sign in sign up
<<
Home , Trypanosoma brucei

478 Research Article Functional genomics in brucei identifies evolutionarily conserved components of motile flagella

Desiree M. Baron1, Katherine S. Ralston1, Zakayi P. Kabututu1 and Kent L. Hill1,2,* 1Department of Microbiology, Immunology, and Molecular Genetics and 2Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA *Author for correspondence (e-mail: [email protected])

Accepted 1 November 2006 120, 478-491 Published by The Company of Biologists 2007 doi:10.1242/jcs.03352

Summary Cilia and flagella are highly conserved, complex organelles knockdown mutants. Epitope tagging, fluorescence involved in a variety of important functions. Flagella are localization and biochemical fractionation demonstrated required for motility of several human and flagellar localization for several TbCMF proteins. Finally, ciliary defects lead to a variety of fatal and debilitating ultrastructural analysis identified a family of novel TbCMF human diseases. Many of the major structural components proteins that function to maintain connections between of cilia and flagella are known, but little is known about outer doublet , suggesting that they are the regulation of flagellar beat. Trypanosoma brucei, the first identified components of nexin links. Overall, our causative agent of African sleeping sickness, provides an results provide insights into the workings of the eukaryotic excellent model for studying flagellar motility. We have , identify several novel human disease gene used comparative genomics to identify a core group of 50 candidates, reveal unique aspects of the trypanosome genes unique to organisms with motile flagella. These flagellum and underscore the value of T. brucei as an genes, referred to as T. brucei components of motile flagella experimental system for studying flagellar biology. (TbCMF) include 30 novel genes, and human homologues of many of the TbCMF genes map to loci associated with human ciliary diseases. To characterize TbCMF protein Supplementary material available online at function we used RNA interference to target 41 TbCMF http://jcs.biologists.org/cgi/content/full/120/3/478/DC1 genes. Sedimentation assays and direct observation Journal of Cell Science demonstrated clear motility defects in a majority of these Key words: Flagellum, Motility, Trypanosome, Nexin

Introduction morphogenesis, and host-parasite interactions Eukaryotic cilia and flagella are complex, highly conserved, (Hill, 2003; Kohl et al., 2003; Ralston et al., 2006). Recent -based structures that are important in a number of work strongly suggests that flagellar motility is required for biological processes. Cilia and flagella drive cellular viability in the bloodstream stage of the T. brucei lifecycle movement of unicellular , including several (Broadhead et al., 2006; Ralston et al., 2006; Ralston and Hill, important human pathogens. Examples include multiflagellate 2006). Therefore, in protozoan parasites the flagellum is organisms such as Giardia lamblia and , essential for disease pathogenesis and parasite development. which respectively, cause epidemic diarrhea and Flagella and cilia are also important for normal human , the latter being the most common, non-viral, physiology and ciliary defects lead to a wide variety of diseases sexually transmitted disease in the world (Rughooputh and and developmental abnormalities (Afzelius, 2004; Pan et al., Greenwell, 2005). Kinetoplastid parasites, which cause 2005; Snell et al., 2004). In humans, one of the most familiar African sleeping sickness, and , flagellum-related disorders is male infertility, in which all depend on a single flagellum for cell motility in their flagellated sperm lack effective motility (Sapiro et al., 2002). vectors and mammalian hosts. Motile flagella also play a Motile cilia are also important for moving fluids across the prominent role in the mating cycle of apicomplexan parasites surface of epithelial cells of the respiratory tract and female that cause and toxoplasmosis (Ferguson, 2002; reproductive tract, as well as ependymal cells that line the Vlachou et al., 2006). Notably, the role of flagellar motility in ventricles of the brain and nodal cells of the developing embryo is not limited to cell movement, but also contributes (Afzelius, 2004). The importance of these functions is to nutrient uptake (Williams et al., 2006) and evidenced by clinical features of immotile cilia syndrome (also (Brown et al., 1999; Ralston et al., 2006). For example in known as primary ciliary dyskinesia, PCD), which include Trypanosoma brucei, the causative agent of African sleeping respiratory deficiencies, hydrocephalus and situs inversus sickness, the flagellum is now recognized to be a (Afzelius et al., 2001). In addition to their role as organelles of multifunctional organelle with crucial roles in cell motility, cell motility, human cilia serve sensory and transport functions and T. brucei components of motile flagella 479

loss of this functionality underlies major human diseases of these 41 TbCMF genes demonstrated a role in flagellar including retinopathies, polycystic kidney disease and Bardet- motility in the majority of cases. Ultrastructural and motility Biedl syndrome (Pan et al., 2005; Pazour and Witman, 2003; analyses identified one family of novel TbCMF proteins that Wang et al., 2006). appear to function as part of the nexin links. The proteins The of motile cilia typically contains nine outer identified in this study are important for understanding doublet microtubules arranged around a pair of singlet flagellar motility in microbial pathogenesis and human microtubules. Dynein arms protruding from one outer doublet physiology. toward the adjacent doublet provide the motive force for flagellar motility (Summers and Gibbons, 1971). Radial spokes Results extend inward from the outer doublets to the central pair Comparative genomics identifies conserved components apparatus and are required for motility (Witman et al., 1978). of motile flagella Connecting the outer doublets to one another are nexin links We performed an ‘in silico’ screen using available (Warner, 1976), which are thought to help maintain outer- databases to identify genes that are conserved uniquely among doublet organization (Cibert, 2001; Lindemann, 2004). In its organisms with motile flagella (Fig. 1). Our screen led to the simplest form, flagellar movement can be described as the identification of 50 predicted T. brucei proteins that are sliding of outer doublet microtubules relative to one another conserved in , major, powered by the dynein arms (Brokaw, 1972; Satir, 1968; Chlamydomonas reinhardtii, Homo sapiens, Mus musculus, Shingyoji et al., 1977; Summers and Gibbons, 1971). Ciona intestinalis and Drosophilia melanogaster, and are not Attachment of doublets to the and to each other found in Caenorhabditis elegans or Arabidopsis thaliana (Fig. limits sliding and this produces a bend, which is propagated 1, supplementary material Table S1). We refer to this group of along the axoneme through the coordinated activation and proteins as TbCMF proteins deactivation of dyneins. Although dynein arms, radial spokes As expected, the set of TbCMF proteins includes and the central pair apparatus have been well studied, homologues of proteins previously demonstrated through relatively little is known about proteins responsible for the genetic and biochemical studies to be bona fide flagellar coordinated regulation of sliding and resisting forces necessary components, as well as proteins identified in recent genomic for wave propagation. Genetic and biochemical studies in and flagellar proteomic analyses (supplementary material Chlamydomonas reinhardtii suggest that regulation is achieved Table S2) (Avidor-Reiss et al., 2004; Broadhead et al., 2006; in part by the dynein regulatory complex (DRC), which acts as Li et al., 2004; Ostrowski et al., 2002; Pazour et al., 2005; a reversible inhibitor of dynein (Huang et al., 1982; Piperno et Smith et al., 2005). Previously characterized flagellar proteins al., 1992; Gardner et al., 1994). Modeling studies suggest that that met the screen criteria include radial spoke proteins (RSP3, beat regulation also involves transverse forces supplied by the 4 and 6), DRC proteins (TPN), protofilament ribbon proteins elastic nexin links in response to axonemal curvature (Cibert, (Rib72 and Rib43a), an MBO2 homologue, and several 2001; Lindemann, 2004). Only one subunit of the DRC is axonemal dynein subunits (IC140, IC138, IC78, LC7a, LC7b known (Ralston et al., 2006; Rupp and Porter, 2003) and the and LC1). A group of predicted dynein heavy chain proteins

Journal of Cell Science composition of nexin links is completely unknown, therefore, were originally identified in the screen, but were not included identification of proteins specifically important for flagellar in the final dataset, because our analysis did not distinguish beat and regulation represents a major challenge for them unambiguously from cytoplasmic dynein sequences. The understanding flagellum function. TbCMF dataset was enriched for proteins containing WD We previously demonstrated that the DRC is an repeats, -rich repeats, EF hand calcium-binding evolutionarily conserved system for dynein regulation and that domains and IQ calmodulin-binding motifs (supplementary trypanin (TPN) functions as part of this system in T. brucei material Table S2), which are commonly found in flagellar (Ralston et al., 2006). TPN is highly conserved among proteins (Li et al., 2004). These results validate the in silico organisms with motile flagella, whereas it is absent in screen as yielding a dataset enriched for flagellar proteins. organisms that contain non-motile flagella or lack flagella Notably, the TbCMF dataset also includes 16 proteins not altogether (Hill et al., 2000; Ralston et al., 2006). We reasoned found in a proteomic analysis of extracted from T. that additional proteins important for motility and regulation brucei, as well as 28 proteins that were not identified in of motility would be similarly conserved and we therefore used previous comparative genomic studies (Avidor-Reiss et al., comparative genomics to identify genes with the same 2004; Broadhead et al., 2006; Li et al., 2004; Ostrowski et al., phylogenetic distribution as TPN. Comparative genomics has 2002). Thirty-three TbCMF proteins had not been functionally proven effective previously for identification of candidate characterized at the time of the screen. Three of these proteins flagellar and basal body genes (Avidor-Reiss et al., 2004; Li et have subsequently been demonstrated by RNAi to be required al., 2004), although previous studies did not exclusively focus for normal flagellar function in T. brucei (Broadhead et al., on organisms with motile flagella. Using this approach we 2006). The remaining 30 proteins have not been examined in identified 50 genes unique to organisms with motile flagella. any organism. This group of genes, referred to as T. brucei components of Since ciliary defects underlie several human diseases motile flagella (TbCMF), encode homologues to 17 previously (Ibanez-Tallon et al., 2003), we asked whether human identified flagellar proteins, three proteins that were homologues of TbCMF genes were associated with ciliary characterized subsequent to our screen (Broadhead et al., 2006) diseases or mapped to disease loci for which the disease gene and 30 novel proteins that have not yet been characterized in is currently unknown. As shown in supplementary material any organism. We analyzed the 30 novel genes and several Table S2, several human TbCMF homologues fall within loci known genes that had not been investigated in T. brucei. RNAi linked to ciliary-based diseases in mammals. In some cases, 480 Journal of Cell Science 120 (3)

Fig. 1. Comparative genomics identifies T. brucei components of motile flagella. (A) Overview of the strategy used to identify and characterize TbCMF genes. of organisms with motile cilia or flagella (H. sapiens, M. musculus, D. melanogaster, L. major, T. cruzi, C. reinhardtii and C. intestinalis) were compared to the T. brucei genome for common genes, then those genes found in organisms that have non-motile flagella or lack flagella (C. elegans and A. thaliana, respectively) were subtracted. Homology was confirmed by protein alignments and reciprocal BLAST analysis. To determine whether the subset of 41 functionally uncharacterized proteins have a role in motility, RNAi mutants were analyzed (see Materials and Methods for details). (B) Amino acid sequence alignment of TbCMF 9 with top BLAST hits from humans (Hs, accession number AAI09127) and C. reinhardtii (Cr, protein ID C_230118). Amino acids highlighted in yellow and blue are identical in all or most sequences, respectively; green highlighting represents conservative substitutions. The RNAi region is underlined and the borders of the SMC in TbCMF 9 are denoted by red arrowheads. Journal of Cell Science T. brucei components of motile flagella 481 Journal of Cell Science

Fig. 2. TbCMF knockdown is specific and mutants can be placed into four phenotypic classes. (A) Classification of TbCMF knockdown mutants. The majority of TbCMF mutants exhibit cytokinesis defects that result in the formation of multicellular clusters (white arrows). Mutants were placed into one of four classes based on the size and time of formation of these clusters. A representative example of each mutant class is shown. Each panel shows a phase-contrast image of the indicated TbCMF knockdown strain grown in the absence (uninduced) or presence (induced) of 0.3 ␮g/ml tetracycline for five days. (B) Northern blots of RNA from two sample TbCMF mutants from each mutant class. RNA samples were probed with the gene targeted by RNAi and with a non-target gene as a loading control. *For TbCMF 2, equal loading was confirmed using rRNA visualized by UV.

this connection has been noted previously. For example, known flagellar genes because the vast majority of these have TbCMF 2, TbCMF 3, TbCMF 4 and TbCMF 34 are only been investigated in C. reinhardtii. The nine remaining homologous to the human Rib72/EFHC1 protein, which has genes were not studied further because they have previously been directly implicated in juvenile myoclonic epilepsy-1 been characterized in T. brucei, have been shown to have a role (Suzuki et al., 2004). In other cases, this connection to human in motility in C. reinhardtii, or will be the focus of future work. disease has not previously been reported, e.g. the human The first level of analysis was to examine each RNAi homologues of TbCMF 10 and TbCMF 56 encode novel knockdown mutant at the whole-culture level by microscopy. proteins and are found at loci associated with retinitis When induced, most TbCMF-knockdown mutants formed pigmentosa and PCD, respectively. clusters of multiple cells that remained physically attached. The severity of clustering and time of onset differed among the RNAi knockdown of TbCMF gene expression mutants and this difference was used to place each mutant into To address the hypothesis that TbCMF genes are required for one of four distinct phenotypic classes (Fig. 2A). Twenty-four flagellar motility, 41 of the 50 genes were targeted for percent of mutants fell into the first class (Class 1), described tetracycline-inducible RNAi knockdown and the resulting as ‘unaffected’, because after six days of induction the induced mutants were assessed for motility defects. We focused our cultures remained indistinguishable from uninduced cultures. attention on uncharacterized genes but also included some Fifteen percent of the mutants exhibited a ‘mild’ clustering 482 Journal of Cell Science 120 (3)

Fig. 3. Class 3 and 4 TbCMF mutants have clear motility defects. The indicated mutants were assessed for cell motility defects using a

Journal of Cell Science sedimentation assay (A) (Bastin et al., 1999; Ralston et al., 2006) and by direct observation using DIC microscopy (B). In both cases, cells were assayed before significant clustering was evident to avoid secondary effects on motility. (A) Sedimentation curves. ⌬OD is the difference between the average OD600 readings for sedimented (S) and resuspended (R) samples. Error bars show a standard deviation of three independent experiments. A trypanin knockdown mutant (Hutchings et al., 2002) is included as a control for samples without (–tet) and with (+tet) motility defects. (B) Time-lapse series taken from a video clip of TbCMF 46 (a Class 3 mutant) grown in the absence (–tet) or presence (+tet) of tetracycline for 30 hours. The black arrow marks the point of origin at the start of the time-lapse series while the white arrow follows the cell. Bar, 10 ␮m. A video of TbCMF 46 motility is provided as supplementary material Movie 2. Additional movies of WT (Movie 5), class 2 (TbCMF 3, Movie 1) and class 4 (TbCMF 5, Movie 4; TbCMF 9, Movie 3) are provided as supplementary material.

phenotype (Class 2 mutants), where small clusters of 5-10 cells Therefore, phenotype severity is not simply a reflection of the appeared within 2 days post induction (dpi), and these clusters degree of knockdown. remained constant in size and remained homogenously distributed. There was no apparent growth defect associated TbCMF genes are required for normal flagellar beat with Class 2 mutants. Approximately 34% of TbCMF mutants We previously demonstrated that flagellar motility defects can exhibited a ‘moderate’ clustering phenotype (Class 3 mutants), interfere with the completion of cytokinesis in T. brucei, where small clusters appeared in the culture at 2 dpi and grew resulting in the accumulation of multicellular clusters (Ralston in size and increased in number over time. Class 3 mutants et al., 2006). Therefore the clustering phenotype of TbCMF were viable, but grew at a reduced rate. Finally, 27% of mutants knockdown mutants supports the idea that these genes are exhibited a ‘severe’ clustering phenotype (Class 4 mutants), required for normal flagellar motility. However, cytokinesis where visible clusters appeared at 1 dpi and increased in size failure can also occur as a result of other primary defects (Das and number over time. Class 4 differed from Class 3 in that et al., 1994; Hammarton et al., 2005). To determine whether these mutants were ultimately lethal. To determine whether TbCMF mutants had true motility defects, sample mutants differences in phenotypic severity were the consequence of from each phenotype class for which mRNA knockdown was different levels of knockdown, northern blots were performed confirmed were selected for more in-depth analysis. These to examine knockdown efficacy in two sample mutants from mutants were examined independently by sedimentation assays each mutant class. Clear and specific knockdown of the (Bastin et al., 1999; Ralston et al., 2006) (Fig. 3A) and direct targeted mRNA was observed in each case (Fig. 2B). microscopic observation (Fig. 3B, Fig. 4, and supplementary T. brucei components of motile flagella 483

material Fig. S1). These assays were initiated before clustering moved away from their point of origin, but only tumbled and became apparent in the culture to ensure that any defect did not run for five contiguous seconds. Cells with this type of observed was due to loss of motility and was not secondary to movement were classified as ‘tumblers’ (T) and were cell clustering. The class 1 mutant TbCMF 15 did not sediment manifested in the trace as tightly wound circling lines. In rare significantly more than uninduced cells over the course of the cases, uninduced cells were immotile (I) and did not move assay, whereas TbCMF 3, a Class 2 mutant, showed mild from their point of origin. Upon knockdown, the number of sedimentation. TbCMF 46 (Class 3) and TbCMF 9 (Class 4) runners decreased with a concomitant increase in the number mutants sedimented extensively, compared with uninduced of tumbling and/or immotile cells. In most of the moderate and cells (Fig. 3A). The extent of sedimentation roughly correlated severe mutants examined, the largest increase was in immotile with the clustering phenotype of these mutants. Though only cells with a moderate increase in tumblers (Fig. 4B). For the four mutants are shown in Fig. 3A, 25% of the TbCMF mutants mild strains tested, the increase in tumbling cells was have been tested in this quantitative assay and this correlation comparable to moderate and severe mutants. The number of has held true for all mutants tested. immotile cells however, was roughly 2.5-fold less than seen in To examine directly how the movement and gross structure severe and moderate mutants. Motility trace analysis of an of the flagellum was affected by loss of TbCMF gene additional eight mutants is shown in supplementary material expression, knockdown mutants were examined by high- Fig. S1. Thus, motility analysis on 14 total mutants, together resolution differential interference contrast (DIC) microscopy. with the sedimentation assays, indicates a direct correlation No gross differences in length or outer morphology of the between increased motility defects and increased clustering flagellum were observed in any of the mutants examined. In severity. This demonstrates directly that these mutants do terms of motility, uninduced cells exhibited normal three- exhibit motility defects and supports earlier reports that dimensional, auger-like movement (Hill, 2003), with extremely motility contributes to cytokinesis in T. brucei (Branche et al., rapid and vigorous flagellar beat as did two Class 1 mutants, 2006; Ralston et al., 2006). TbCMF 15 and TbCMF 33 (not shown). TbCMF 3 (supplementary material Movie 1) and TbCMF 2 (not shown) TbCMF 9 and TbCMF 76b are required for maintaining also displayed normal flagellar movement, although slight connections between outer doublet microtubules irregularities may not have been detectable. In TbCMF 46 To understand the molecular mechanisms behind TbCMF (supplementary material Movie 2) and TbCMF 49 (not shown), motility defects, mutants were examined for ultrastructural flagellar motility was partially restricted, because a waveform defects using transmission electron microscopy. Several could be produced and propagated, but flagellar movement was mutants were examined and will be the topic of future work. limited to two dimensions. In TbCMF 5 and TbCMF 9, the Here we have focused on two class 4 mutants, TbCMF 5 and majority of cells displayed extremely deficient flagellar TbCMF 9, which revealed two important findings. First, no movement. For TbCMF 9, there was a gradient of flagellar gross defects were observed in TbCMF 5 mutants (Fig. 5A-C), defects. Most of the TbCMF 9 mutant cells displayed almost suggesting that the severe motility defect in this mutant may complete flagellar paralysis, with only a slight twitch of the be due regulatory problems rather than gross structural

Journal of Cell Science flagellum that was restricted to a segment of the flagellum and abnormalities. Notably, central pair orientation, which is not propagated (supplementary material Movie 3). A few cells randomized in the central pair mutants PF16 and PF20 (Ralston had rapidly beating flagella, although movement was only two- et al., 2006), remained fixed in all mutants examined as it does dimensional and did not effectively propel the cell. The in wild-type trypanosomes (Ralston et al., 2006). The fact that phenotype of TbCMF 5 mutants (supplementary material several mutants with varying degrees of flagellar beat defects Movie 4) was more homogenous, where motility consisted of have normal central pair orientation, suggests that randomized a strong two-dimensional beat at the distal tip of the flagellum, central pair orientation is not a general consequence of with severely decreased amplitude at the proximal region of abnormal flagellar beat. Thus PF16 and PF20 are likely to the flagellum. Also, the posterior end of these cells became function specifically in maintenance of central pair orientation. twisted like a corkscrew, which resulted in a slow spinning This conclusion is also supported by recent independent motion. Thus, detailed analysis revealed that although the exact studies (Gadelha et al., 2006). beat defects may differ between mutants within a given class, The second finding was a striking axonemal defect observed the Class 3 and Class 4 mutants examined had dramatically in TbCMF 9 mutants. These mutants exhibited disordered and impaired wave propagation along the flagellum leading to fractured axonemes in 44% of the sections examined, which in complete loss of propulsive cellular movement (Fig. 3B). the mildest cases showed doublets falling away or completely separated from the rest of the axoneme (Fig. 5D,E). In more TbCMF mutants lack propulsive movement severe cases, the axoneme had split and one half of the To provide a quantitative assessment of propulsive movement axoneme had twisted around to sit ‘piggyback’ on the other in TbCMF mutants, we developed an assay that allowed for outer doublets (Fig. 5F). This gradient of structural automated tracking of a large number of individual abnormalities correlated with the range of mutant phenotypes trypanosomes. A representative trace is shown in Fig. 4A for seen with DIC, suggesting a direct link between the extent of TbCMF 46 grown in the presence or absence of tetracycline. doublet abnormalities and the motility defect. To our All uninduced cultures examined exhibited essentially the knowledge, the ultrastructural defect of TbCMF 9 has not been same result. In each case, the majority of uninduced cells were previously reported for any flagellar mutant in any organism. vigorously motile and progressed forward along a curvilinear As discussed below, TbCMF 9 is part of a novel family of path for 5 seconds or more. These were classified as ‘runners’ proteins that includes two other closely related proteins in T. (R). In some cases, uninduced cells were clearly motile and brucei, TbCMF 76 and TbCMF 76b, with BLASTP expect 484 Journal of Cell Science 120 (3) Journal of Cell Science

Fig. 4. Quantitative analysis of TbCMF cell motility. (A) Cell traces of uninduced and induced (30 hpi) TbCMF 46 cells. Analysis was done as described in Materials and Methods. Running cells (R) were classified as having sustained propulsive movement for at least 5 seconds. Tumbling cells (T) had visible movement, but did not sustain a run for 5 seconds or more. Immotile cells (I) were classified as cells where movement was visible at the cellular level, but the cell did not move from the point of origin. (B) Graphical representation of motility differences between uninduced and induced mutant cells. Class 2, TbCMF 2 and TbCMF 3; Class 3, TbCMF 46 and TbCMF 76b; Class 4, TbCMF 5 and TbCMF 9. n=49-53 cells; 30-second interval. Blue, immotile cells; red, tumbler cells; yellow, runner cells. Bars, 50 ␮m.

values of 10e–59 and 10e–69, respectively. To determine whether required for maintaining the stability of the connections these proteins have similar functions as well as sequence between outer doublet microtubules. Alternatively, misplaced homology, we examined axoneme ultrastructure in TbCMF- doublets might be a consequence of a templating defect, i.e. 76b knockdown mutants. As shown in Fig. 5G-I, the TbCMF doublet defects in the axoneme arising from basal body 76b mutant exhibited the same range of ultrastructural defects defects. We distinguished between these possibilities in two observed for TbCMF 9, including abnormal outer-doublet ways. First, transverse sections through the basal body and spacing (5G), displaced outer doublets (5H) and piggybacked transition zone of TbCMF 9 mutants revealed normal hemi-axonemes (5I). These ultrastructural defects were microtubule arrangements in all cases (not shown). Second, observed in 40% of sections. longitudinal sections from both mutants showed outer doublet These findings suggest that TbCMF 9 and TbCMF 76b are microtubules that were bent away from the main axis of the T. brucei components of motile flagella 485

Fig. 5. TbCMF 9 and TbCMF 76b are part of a novel family of axonemal stabilizing proteins. Transmission EM images of flagella from the indicated TbCMF mutants. (A-C) Knockdown of TbCMF 5 does not significantly perturb the axoneme or PFR structure including the orientation of central pair microtubules. (D-F) TbCMF 9 knockdown causes a range of defects in outer-doublet arrangement. Axonemes ranged from outer doublets falling away from the axoneme (D, arrow), to groups of doublets that are completely separated from the axoneme (E, arrow), to split ‘hemi-axonemes’, in which half of the axoneme has twisted around to a piggyback arrangement on the other half (F). This range of structural defects was observed in 44% of sections, 68% displayed doublets in disarray and 32% displayed a piggyback configuration. A total of 100 sections were examined. (G-I) Knockdown of TbCMF 76b, a protein related to TbCMF 9, results in similar ultrastructural defects. These are observed in ~40% of sections, 81% of which show aberrant outer-doublet arrangement and 19% show the hemi- axoneme arrangement. A total of 75 sections were examined. (J) Longitudinal Journal of Cell Science sections of TbCMF 9 and 76b flagella. Doublets that have lost their linkages with the rest of the doublets bow out from the axoneme (arrows). Longitudinal images were taken at the same time point as the cross-sections shown in A-I.

TbCMF 9 is localized to the flagellum If TbCMF 9 functions to maintain connections between outer doublet microtubules, it should be localized along the length of the axoneme. We therefore used GFP tagging to determine the subcellular localization of TbCMF 9, as well as TbCMF proteins from other mutant classes. Since overexpression can interfere with protein localization, we used the pLEW100 tet- inducible expression (Wirtz et al., 1999) to drive low- level expression of GFP fusion proteins. We first used GFP- tagged TPN as a control. As shown in Fig. 6A, the TPN-GFP fusion protein was expressed only in the presence of tetracycline. Importantly, the fusion protein was expressed at a level roughly equivalent to the endogenous protein and, like axoneme (Fig. 5J, arrows) providing direct evidence that the endogenous TPN (Hill et al., 2000), was quantitatively outer-doublet defects arise within the axoneme and are not the associated with detergent-extracted cytoskeletons (Fig. 6A). result of a templating defect. Together, the data demonstrate When live cells were viewed by fluorescence microscopy the the presence of a novel family of proteins in T. brucei TPN-GFP fusion protein was localized along the flagellum responsible for maintaining the structural integrity of the outer- (Fig. 6C arrows). There was a low level of autofluorescence in doublet array in motile flagella. the cell body that was also evident in uninduced cells (Fig. 6C) 486 Journal of Cell Science 120 (3) Journal of Cell Science

Fig. 6. TbCMF proteins localize along, and are stably associated with, the flagellum. (A) Anti-TPN western blot on whole-cell lysates (L), detergent-solubilized proteins (S1) and detergent-extracted cytoskeletons prepared (Hill et al., 2000) from a trypanosome cell line harboring a Tet-inducible TPN-GFP fusion protein. The TPN- GFP fusion protein is expressed at levels equivalent to endogenous TPN and is quantitatively associated with the cytoskeleton. (B-F) Fluorescence microscopy shows that TPN-GFP is correctly localized to the flagellum (arrows) in live cells (C-D) and in cytoskeletons (E- F). (B) Live cells exhibit background autofluorescence that does not overlap with the flagellum and is also evident in the 29-13 parent cell line. As a negative control, GFP alone was used. Although fluorescence was evident in the cell body of live cells, there was no and untransfected cells (Fig. 6B). This autofluorescence fluorescence in the flagellum of detergent-extracted cytoskeletons overlapped with mitochondrial markers (not shown) and was (supplementary material Fig. S2). (G) Fluorescence microscopy of lost upon detergent extraction (Fig. 6E). GFP alone was not detergent-extracted cytoskeletons prepared from cell lines harboring the indicated Tet-inducible TbCMF-GFP fusion proteins. In all cases, specifically localized to the flagellum (supplementary material the GFP fusions are localized along the flagellum (arrows). (H) Anti- Fig. S2). The localization of TPN-GFP demonstrates for the GFP western blots of whole cell lysates (L), detergent-solubilized first time that TPN is localized to the flagellum in live cells proteins (S1) and detergent-extracted cytoskeletons (P1) prepared and establishes this system as a reliable means to examine from induced (+) and uninduced (–) TbCMF-GFP strains. The flagellar protein localization in T. brucei. asterisk indicates a secondary band at ~100 kDa that reacts with the We next constructed GFP fusions for TbCMF 9, as well as anti-GFP primary . Bars, 5 ␮m (B); 10 ␮m (G). T. brucei components of motile flagella 487

sample TbCMF Class 1 (TbCMF 19), Class 2 (TbCMF 40) and have direct relevance to infectious and inherited human Class 3 (TbCMF 46) proteins. TbCMF 9, TbCMF 40 and diseases. TbCMF 46 are novel proteins of unknown function. TbCMF 19 is a T. brucei homologue of a characterized C. reinhardtii The TbCMF dataset flagellar protofilament ribbon protein, Rib43a (supplementary The TbCMF dataset contains subunits of axonemal complexes material Table S1). In all cases TbCMF-GFP fusion proteins that are established components of motile flagella including were localized to the flagellum in live cells (not shown) and radial spokes, the dynein regulatory complex, and inner and remained stably associated with the flagellum upon detergent outer dynein arms. Approximately 76% of TbCMF proteins are extraction (Fig. 6G). The few spots observed in the cytoplasm also represented in one or more recently completed proteomic of TbCMF-9-GFP cells probably correspond to aggregated analyses of eukaryotic flagella (Broadhead et al., 2006; protein, because these did not occur in consistent number or Gibbons, 1963; Ostrowski et al., 2002; Pazour et al., 2005; placement. Western blots on whole-cell lysates, detergent- Smith et al., 2005). We also identified several proteins not soluble proteins, and detergent-insoluble cytoskeletons found in earlier proteomic and genomic studies, adding to the demonstrated that TbCMF 9, TbCMF 19 and TbCMF 46 were repertoire of proteins important for flagellar motility. Notably quantitatively associated with the cytoskeleton (Fig. 6H). A absent from the TbCMF dataset are components of the central significant fraction of TbCMF 40 was released into the pair apparatus, such as PF16 and PF20 (Smith and Lefebvre, detergent-soluble fraction. It is not known whether the soluble 1996; Smith and Lefebvre, 1997). Using T. brucei PF16 as a fraction represents non-flagellar protein, or protein released query, we found a hit in the A. thaliana predicted from the flagellar compartment. Overall, this localization data with an E-value of 10e–15, which would have excluded this supports the proposal that the proteins identified in this study gene from our dataset. Direct protein alignments indicate that represent conserved components of motile flagella. More this A. thaliana protein is not likely to be a true PF16 specifically, it supports a function for TbCMF 9 as an outer- orthologue and this reflects an inherent limitation of doublet connector along the length of the axoneme. comparative genomics, namely that some bona fide flagellar proteins might be missed. Nonetheless, the majority of known Discussion proteins expected to be unique to motile flagella are in the The mechanism and regulation of flagellar motility is not fully TbCMF dataset, as are several novel proteins for which understood, thus it is important to identify proteins important functional analysis demonstrated a clear role in flagellar for proper function and regulation of flagellar beat. To motility. accomplish this we have used the African trypanosome, T. Several TbCMF proteins are represented as families of two brucei, as a model system to identify motility genes via or more related sequences (e.g. TbCMF 2/3/4/34, TbCMF 5/6, comparative genomics and functional analysis. We identified TbCMF 40/40a and TbCMF 9/76/76b) and often the family is 50 genes that are unique to organisms with motile flagella, of expanded in T. brucei. For example, there is a single TPN which 30 are novel. We used RNAi to investigate the function homologue in mammals and C. reinhardtii, but there are two of these novel genes, as well as several others that had not been paralogues in T. brucei, referred to as TPN and trypanin-related

Journal of Cell Science functionally characterized in T. brucei previously. The majority protein (TRP) (K.L.H. and J. E. Donelson, unpublished of these genes are required for normal cell motility. For 30 of observation) (Ralston et al., 2006). Likewise, a single gene in these genes, our study represents the first functional C. reinhardtii encodes the Rib72 protein (Ikeda et al., 2003), characterization in any organism. Taken together, our analyses but there are four related sequences (TbCMF 2/3/4/34) in T. of motility defects, protein localization and ultrastructural brucei. Pair-wise alignments of TbCMF 9, TbCMF 76 and defects demonstrate that we have identified a novel set of T. TbCMF 76b indicate that they are part of a family that includes brucei proteins that represent conserved components of motile two closely related proteins in C. reinhardtii and a single flagella, referred to as TbCMF proteins. protein in humans. TbCMF 9, TbCMF 76 and TbCMF 76b are Several recent studies have used proteomic and comparative distantly related to TbCMF 8, which is the T. brucei homologue genomic approaches to identify candidate flagellar components of MBO2 from C. reinhardtii (Segal et al., 1984). This (Avidor-Reiss et al., 2004; Broadhead et al., 2006; Li et al., expansion might reflect unique aspects of flagellar beat in 2004; Ostrowski et al., 2002; Pazour et al., 2005; Smith et al., trypanosomes (Hill, 2003) or might be a manifestation of the 2005). Our effort is distinguished by focusing specifically on asymmetric architecture of the trypanosome flagellum, in organisms with motile flagella and by taking advantage of the which a paracrystalline rod is anchored to outer doublets 4-7 powerful tools available in T. brucei for probing the function along the length of the axoneme (Cachon et al., 1988). This of the candidate flagellar proteins identified. The impetus for arrangement imposes unique constraints on systems for our screen stemmed from the observation that trypanin, which regulation and propagation of flagellar beat and these demands is part of an evolutionarily conserved axonemal dynein might be met by using distinct but functionally related regulatory system (Ralston et al., 2006), is conserved only in regulatory systems on each side of the axoneme (Ralston et al., organisms with motile flagella. Coordinated regulation of 2006). axonemal dyneins represents one of the least understood aspects of flagellar function and we reasoned that additional Human disease genes important for axoneme motility and regulation of Motile cilia are crucial for normal human development and motility would exhibit a similar phylogenetic distribution. As physiology and human homologues of TbCMF genes might motile cilia and flagella are important for human physiology represent novel disease gene candidates. In support of this, and essential for viability of bloodstream-form T. brucei many human CMF genes have been directly implicated in (Broadhead et al., 2006; Ralston and Hill, 2006), our findings human disease, or map to loci that are linked to diseases that 488 Journal of Cell Science 120 (3)

are known to be, or are suspected to be, caused by ciliary gill cilia after detergent and protease treatment to remove nexin dysfunction (supplementary material Table S2). In 70% of links and might also reflect differential activation of specific these cases, this represents the first time that a connection has dynein subsets (Satir and Matsuoka, 1989). been made between this gene and a human disease. The doublet abnormalities of TbCMF 9 and TbCMF 76b can Identification of candidate disease genes is especially crucial be explained if they function as part of the nexin links in in the case of PCD, which is genetically heterogeneous, maintaining the integrity of the outer doublet microtubule making disease gene identification through linkage analysis array. Nexins are elastic structures that link adjacent outer extremely difficult. Indeed, the only three genes so far doublets (Warner, 1976). They provide support and identified as causal in PCD were identified by specifically organization to the outer-doublet array and contribute to looking for mutations in candidate disease genes that encode transverse elastic forces postulated to help regulate dynein axonemal dynein subunits (Ibanez-Tallon et al., 2003). Juvenile activity in response to microtubule sliding and curvature myoclonic epilepsy (JME) is caused by mutations in the (Cibert, 2001; Lindemann, 2004). These structures have been flagellar protofilament ribbon protein Rib72 (Ikeda et al., identified by electron microscopy (Gibbons, 1965; Olson and 2005). Although biochemical fractionation demonstrates that Linck, 1977; Warner, 1976) and axonemal fractionation Rib72 is an integral component of the axoneme (Ikeda et al., (Stephens, 1970; Stephens and Edds, 1976), although they have 2003; Patel-King et al., 2002), the function of Rib72 in never been characterized directly by mutational analysis. If flagellar motility has not previously been examined. Therefore, nexin links are absent or compromised, one predicts that, as our results (Fig. 4) provide the first demonstration that Rib72 the flagellum beats, the resulting forces would cause outer is required for flagellar beat, suggesting that JME results from doublets to separate and fall away from the outer-doublet ring. defects in motility functions of the cilium. This is precisely what was seen in TbCMF 9 and TbCMF 76b mutants. Mutants with this defect have not been observed Functional analysis of TbCMF proteins previously and this was not observed in any of the other We used RNAi to investigate the function of 41 of the 50 TbCMF mutants examined in this study. As motility becomes TbCMF genes identified. Thirty of these had not previously increasingly aberrant, forces within the structurally impaired been characterized in any organism and our data provide the axoneme may become strong and erratic enough to shift the first demonstration that these proteins play a role in flagellar loose doublets, resulting in the piggyback arrangement motility. Most TbCMF mutants (76%) accumulate as observed. Loss of the canonical 9+2 axonemal arrangement is multicellular clusters that fail to complete cell separation. In at expected to have a severe impact on flagellar beat. If breaks least fourteen cases where motility was examined are localized to a single region, one might expect a localized independently by sedimentation and/or direct microscopic beat that is not propagated along the axoneme. In more severe observations the severity of this phenotype correlates with the cases, the entire flagellum might become paralyzed, which is severity of the motility defect and thus further supports the idea again what we observed in the TbCMF 9 mutant. An alternative that flagellar motility is required for normal cell division in T. hypothesis is that axonemal splitting results from brucei (Ralston et al., 2006). Thus, as shown by indirect and asynchronous dynein activation (Satir and Matsuoka, 1989)

Journal of Cell Science direct methods, TbCMF genes are confirmed in vivo to be and these hypotheses are not mutually exclusive. functionally important for flagellar motility. The protein composition of nexins is unknown and several lines of evidence suggest that TbCMF 9 and TbCMF 76b are TbCMF 9 and TbCMF 76b are part of a novel protein the first ever identified components of these linkages. First, family required for maintaining linkages between outer nexins are prominent features of motile axonemes (Bozkurt doublet microtubules and Woolley, 1993; Gibbons, 1963), but are not present in Functional analysis of TbCMF 9 and TbCMF 76b, both immotile sensory cilia of C. elegans (Perkins et al., 1986; members of a novel protein family, demonstrates functional Signor et al., 1999), thus, nexin genes are expected to be analogy among these proteins. Both TbCMF 9 and TbCMF recovered in our screen. Whether nexins, or other CMF 76b were identified in a recent axonemal proteomic analysis proteins are also present in non-motile cilia in organisms that (Broadhead et al., 2006), although only TbCMF 9 was assemble both motile and non-motile cilia remains to be functionally characterized in that study. When TbCMF 9 (or determined and their function investigated. For instance, the MENG) was knocked down by RNAi, the cells exhibited human homologue of trypanin, Gas11, is expressed in reduced motility, but no apparent cytokinesis defect or loss of mammalian cell types that contain motile cilia, non-motile viability (Broadhead et al., 2006). This result differs from our primary cilia or lack cilia (Colantonio et al., 2006; Whitmore findings; however, the reason for this is not presently clear. et al., 1998; Yeh et al., 2002), and appears to have been adapted Broadhead and colleagues did not examine the extent of RNAi to serve multiple functions in mammals (Colantonio et al., knockdown, thus knockdown of MENG might not have been 2006). Second, aberrant arrangement of outer doublet as complete as we observed for TbCMF 9 (Fig. 2B). Both microtubules, as well as the paralyzed and localized flagellar TbCMF 9 and TbCMF 76b mutants exhibit motility and beat defects observed in TbCMF 9 and TbCMF 76b mutants cytokinesis defects and share a novel axonemal ultrastructural are precisely what one would expect for nexin mutants. Indeed, defect. The affected doublets are most often those not adjacent the outer-doublet defect of TbCMF 9 and TbCMF 76b is to the PFR, suggesting that attachment of microtubules to the reminiscent of that seen in cilia following protease and PFR might provide enough residual stability to keep those chemical treatments to solubilize the nexin links and other doublets properly positioned. In fact ‘hot spots’ for breakage mechanical impediments to axoneme disintegration (Linck, appear to be between doublets 3-4 and 6-7 on either side of the 1973a; Linck, 1973b; Lindemann et al., 1992; Satir and PFR. Hot spots of breakage are also observed in disintegrating Matsuoka, 1989). Third, nexin proteins are expected to be T. brucei components of motile flagella 489

localized along the length of the axoneme, as we observed for identified by the Trypanofan RNAit algorithm (Redmond et al., 2003) TbCMF 9. Fourth, nexin links in T. brucei are retained (http://trypanofan.path.cam.ac.uk/cgi-bin/rnait.org). The TbCMF gene targets were ligated into the p2T7-Ti/B-RNAi vector, which is a tetracycline-controlled following detergent and salt-extraction of flagellar axonemes expression vector with opposing T7 promoters (LaCount et al., 2002). Inserts were (not shown) and biochemical fractionation demonstrates that verified by sequencing at the UCLA genomics center. TbCMF 9 is also retained in axonemal preparations following For GFP tagging, full-length TbCMF genes were amplified from 29-13 (Wirtz detergent extraction (Fig. 6H) and salt extraction (not shown). et al., 1999) genomic DNA with primers containing the appropriate restriction sites and ligated into pKH10 (N-terminal tag) or pKH12 (C-terminal tag). For C- Finally, TbCMF 9 and TbCMF 76b contain domains with terminal tags, TbCMF genes were cloned into the HindIII and XbaI sites at the 5Ј homology to domains found in structural maintenance of end of the GFP ORF in pKH12. pKH12 was generated by removing the GFP (SMC) proteins, which have functional cassette from pHD:HX-GFPm3 (Hill et al., 1999) as a HindIII-BamHI fragment and inserting it into the HindIII-BamHI sites of pLEW100 (Wirtz et al., 1999). For characteristics similar to what one would hypothesize for a N-terminal tagging, TbCMF genes were cloned into XbaI and BamHI sites at the nexin. SMC proteins are known for their role in the structural 3Ј end of the GFP ORF in the pKH10 expression vector. pKH10 is a derivative of and functional organization of chromosomes (Hirano, 2006; pLEW100 (Wirtz et al., 1999) in which the GFP ORF is flanked with HindIII and XbaI-BamHI restrictions sites at its 5Ј and 3Ј end, respectively. pKH10 was Nasmyth and Haering, 2005), but also play a ciliary role, generated by PCR amplification of the GFP-coding sequence minus its stop codon because SMC1 and SMC3 have been localized to retinal and from pHD496-GFP (Biebinger et al., 1997), using primers to introduce a 5Ј HindIII renal cilia (Khanna et al., 2005). SMC proteins are large (110- restriction site and 3Ј XbaI and BamHI restriction sites. The amplified GFP product 170 kDa) and very flexible, which together with ATP binding was inserted into HindIII and BamHI restriction sites of pKH12. All sequences were verified by DNA sequencing at the UCLA genomics center. For GFP alone (Hirano, 2006), could provide the elastic and dynamic in pLEW82 (see supplementary material Fig. S1), the GFP cassette was removed functions ascribed to nexins. SMC proteins generally function from pHD:HX-GFPm3 (Hill et al., 1999) as a HindIII-BamHI fragment and as heterodimers of closely related proteins, which might inserting it into the HindIII-BamHI sites of pLEW82 (Wirtz et al., 1998) to generate explain why TbCMF 9 and TbCMF 76b are part of a gene pKH15. family. Since nexins are intimately associated with the DRC Trypanosome transfection and cell maintenance (Mastronarde et al., 1992; Nicastro et al., 2006; Woolley, Procyclic 29-13 cells (Wirtz et al., 1999) were used to create RNAi and GFP-tagged 1997), it would not be surprising if nexin genes exhibit the TbCMF strains. Cells were maintained and transfected as described previously (Hill et al., 1999; Hutchings et al., 2002) and clonal lines were obtained by limiting same phylogenetic distribution as DRC genes. A major finding dilution. Knockdown mutants were induced with 0.3-1 ␮g/ml tetracycline. For our to some recent cryoelectron studies (Nicastro et al., 2006) is initial analysis, at least three clonal lines for each knockdown mutant were induced that novel linkers connect outer-arm dyneins with each other to assess clone-to-clone variability. Very little variation was observed and a single clone for each knockdown was selected for further analysis. For expression of GFP- and with the inner-arm dyneins and DRC. These linkers are TbCMF fusion proteins cells were induced with 1 ␮g/ml tetracycline for 24 hours postulated to provide a mechanism for coordinating dynein and viewed on a Zeiss Axioskop II compound fluorescent microscope using a 63ϫ activity and it will be of interest to determine whether any of oil objective. the novel CMF proteins encode components of these linkers. Northern blots RNA was isolated from induced (same dpi as in motility assays) and uninduced Materials and Methods TbCMF strains using a Qiagen RNeasy Miniprep kit according to the Bioinformatic analysis manufacturer’s instructions. Northern blots (Hill et al., 1991) using 5 ␮g total RNA Dutcher and colleagues (Li et al., 2004) recently described a group of predicted were probed with 32P-labeled DNA fragments corresponding to the region used for proteins that are conserved in flagellated organisms, namely C. reinhardtii, H. sapiens, RNAi knockdown. Journal of Cell Science M. musculus, C. intestinalis and D. melanogaster, but are not found in organisms lacking flagella, namely A. thaliana (Table S1 in supplementary material) (Li et al., Motility assays 2004). This includes 81 proteins that are also not found in C. elegans, which contains Unless otherwise stated, strains were examined at a time point before clustering non-motile sensory cilia. To identify T. brucei proteins that are restricted to organisms became apparent in the culture, typically 24 hours post induction for Class 3 and with motile flagella, the ‘best matched’ accession number for these proteins was used 4 strains and 48 hours for Class 1 and 2 strains. Digital images and movies were to procure the corresponding protein sequence from the NCBI database. These were captured using a Sony handycam and directly imported using Adobe Premiere then compared (BLASTp; BLOSUM 62) (Altschul et al., 1990) against the T. brucei Elements Software. Sedimentation assays were carried out essentially as described predicted proteome at the Gene Database (GeneDB, http://www.genedb.org/) (Hertz- previously (Bastin et al., 1999; Ralston et al., 2006) except that OD measurements Fowler et al., 2004). T. brucei hits with an expected value of 1ϫ10–20 or less were were done in triplicate and were taken every hour for 10 hours. Whole culture retained and identical entries were removed, yielding a total of 38 proteins. To observations of the induced TbCMF cultures in flasks were made every 24 hours supplement this dataset, we used the proteome comparison (Procom) tool developed for 6 days post induction on a Zeiss Axiovert 200 inverted microscope using a 5ϫ by Stormo and colleagues (Li et al., 2005). Procom is a web-based tool that allows objective. Cells were observed using a 63ϫ oil objective on a Zeiss Axiovert 200 for rapid, automated comparison of predicted eukaryotic . T. brucei was inverted microscope in polyglutamate-coated slide chambers (Gadelha et al., used as the anchor organism, H. sapiens, M. musculus, D. melanogaster, L. major, T. 2005). Log-phase cells of uninduced and induced TbCMF cells were diluted to cruzi, C. reinhardtii and C. intestinalis were used as intersection organisms, whereas 1ϫ106 cells/ml and added to a polyglutamate-coated slide chamber (Gadelha et C. elegans and A. thaliana were used as subtraction organisms. An expected value of al., 2005) (~40 ␮l) before the sides were sealed with a thin layer of Vaseline. The 1ϫ10–10 was used for intersect and subtract cut-off values. This analysis yielded 82 cells were viewed under dark-field illumination on a Zeiss Axioskop II compound entries from the T. brucei predicted proteome. Sequences for each of these 120 microscope using a 10ϫ objective. Approximately 30 seconds of video from proteins (38 + 82) were obtained from GeneDB and redundant entries removed, for separate regions on each slide was captured. Motility traces were generated using a preliminary total of 88 proteins. As a further refinement, we used reciprocal best Metamorph software (Molecular Devices). Cells that were not tracked for the full BLAST to determine whether the top human homologue for each T. brucei protein 30 seconds, e.g. as a result of leaving the field or plane of focus, were not used in returned the original T. brucei entry as the top hit when compared against the T. brucei the analysis. predicted proteome. Individual protein sequences were also evaluated by direct alignment. This reduced our dataset to 46 proteins. Reciprocal BLAST analysis also Trypanosome cellular fractionation and western blotting identified another four T. brucei proteins as paralogues to proteins already on the list. Trypanosome lysates, detergent-soluble proteins, and cell cytoskeletons were Thus, the final dataset contains 50 proteins. The final TbCMF dataset was compared prepared as described previously (Hill et al., 2000) and western blotted (Hill et al., against the C. briggsiae genome using the same methods listed above and no 1999). Primary antibody dilutions were as follows, mGFP 1:500 (Clontech 632380) homologues were identified. Trypanin-related protein, TRP (GeneDB ID and mTPN 1:5000 described previously (Ralston et al., 2006). Tb09.244.2800), identified in this screen is 28% identical and 43% similar to TPN at the amino acid level and will be described elsewhere (K.H., unpublished observation). Transmission electron microscopy Transmission electron microscopy was performed as described previously Cloning of TbCMF RNAi and GFP-tagged constructs (Hutchings et al., 2002). Class 3 and 4 mutants were analyzed 32-48 hours post The gene targets for RNAi (400-600bp) were amplified by PCR from 29-13 (Wirtz induction, a time point at which the mRNA is clearly knocked down, and motility et al., 1999) genomic DNA using primers specific to an RNAi target sequence defects are evident. 490 Journal of Cell Science 120 (3)

We thank Randy Nessler (University of Iowa) for his assistance targeting domain directs proteins to the anterior cytoplasmic face of the flagellar pocket with electron microscopy. We are grateful to George Cross in African trypanosomes. J. Cell Sci. 112, 3091-3101. (Rockefeller University) for the 29-13 cell line. We also appreciate Hill, K. L., Hutchings, N. R., Grandgenett, P. M. and Donelson, J. E. (2000). T Lymphocyte triggering factor of African trypanosomes is associated with the flagellar the helpful comments and discussions from Nancy Sturm. This work fraction of the cytoskeleton and represents a new family of proteins that are present in was supported by grants from the National Institutes of Health several divergent eukaryotes. J. Biol. Chem. 275, 39369-39378. (R01AI52348), Ellison Medical Foundation (ID-NS-0148-03) and Hirano, T. (2006). At the heart of the : SMC proteins in action. Nat. Rev. Beckman Young Investigator Program to K.L.H. D.M.B. is the Mol. Cell Biol. 7, 311-322. Huang, B., Ramanis, Z. and Luck, D. J. (1982). Suppressor mutations in recipient of a USPHS National Research Service Award in Microbial Chlamydomonas reveal a regulatory mechanism for Flagellar function. Cell 28, 115- Pathogenesis (2-T32-AI-07323) and the UCLA Graduate Dissertation 124. Year Fellowship for 2006-2007. K.S.R. is the recipient of a USPHS Hutchings, N. R., Donelson, J. E. and Hill, K. L. (2002). Trypanin is a cytoskeletal National Research Service Award in Genetics (GM07104). linker protein and is required for cell motility in African trypanosomes. J. Cell Biol. 156, 867-877. Ibanez-Tallon, I., Heintz, N. and Omran, H. (2003). To beat or not to beat: roles of References cilia in development and disease. Hum. Mol. Genet. 12, R27-R35. Afzelius, B. A. (2004). Cilia-related diseases. J. Pathol. 204, 470-477. Ikeda, K., Brown, J. A., Yagi, T., Norrander, J. M., Hirono, M., Eccleston, E., Afzelius, B., Mossberg, B. and Bergstrom, S. E. (2001). Immotile Cilia Syndrome Kamiya, R. and Linck, R. W. (2003). Rib72, a conserved protein associated with the (Primary Ciliary Dyskinesis), including Kartagener Syndrome. New York: McGraw- ribbon compartment of flagellar A-microtubules and potentially involved in the linkage Hill. between outer doublet microtubules. J. Biol. Chem. 278, 7725-7734. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990). Basic Ikeda, T., Ikeda, K., Enomoto, M., Park, M. K., Hirono, M. and Kamiya, R. (2005). local alignment search tool. J. Mol. Biol. 215, 403-410. The mouse ortholog of EFHC1 implicated in juvenile myoclonic epilepsy is an Avidor-Reiss, T., Maer, A. M., Koundakjian, E., Polyanovsky, A., Keil, T., axonemal protein widely conserved among organisms with motile cilia and flagella. Subramaniam, S. and Zuker, C. S. (2004). Decoding cilia function: defining FEBS Lett. 579, 819-822. specialized genes required for compartmentalized cilia biogenesis. Cell 117, 527-539. Khanna, H., Hurd, T. W., Lillo, C., Shu, X., Parapuram, S. K., He, S., Akimoto, M., Bastin, P., Pullen, T. J., Sherwin, T. and Gull, K. (1999). Protein transport and flagellum Wright, A. F., Margolis, B., Williams, D. S. et al. (2005). RPGR-ORF15, which is assembly dynamics revealed by analysis of the paralysed trypanosome mutant snl-1. mutated in retinitis pigmentosa, associates with SMC1, SMC3, and microtubule J. Cell Sci. 112, 3769-3777. transport proteins. J. Biol. Chem. 280, 33580-33587. Biebinger, S., Wirtz, L. E., Lorenz, P. and Clayton, C. (1997). Vectors for inducible Kohl, L., Robinson, D. and Bastin, P. (2003). Novel roles for the flagellum in cell expression of toxic gene products in bloodstream and procyclic Trypanosoma brucei. morphogenesis and cytokinesis of trypanosomes. EMBO J. 22, 5336-5346. Mol. Biochem. Parasitol. 85, 99-112. LaCount, D. J., Barrett, B. and Donelson, J. E. (2002). Trypanosoma brucei FLA1 is Bozkurt, H. H. and Woolley, D. M. (1993). Morphology of nexin links in relation to required for flagellum attachment and cytokinesis. J. Biol. Chem. 277, 17580-17588. interdoublet sliding in the sperm flagellum. Cell Motil. Cytoskeleton 24, 109-118. Li, J. B., Gerdes, J. M., Haycraft, C. J., Fan, Y., Teslovich, T. M., May-Simera, H., Branche, C., Kohl, L., Toutirais, G., Buisson, J., Cosson, J. and Bastin, P. (2006). Li, H., Blacque, O. E., Li, L., Leitch, C. C. et al. (2004). Comparative genomics Conserved and specific functions of axoneme components in trypanosome motility. J. identifies a flagellar and basal body proteome that includes the BBS5 human disease Cell Sci. 119, 3443-3455. gene. Cell 117, 541-552. Broadhead, R., Dawe, H. R., Farr, H., Griffiths, S., Hart, S. R., Portman, N., Shaw, Li, J. B., Zhang, M., Dutcher, S. K. and Stormo, G. D. (2005). Procom: a web-based M. K., Ginger, M. L., Gaskell, S. J., McKean, P. G. et al. (2006). Flagellar motility tool to compare multiple eukaryotic proteomes. Bioinformatics 21, 1693-1694. is required for the viability of the bloodstream trypanosome. 440, 224-227. Linck, R. W. (1973a). Chemical and structural differences between cilia and flagella from Brokaw, C. J. (1972). Flagellar movement: a sliding filament model. Science 178, 455- the lamellibranch mollusc, Aequipecten irradians. J. Cell Sci. 12, 951-981. 462. Linck, R. W. (1973b). Comparative isolation of cilia and flagella from the lamellibranch Brown, J. M., Hardin, C. and Gaertig, J. (1999). Rotokinesis, a novel phenomenon of mollusc, Aequipecten irradians. J. Cell Sci. 12, 345-367. cell locomotion-assisted cytokinesis in the Tetrahymena thermophila. Cell Biol. Lindemann, C. B. (2004). Testing the geometric clutch hypothesis. Biol. Cell 96, 681- Int. 23, 841-848. 690. Cachon, J., Cachon, M., Cosson, M.-P. and Cosson, J. (1988). The paraflagellar rod: Lindemann, C. B., Orlando, A. and Kanous, K. S. (1992). The flagellar beat of rat a structure in search of a function. Biol. Cell 63, 169-181. sperm is organized by the interaction of two functionally distinct populations of dynein Cibert, C. (2001). Elastic extension and jump of the flagellar nexin links: a theoretical bridges with a stable central axonemal partition. J. Cell Sci. 102, 249-260. Journal of Cell Science mechanical cycle. Cell Motil. Cytoskeleton 49, 161-175. Mastronarde, D. N., O’Toole, E. T., McDonald, K. L., McIntosh, J. R. and Porter, Colantonio, J. R., Bekker, J. M., Kim, S. J., Morrissey, K. M., Crosbie, R. H. and M. E. (1992). Arrangement of inner dynein arms in wild-type and mutant flagella of Hill, K. L. (2006). Expanding the role of the dynein regulatory complex to non- Chlamydomonas. J. Cell Biol. 118, 1145-1162. axonemal functions: association of GAS11 with the . Traffic 7, 538- Nasmyth, K. and Haering, C. H. (2005). The structure and function of SMC and kleisin 548. complexes. Annu. Rev. Biochem. 74, 595-648. Das, A., Gale, M., Jr, Carter, V. and Parsons, M. (1994). The protein phosphatase Nicastro, D., Schwartz, C., Pierson, J., Gaudette, R., Porter, M. E. and McIntosh, J. inhibitor okadaic acid induces defects in cytokinesis and organellar genome segregation R. (2006). The molecular architecture of axonemes revealed by cryoelectron in Trypanosoma brucei. J. Cell Sci. 107, 3477-3483. tomography. Science 313, 944-948. Ferguson, D. J. (2002). Toxoplasma gondii and sex: essential or optional extra? Trends Olson, G. E. and Linck, R. W. (1977). Observations of the structural components of Parasitol. 18, 355-359. flagellar axonemes and central pair microtubules from rat sperm. J. Ultrastruct. 21-43. Gadelha, C., Wickstead, B., de Souza, W., Gull, K. and Cunha-e-Silva, N. (2005). Ostrowski, L. E., Blackburn, K., Radde, K. M., Moyer, M. B., Schlatzer, D. M., Cryptic paraflagellar rod in -containing kinetoplastid protozoa. Moseley, A. and Boucher, R. C. (2002). A proteomic analysis of human cilia: Eukaryotic Cell 4, 516-525. identification of novel components. Mol. Cell. Proteomics 1, 451-465. Gadelha, C., Wickstead, B., McKean, P. G. and Gull, K. (2006). Basal body and Pan, J., Wang, Q. and Snell, W. J. (2005). Cilium-generated signaling and cilia-related flagellum mutants reveal a rotational constraint of the central pair microtubules in the disorders. Lab. Invest. 85, 452-463. axonemes of trypanosomes. J. Cell Sci. 119, 2405-2413. Patel-King, R. S., Benashski, S. E. and King, S. M. (2002). A bipartite Ca2+-regulated Gardner, L. C., O’Toole, E., Perrone, C. A., Giddings, T. and Porter, M. E. (1994). nucleoside-diphosphate kinase system within the Chlamydomonas flagellum. The Components of a ‘dynein regulatory complex’ are located at the junction between the regulatory subunit p72. J. Biol. Chem. 277, 34271-34279. radial spokes and the dynein arms in Chlamydomonas flagella. J. Cell Biol. 127, 1311- Pazour, G. J. and Witman, G. B. (2003). The vertebrate primary cilium is a sensory 1325. organelle. Curr. Opin. Cell Biol. 15, 105-110. Gibbons, I. R. (1963). Studies on the protein components of cilia from tetrahymena Pazour, G. J., Agrin, N., Leszyk, J. and Witman, G. B. (2005). Proteomic analysis of pyriformis. Proc. Natl. Acad. Sci. USA 50, 1002-1010. a eukaryotic cilium. J. Cell Biol. 170, 103-113. Gibbons, I. R. (1965). Chemical dissection of cilia. Arch. Biol. Liege 76, 317-352. Perkins, L. A., Hedgecock, E. M., Thomson, J. N. and Culotti, J. G. (1986). Mutant Hammarton, T. C., Lillico, S. G., Welburn, S. C. and Mottram, J. C. (2005). sensory cilia in the nematode Caenorhabditis elegans. Dev. Biol. 117, 456-487. Trypanosoma brucei MOB1 is required for accurate and efficient cytokinesis but not Piperno, G., Mead, K. and Shestak, W. (1992). The inner dynein arms I2 interact with for exit from mitosis. Mol. Microbiol. 56, 104-116. a ‘dynein regulatory complex’ in Chlamydomonas flagella. J. Cell Biol. 118, 1455- Hertz-Fowler, C., Peacock, C. S., Wood, V., Aslett, M., Kerhornou, A., Mooney, P., 1463. Tivey, A., Berriman, M., Hall, N., Rutherford, K. et al. (2004). GeneDB: a resource Ralston, K. S. and Hill, K. L. (2006). Trypanin, a component of a flagellar dynein for prokaryotic and eukaryotic organisms. Nucleic Acids Res. 32, D339-D343. regulatory complex, is essential in bloodstream-form Trypanosoma brucei. PLoS Hill, K. L. (2003). Mechanism and biology of trypanosome cell motility. Eukaryotic Cell Pathog. 2, e101. 2, 200-208. Ralston, K. S., Lerner, A. G., Diener, D. R. and Hill, K. L. (2006). Flagellar motility Hill, K. L., Li, H. H., Singer, J. and Merchant, S. (1991). Isolation and structural contributes to cytokinesis in Trypanosoma brucei and is modulated by an evolutionarily characterization of the Chlamydomonas reinhardtii gene for cytochrome c6. Analysis conserved dynein regulatory system. Eukaryotic Cell 5, 696-711. of the kinetics and metal specificity of its copper-responsive expression. J. Biol. Chem. Redmond, S., Vadivelu, J. and Field, M. C. (2003). RNAit: an automated web-based 266, 15060-15067. tool for the selection of RNAi targets in Trypanosoma brucei. Mol. Biochem. Parasitol. Hill, K. L., Hutchings, N. R., Russell, D. G. and Donelson, J. E. (1999). A novel protein 128, 115-118. T. brucei components of motile flagella 491

Rughooputh, S. and Greenwell, P. (2005). Trichomonas vaginalis: paradigm of a Summers, K. E. and Gibbons, I. R. (1971). Adenosine triphosphate-induced sliding of successful sexually transmitted organism. Br. J. Biomed. Sci. 62, 193-200. tubules in trypsin-treated flagella of sea-urchin sperm. Proc. Natl. Acad. Sci. USA 68, Rupp, G. and Porter, M. E. (2003). A subunit of the dynein regulatory complex in 3092-3096. Chlamydomonas is a homologue of a growth arrest-specific gene product. J. Cell Biol. Suzuki, T., Delgado-Escueta, A. V., Aguan, K., Alonso, M. E., Shi, J., Hara, Y., 162, 47-57. Nishida, M., Numata, T., Medina, M. T., Takeuchi, T. et al. (2004). Mutations in Sapiro, R., Kostetskii, I., Olds-Clarke, P., Gerton, G. L., Radice, G. L. and Strauss, EFHC1 cause juvenile myoclonic epilepsy. Nat. Genet. 36, 842-849. I. J. (2002). Male infertility, impaired sperm motility, and hydrocephalus in mice Vlachou, D., Schlegelmilch, T., Runn, E., Mendes, A. and Kafatos, F. C. (2006). The deficient in sperm-associated antigen 6. Mol. Cell. Biol. 22, 6298-6305. developmental migration of Plasmodium in mosquitoes. Curr. Opin. Genet. Dev. 16, Satir, P. (1968). Studies on cilia. 3. Further studies on the cilium tip and a “sliding 384-391. filament” model of ciliary motility. J. Cell Biol. 39, 77-94. Wang, Q., Pan, J. and Snell, W. J. (2006). Intraflagellar transport particles Satir, P. and Matsuoka, T. (1989). Splitting the ciliary axoneme: implications for a participate directly in cilium-generated signaling in Chlamydomonas. Cell 125, 549- “switch-point” model of dynein arm activity in ciliary motion. Cell Motil. Cytoskeleton 562. 14, 345-358. Warner, F. D. (1976). Ciliary inter-microtubule bridges. J. Cell Sci. 20, 101-114. Segal, R. A., Huang, B., Ramanis, Z. and Luck, D. J. (1984). Mutant strains of Whitmore, S. A., Settasatian, C., Crawford, J., Lower, K. M., McCallum, B., Chlamydomonas reinhardtii that move backwards only. J. Cell Biol. 98, 2026-2034. Seshadri, R., Cornelisse, C. J., Moerland, E. W., Cleton-Jansen, A. M., Tipping, Shingyoji, C., Murakami, A. and Takahashi, K. (1977). Local reactivation of Triton- A. J. et al. (1998). Characterization and screening for mutations of the growth arrest- extracted flagella by iontophoretic application of ATP. Nature 265, 269-270. specific 11 (GAS11) and C16orf3 genes at 16q24.3 in breast cancer. Genomics 52, 325- Signor, D., Wedaman, K. P., Rose, L. S. and Scholey, J. M. (1999). Two heteromeric 331. kinesin complexes in chemosensory neurons and sensory cilia of Caenorhabditis Williams, N. E., Tsao, C. C., Bowen, J., Hehman, G. L., Williams, R. J. and Frankel, elegans. Mol. Biol. Cell 10, 345-360. J. (2006). The actin gene ACT1 is required for phagocytosis, motility, and cell Smith, E. F. and Lefebvre, P. A. (1996). PF16 encodes a protein with armadillo repeats separation of Tetrahymena thermophila. Eukaryotic Cell 5, 555-567. and localizes to a single microtubule of the central apparatus in Chlamydomonas Wirtz, E., Hoek, M. and Cross, G. A. (1998). Regulated processive transcription of flagella. J. Cell Biol. 132, 359-370. chromatin by T7 RNA polymerase in Trypanosoma brucei. Nucleic Acids Res. 26, Smith, E. F. and Lefebvre, P. A. (1997). PF20 gene product contains WD repeats and 4626-4634. localizes to the intermicrotubule bridges in Chlamydomonas flagella. Mol. Biol. Cell Wirtz, E., Leal, S., Ochatt, C. and Cross, G. A. (1999). A tightly regulated inducible 8, 455-467. expression system for conditional gene knock-outs and dominant-negative genetics in Smith, J. C., Northey, J. G., Garg, J., Pearlman, R. E. and Siu, K. W. (2005). Robust Trypanosoma brucei. Mol. Biochem. Parasitol. 99, 89-101. method for proteome analysis by MS/MS using an entire translated genome: Witman, G. B., Plummer, J. and Sander, G. (1978). Chlamydomonas flagellar mutants demonstration on the ciliome of Tetrahymena thermophila. J. Proteome Res. 4, 909-919. lacking radial spokes and central tubules. Structure, composition, and function of Snell, W. J., Pan, J. and Wang, Q. (2004). Cilia and flagella revealed: from flagellar specific axonemal components. J. Cell Biol. 76, 729-747. assembly in Chlamydomonas to human obesity disorders. Cell 117, 693-697. Woolley, D. M. (1997). Studies on the eel sperm flagellum. I. The structure of the inner Stephens, R. E. (1970). Isolation of nexin – the linkage protein responsible for the dynein arm complex. J. Cell Sci. 110, 85-94. maintenance of the nine-fold configuration of flagellar axonemes. Biol. Bull. Mar. Biol. Yeh, S. D., Chen, Y. J., Chang, A. C., Ray, R., She, B. R., Lee, W. S., Chiang, H. S., Lab. Woods Hole, 139, 438. Cohen, S. N. and Lin-Chao, S. (2002). Isolation and properties of Gas8, a growth Stephens, R. E. and Edds, K. T. (1976). Microtubules: structure, chemistry, and function. arrest-specific gene regulated during male gametogenesis to produce a protein Physiol. Rev. 56, 709-777. associated with the sperm motility apparatus. J. Biol. Chem. 277, 6311-6317. Journal of Cell Science