3 dimer in RS precursors to form a rod at the RS base, RS ASSEMBLY promoting RSP3’s phosphorylation and dock- RSPs are assembled into RSs in two phases. ing to the outer doublets (Yang et al. 2009; Fractionations of cell body extracts and the fla- Gupta et al. 2012). gellar membrane matrix suggest that the cell RSP3 phosphorylation istightly linked to RS body only makes partially furbished RSs that docking. RSP3 is hypophosphorylated in RS sediment as 12S particles in the sucrose gradient precursors and in mutants with reduced RSs, (Qin et al. 2004). After delivery into flagella by such as fla14 that lacks LC8 and pf27 (Yang and intraflagellar transport (IFT), 12S precursors Yang2006; Guptaet al. 2012), suggesting that the are converted into 20S mature RSs. The direct RSs in pf27,asinfla14, are not assembled via a interaction of human orthologs of NDK5 and normal process that involves phosphorylation. RSP2 at the neck (e.g., Rual et al. 2005) suggests The PF27 gene has not been identified but likely

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The Basic and Add-Ons of Radial Spokes

encodes a protein outside of flagella (Huang tions. They are paralyzed if RS-CP contacts are et al. 1981). Interestingly, RSs in pf27 preferen- absent because they lack the entire RS, the spoke tially distribute toward the base of flagella (Al- head, or spoke head and neck. Flagella lacking ford et al. 2013). It is proposed that PF27 is an spoke HSP40 jerk constantly. In contrast, the adaptor linking spoke precursors to IFT. paralysis of the spoke head mutant pf26 (RSP6) is conditional. In the healthy log phase cultures at room temperature, the motility and the other RS OUTER DOUBLET CONNECTIONS spoke head proteins appear normal (Huang et Much progress has been made in elucidating the al. 1981; Weiet al. 2010). However, in stationary distinct structural linkages of the three RSs. phase cultures, pf26 cells are largely paralyzed, Three proteins (CaM-IP2, IP3, and IP4) cosedi- lacking nearly all spoke head proteins in flagella. ment with the 20S RS fraction but were not In an intermediate condition, a culture will con- abundant enough for identification. Instead, tain swimmers, immotile cells, and cells with they were identified in the pull-down of the jerky (uncoordinated) flagella. Therefore, al- anticalmodulin antibody. Knockdown of CSC though RSP26 is dispensable in the laboratory, proteins impaired RS2, RS3S, N-DRC, and a it is presumably needed in Chlamydomonas’nat- single-headed dynein, e (Dymek et al. 2011; ural environment in which the temperature and Heuser et al. 2012), suggesting that the CSC is nutrient availability are expected to fluctuate. among the base of RS2 and RS3, N-DRC, and Similar motility phenotypes are shown by pf25 the nearby dynein. Consistent with this, the an- that lacks RSP11 (Yangand Yang2006) and par- tibody for a CSC protein inhibits tially rescued RS mutants (Gupta et al. 2012). sliding velocity as the reduced microtubule slid- Similar scenarios were noted in other spe- ing velocity of pf14 axonemes. And yet the cies. Tetrahymena FAP206 knockout could seemingly normal CSC in pf14 axonemes indi- swim despite diminished RS2. Its velocity is re- cates that the CSC assembly is independent of duced, attributed to altered waveform, likely the rest of RSs (Dymek and Smith 2007). because of the deficit of inner dynein arms (Va- A pull-down of the spoke stalk recovered the sudevan et al. 2015) and uncoordinated beat as CSC and another protein that associated with expected of minor RS deficits. Unexpectedly, RS2, FAP206 (Gupta et al. 2012). FAP206 is cilia of human RS PCD patients beat rhythmi- reduced in pf14 axonemes (Lin et al. 2011). cally with helical waveform—amid asynchro- Knockout of Tetrahymena FAP206 also impaired nous beating ones in some cases—despite lack- RS2 specifically, and indicated that FAP206 ing spoke heads in RS1 and RS2 or most of RSs linked the proximal end of RS2 base to the sin- (Burgoyne et al. 2014; Jeanson et al. 2015). The gle-headed dynein, c (Vasudevan et al. 2015). helical movement is attributed to the presence Therefore, FAP206 and the CSC appear to link of RS3, because Chlamydomonas flagella with the base of RS2 to different single-headed dyne- same genetic defect and lacking RS3 are immo- ins aligned in tandem. The biochemical evi- tile. However, one should bear in mind that dence for direct coupling among RS1, the MIA natural 9þ0 cilia and flagella generate rhythmic complex, and I1 has not yet emerged. beating with helical waveform without any RS (Nonaka et al. 2002). Therefore, phenotype polymorphism, cell conditions, and evolution- THE SPECTRUM OF MOTILITY ANOMALIES ary divergence should be considered in pheno- OF RS MUTANTS typing RS mutants. It is important to note that not all RS mutants are immotile. Although Chlamydomonas RS ROLES OF RSS mutants are known for paralyzed flagella, they actually show a spectrum of motility anomalies, It will be easier to appreciate RSs if one consid- depending on the defective RSP, the nature of ers the 9þ2 cilia and flagella as reversible bio- mutations, and, surprisingly, the culture condi- logical nanomachines.

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Generation of Planar Waveform right asymmetry (Nonaka et al. 2002; Shinohara et al. 2015). By the same token, 3D-bihelical This is not absolute. The 9þ0 motile cilia is movement is what Trypanosome adopt to navi- rare, such as that found in the node or certain gate in the viscous bloodstream. eel and horseshoe crab sperm (Gibbons et al. 1985; Ishijima et al. 1988). As such, they con- note an inferior perception. However, these Coordination of Molecular Motors by Mechanical Feedback sperm swim rapidly with a 9þ0 flagellum of a high beat frequency. Importantly, the typical Diverse evidence suggests that the RS-CP system waveform is helical, similar to the movement overrides the inherent helical movement of the of the respiratory cilia of RS PCD patients outer doublet bundle by serving as a venue for (e.g., Castleman et al. 2009; Jeanson et al. mechanical feedback, perhaps of forces or 2015) and reactivated axonemes of paralyzed tensions. Although Chlamydomonas RS or CP Chlamydomonas RS or CP mutants under al- mutants have paralyzed flagella, their axonemes tered conditions (e.g., Yagi and Kamiya 2000; are able to vibrate in reactivation conditions, and see Loreng and Smith 2016). suggesting that dynein motors are active but In contrast, 9þ2 cilia usually display planar are not coordinated (Yagi et al. 1994). An waveform of a large amplitude, but retain the additional mutation in outer dynein or N- ability to generate helical waveform. For Chla- DRC partially rescues the paralysis (e.g., Huang mydomonas, power strokes are largely planar, et al. 1982; Porter et al. 1994; Rupp et al. 1996). but recovery strokes are helical. The planar This leads to the prediction that the RS-CP waveform with large amplitude appears to be system coordinates the activation of dynein crucial for respiratory cilia to generate sufficient motors via N-DRC—to generate planar wave- propulsive forces, because the helical movement form in most cases. of cilia in RS PCD patients does not offer suffi- Generation and propagation of a planar cient despite a normal waveform are a result of alternate sliding of op- beat frequency. An exception is the 9þ2axo- posing subsets of outer doublets at any par- neme of Trypanosome that generates 3D bihel- ticular moment. The selection appears deter- ical movements, perhaps because of the para- mined by the CP with asymmetric projections flagellar rod, a cytoskeletal system unique to (see Loreng and Smith 2016). The geometric kinoplastid parasites (Portman and Gull 2010; clutch model (Lindemann and Lesich 2015) Koyfman et al. 2011). Interestingly, helical and offers an explicit explanation of mechanical planar waveforms of both 9þ0 and 9þ2 flagella feedback. It posits that the CP and RSs, which could be switched by changes in physical prop- contact intermittently during each beat cycle erties of the aqueous environment, such (Warner and Satir 1974), transmit transverse as surface tension and viscosity (Ishijima et forces developed from flagellar bend, which in al. 1988; Woolley and Vernon 2001). Therefore, turn differentially alter the distances among nine outer doublets are inherently switchable to doublets confined by the base, tip, and N- various waveforms. DRC links. Like the action of an automobile We envisage that the RS-CP system is a de- clutch, increasing and reducing interdoublet vice for swift reordering of the activation se- distances would, respectively, disengage and en- quence of motors in the nine outer doublets. gage distinct subsets of dynein motors, leading The resulting high-frequency beating of planar to bend propagation and rhythmic beating. waveform is hydrodynamically favorable in This theory is concordant with elliptic versus most circumstances. But such movement may circular cross sections of motile and immotile not be appropriate for a select few. The low- axonemes, and oscillation of axoneme diame- frequency helical beat of 9þ0 nodal cilia may ters during rhythmic beating (Warner 1978; Sa- be just right for establishing a gradient of mor- kakibara et al. 2004; Lindemann and Mitchell phogens that direct the development of left– 2007; Yang et al. 2008).

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The Basic and Add-Ons of Radial Spokes

Mechanical feedback is also supported by structure corridor from the CP to dynein motors perturbations that paralyze motile flagella or (see Loreng and Smith 2016). Indeed, the mu- rescue the paralyzed. For example, simply re- tant lacking the calmodulin-binding region in moving HSP40 at a spoke neck resulted in jerky RSP2 is capable of normal swimming and both flagella with uncoordinated bend initiation, light-induced responses, but cannot steer under propagation, and switching, presumably be- bright light (Gopal et al. 2012). It is likely that cause of weakening of RS rigidity necessary for motility control is via concerted actions of mul- force transmission (Yang et al. 2008). Converse- tiple calcium sensors, rather than a handful of ly, forced bending could jolt paralyzed flagella to calcium switches. RSs may be involved in this via beat rhythmically for a brief period (Hayashibe the RSPs with calcium- and calmodulin-bind- et al. 1997). Finally, adding a large tag to the ing motifs. If calmodulin binding of Tetrahyme- spoke head rescued paralyzed flagella of a CP na RSP4 and RSP6 (Ueno et al. 2006) proves mutant, probably complementing a missing physiologically relevant, these two conserved CP projection to restore structural contact nec- molecules are appealing candidates for universal essary for mechanical signaling (Oda et al. major calcium switch. 2014b). Suppressor mutations may alter N- Independent approaches strongly suggest DRC or motors as to bypasses the defective me- that RSs also regulate dynein motors via phos- chanical feedback, or return toward the 9þ0 phorylation—of the inner dynein arm, I1. machinery. While RS mutant flagella are paralyzed, their axoneme can undergo interdoublet sliding al- beit with a reduced velocity (Witman et al. 1978; Chemical Signaling Smith and Sale 1992). The inhibition stems The RS-CP system is integral to the modulation from phosphorylation mediated by kinases of cilia and flagella motility by calcium, cal- such as PKA and CK1 (Howard et al. 1994; modulin, cyclic nucleotides, and phospho- Yang and Sale 2000) when RSs are defective, rylation. Consistent with this, the calcium- and could be antagonized by calcium-indepen- dependent waveform changes appeared missing dent phosphatases (Habermacher and Sale in 9þ0 sperm flagella (Gibbons et al. 1985). 1996). The key substrate of these enzymes is Contrary to the expected universal principle the intermediate chain IC138 at I1 base, near of mechanical feedback, the mechanisms of RS1 (Habermacher and Sale 1997). IC138 hy- chemical signaling may differ substantially perphosphorylation as well as defects in I1 and for Chlamydomonas, which use two relatively the MIA complex correlate with reduced sliding short flagella to steer, and for sperm’s long fla- velocity (Yamamoto et al. 2013) and, interest- gella, which are activated by Wnt signaling ingly, ineffective phototaxis (King and Dutcher (Koch et al. 2015). 1997; Okita et al. 2005). Therefore, I1 is a key The biflagellate Chlamydomonasis known to effector that enables the calcium-dependent show two calcium-dependent responses: awave- of one flagellum for turning the tra- form switch when encountering barriers or jectory during phototaxis. RS may operate to stimulated by intense white light (photoshock), suppress IC138 phosphorylation that may oth- and phototaxis in an environment with differ- erwise hinder such responses. ential light intensity (Yangand Smith 2008). The RS-CP system is proposed to be involved in the Maintenance of 9þ2 Axoneme Structural former because of the symmetric waveform of Stability the suppressor of RS mutants (Brokaw et al. 1982). While this is likely, calcium signaling This role is not as well known as motility con- pathways are far more complicated given more trol, and is less obvious in Chlamydomonas than than 80 proteins in algal flagella are capable of in some other organisms. First of all, RSs nudge binding calcium or calmodulin (Pazour et al. the CP to its central location (e.g., Sivadas et al. 2005), including multiple ones residing in the 2012). The CP shifts from the central location

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X. Zhu et al.

when RSs are defective. In addition, RSs en- mediated by PKA and CK1 that also regulate hance the stability of the CP and the entire ax- critical events occurring at the area oneme. Cilia EMs from RS PCD patients often in diverse organisms (Briscoe and Therond show an array of structural anomalies such as 2013). Although Chlamydomonas, like plants, two CPs and transposition—the replacement of do not have the typical PKA tetramer common the CP by an outer doublet (e.g., Castleman in animals, PKA activity is indisputable (Hase- et al. 2009; Kott et al. 2013). Cilia tomography gawa et al 1987; Howard et al. 1994; Yang and suggests that with defective RSs, the CP actually Sale 2000). Identification of PKA in this organ- is assembled but tends to disassemble, leading ism and elucidation of how anchored PKA and to transposition (Burgoyne et al. 2014). Tet ra- CK1 phosphorylate IC138 is central to eluci- hymena axonemes with diminished RS2 are also date RS-mediated regulation. Related, but not susceptible to deformation (Vasudevan et al. identical, to this are the questions about how 2015). Eel 9þ0 axoneme is exceptionally fragile RSPs become phosphorylated and whether the when the membrane is removed (Gibbons et al. phosphorylation is a consequence or a cause of 1985). Conversely, the perceived structural RS assembly. The third is RS assembly, likely via stability of spokeless axonemes in Chlamydo- spoke-unique mechanisms because of drastic monas RS mutant may be caused by multiple differences between spoke precursor and mature factors (LeDizet and Piperno 1995; Kubo et al. RSs, the multiple -like spoke sub- 2015). Nonetheless, the structural instability units, and subunits of a low stoichiometry (Pi- surfaces as short flagella of double mutants perno et al. 1981). The fourth is characterization lacking both RSs and the CP, likely caused by of RS3. Aside from satisfying curiosity, the find- an increasing disassembly rate. ings may lead to new insight of RS evolution— While all major axonemal complexes may how Chlamydomonas managed to lose RS3, and confer stability to the axoneme that is built on what are the expense and benefits to retain a inherently unstable (Kubo et al. distinct RS3. Last, but not least, is to elucidate 2015), we take the liberty to speculate that the RSs of mammals and phenotype RS PCD pa- RS-CP system is particularly so by filling up the tients. With the advance of technologies and hollow center, and by the transient contact that reagents (Lin et al. 2014; Frommer et al. 2015), dissipates forces and tensions, which the axo- the challenges inherent to these systems (Papon neme endures as it repetitively sweeps through et al. 2010) will be overcome and the findings fluid of relatively high viscosity. This may ex- will complement the limitations of simple mod- plain the nearly identical dimensions of RSs el systems. and thus the diameter of 9þ2 axonemes across all sources. Interestingly, the ratio of the dimen- sions of the CP and RS versus the CP in cross ACKNOWLEDGMENTS sections is strikingly similar to the golden ratio We thank Dr. James Courtright (Marquette (w). Perhaps the 9þ2 axonemal platform of the University) for his advice and editing. last common ancestor of is as good as it gets. Much of the subsequent changes are all but individual preferences. REFERENCES Reference is also in this collection.

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The Basic and Add-Ons of Radial Spokes

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The Basic and Add-Ons of Radial Spokes

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Advanced Online Article. Cite this article as Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a028126 13 Downloaded from http://cshperspectives.cshlp.org/ at MARQUETTE UNIV on January 24, 2017 - Published by Cold Spring Harbor Laboratory Press

Radial Spokes−−A Snapshot of the Motility Regulation, Assembly, and Evolution of Cilia and Flagella

Xiaoyan Zhu, Yi Liu and Pinfen Yang

Cold Spring Harb Perspect Biol published online December 9, 2016

Subject Collection Cilia

Transition Zone Migration: A Mechanism for Open Sesame: How Transition Fibers and the Cytoplasmic and Postaxonemal Transition Zone Control Ciliary Composition Elongation Francesc R. Garcia-Gonzalo and Jeremy F. Reiter Tomer Avidor-Reiss, Andrew Ha and Marcus L. Basiri The Central Apparatus of Cilia and Eukaryotic Cilia and Obesity Flagella Christian Vaisse, Jeremy F. Reiter and Nicolas F. Thomas D. Loreng and Elizabeth F. Smith Berbari Sperm Sensory Signaling Posttranslational Modifications of Tubulin and Dagmar Wachten, Jan F. Jikeli and U. Benjamin Cilia Kaupp Dorota Wloga, Ewa Joachimiak, Panagiota Louka, et al. Radial Spokes−−A Snapshot of the Motility Multiciliated Cells in Animals Regulation, Assembly, and Evolution of Cilia and Alice Meunier and Juliette Azimzadeh Flagella Xiaoyan Zhu, Yi Liu and Pinfen Yang Primary Cilia and Mammalian Hedgehog Signaling Cilia and Mucociliary Clearance Fiona Bangs and Kathryn V. Anderson Ximena M. Bustamante-Marin and Lawrence E. Ostrowski Axonemal Dynein Arms Stephen M. King Daniela A. Braun and Friedhelm Hildebrandt The Machinery Evolution of Cilia Michael Taschner and Esben Lorentzen David R. Mitchell Primary Cilia and Coordination of Receptor Axoneme Structure from Motile Cilia Tyrosine Kinase (RTK) and Transforming Growth Takashi Ishikawa Factor β (TGF-β) Signaling Søren T. Christensen, Stine K. Morthorst, Johanne B. Mogensen, et al.

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