1513Short Report

IntroductionFlagella and cilia (eukaryotic cilia and flagella are structurallyand functionally analogous and we will use these termsinterchangeably) are dynamic organelles involved in severalbiological processes including sensing the surroundingenvironment, cellular adhesion, mating (Pazour and Witman,2003; Solter and Gibor, 1977; Vickerman et al., 1988) and mostnotably, motility. For example, in mammals, motile cilia arerequired for proper reproduction, left-right axis determination,brain and respiratory function. As such, ciliary defects can leadto serious diseases including infertility, situs inversus,hydrocephalus and respiratory malfunction (Ibanez-Tallon etal., 2003; Pan et al., 2005). Flagella are also the primary modeof locomotion for many pathogenic protozoa, such asTrypanosoma brucei, Giardia lamblia and Trichomonasvaginalis. For many pathogens, motility is believed to be vitalfor disease pathogenesis and in some cases, flagella serveadditional, essential roles beyond their role in cell motility(Baron et al., 2007; Broadhead et al., 2006; Kohl et al., 2003;Ralston and Hill, 2006; Ralston et al., 2006). Thereforeflagellar motility impacts infectious and heritable diseases inhumans. At the heart of motile flagella and cilia is the

conserved 9+2 axoneme, which is composed of approximately250 proteins (Luck et al., 1977; Pazour et al., 2005). It is thecoordinated activation of dynein molecular motors on adjacentouter doublets of the axoneme that is the driving force behindthe motility of this structure. The axoneme contains severaldiscrete populations of dyneins having distinct subunitcompositions and specialized roles in flagellar beat.Determining the contribution of these subunits to dyneinassembly and function is critical for understanding themechanism and regulation of flagellar motility in normal anddiseased states.

Dyneins are ATP-driven, microtubule-based molecularmotors. These massive, multi-subunit complexes contain oneor more catalytic heavy chains associated with several light andintermediate chains (Fig. 1) (King, 2000). The heavy chain(HC) is divided into three regions: the narrow neck, wherecargo binding occurs, the globular head, which corresponds tothe motor domain, and the stalk, which extends from theglobular head and is responsible for ATP-dependentmicrotubule binding. The motor domain contains six AAA+ATPase domains, of which one (P1) binds and hydrolyzes ATP(Neuwald et al., 1999; Ogawa, 1991). In dyneins containing

Axonemal dyneins are multisubunit molecular motors thatprovide the driving force for flagellar motility. Dynein lightchain 1 (LC1) has been well studied in Chlamydomonasreinhardtii and is unique among all dynein components asthe only protein known to bind directly to the catalyticmotor domain of the dynein heavy chain. However, the roleof LC1 in dynein assembly and/or function is unknownbecause no mutants have previously been available. Weidentified an LC1 homologue (TbLC1) in Trypanosomabrucei and have investigated its role in trypanosomeflagellar motility using epitope tagging and RNAi studies.TbLC1 is localized along the length of the flagellum andpartitions between the axoneme and soluble fractionsfollowing detergent and salt extraction. RNAi silencing ofTbLC1 gene expression results in the complete loss of thedominant tip-to-base beat that is a hallmark oftrypanosome flagellar motility and the concomitantemergence of a sustained reverse beat that propagates base-to-tip and drives cell movement in reverse. Ultrastructure

analysis revealed that outer arm dyneins are disrupted inTbLC1 mutants. Therefore LC1 is required for stabledynein assembly and forward motility in T. brucei. Ourwork provides the first functional analysis of LC1 in anyorganism. Together with the recent findings in T. bruceiDNAI1 mutants [Branche et al. (2006). Conserved andspecific functions of axoneme components in trypanosomemotility. J. Cell Sci. 119, 3443-3455], our data indicatefunctionally specialized roles for outer arm dyneins in T.brucei and C. reinhardtii. Understanding these differenceswill provide a more robust description of the fundamentalmechanisms underlying flagellar motility and will aidefforts to exploit the trypanosome flagellum as a drugtarget.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/120/9/1513/DC1

Key words: LC1, Motility regulation, Dynein

Summary

Stuck in reverse: loss of LC1 in Trypanosoma bruceidisrupts outer dynein arms and leads to reverseflagellar beat and backward movementDesiree M. Baron1, Zakayi P. Kabututu1 and Kent L. Hill1,2,*1Department of Microbiology, Immunology, and Molecular Genetics and 2Molecular Biology Institute, University of California, Los Angeles, CA90095, USA*Author for correspondence (e-mail: kenthill@mednet.ucla.edu)

Accepted 12 March 2007Journal of Cell Science 120, 1513-1520 Published by The Company of Biologists 2007doi:10.1242/jcs.004846

Jour

nal o

f Cel

l Sci

ence

Jour

nal o

f Cel

l Sci

ence

1514

two or more HCs, a complex at the base of the HC, composedof intermediate chains (IC) and light chains (LC), isresponsible for matching the appropriate cargo to the motorenzyme (DiBella and King, 2001; King et al., 1995; King andWitman, 1990; Wilkerson et al., 1995). Most of these accessoryproteins are also required for dynein assembly and aresuspected to play regulatory roles (King, 2003; Sakato andKing, 2004). The extensively studied outer dynein arms of theChlamydomonas flagellar axoneme are comprised of threeHCs (�, �, �), two intermediate chains (IC1 and IC2) and ninelight chains (LC1-LC9). LC2-LC9 are associated with theintermediate chains bound to the neck region (Fig. 1A) (King,2003; Sakato and King, 2004). LC1, which binds to the �HC,is unique in that it is the only protein known to bind directlyto a HC catalytic motor domain, rather than the cargo bindingneck (Benashski et al., 1999). This interaction is mediated bya hydrophobic patch on the �-sheet face of LC1, which binds

Journal of Cell Science 120 (9)

at or near the P1 AAA+ loop (Wu et al., 2000). Two basicresidues at the carboxyl end of the protein are thought to makeionic contacts within the ATP-hydrolyzing site of the P1 AAA+domain, suggesting that LC1 is directly involved in regulatingmotor activity (Benashski et al., 1999). Despite the unique andinteresting aspects of LC1-dynein interactions, the role of thisprotein in dynein function and/or assembly is unknown,because no mutants have been available for analysis.

Previously, we identified a candidate T. brucei LC1homologue (TbLC1) in a screen for conserved components ofmotile flagella (Baron et al., 2007). Taking advantage of thetools available for reverse genetics in T. brucei, we created anLC1 knockdown mutant by RNAi. Data from direct andindirect assays demonstrate that TbLC1 is necessary for properforward flagellar motility. The TbLC1 mutant presents a novelmotility phenotype with nearly all of the mutant cellsdisplaying a slow backward propulsion and a reverse flagellar

Fig. 1. TbLC1 has sequence andstructural homology to CrLC1. (A) Schematic diagram of theChlamydomonas outer arm dyneincomplex [adapted from King(King, 2003) with permission]. It iscomposed of three heavy chains(HC), each associated with at leastone light chain (LC). HCs havethree regions, a globular headmotor domain, a microtubule-binding stalk and a neck domainthat contributes to correct cargobinding. (B) Sequence alignmentbetween TbLC1 and LC1homologues (accession numberslisted in Materials and Methods).There is strong conservation,particularly in the LRR region(underlined residues are 40.5%identical, 94.6% similar). Keyresidues are conserved includingthose predicted to bind the �HC(#), p45 (*) and two C-terminalbasic residues (x) thought tocontact the ATP-hydrolyzing site inthe motor domain. Yellow and bluehighlighted amino acids areidentical between all and mostorganisms, respectively. Greenhighlighted amino acids representconservative substitutions.(C) Space filling model of CrLC1compared with TbLC1. The TbLC1structure was predicted bysequence comparison to CrLC1 andmodeling on the confirmed CrLC1structure (Wu et al., 2000) asdescribed in the Materials andMethods. CrLC1 residues predictedto bind the �HC (green) as well asthe basic residues in the C-terminus(blue) are conserved in the T.brucei protein. Red, �-helices;yellow, �-sheets.

Jour

nal o

f Cel

l Sci

ence

Jour

nal o

f Cel

l Sci

ence

1515Functional analysis of LC1 in T. brucei

beat. Additionally, loss of TbLC1 destabilizes the outer armdyneins, which is remarkable given its distal position in thedynein motor complex. Our data are therefore consistent withthe findings of Branche and co-workers (Branche et al., 2006)supporting the idea that tip-to-base wave propagation requiresouter arm dyneins. By understanding how each of thesubcomponents of the dynein motor complex contribute tooverall dynein function, a better comprehension of the role ofdynein in axonemal motility can be achieved.

Results and DiscussionAlignment of TbLC1 with CrLC1 and homologous sequencesfrom other kinetoplastids and humans demonstrates strongsequence conservation (Fig. 1B). Overall, TbLC1 is 46.1%identical to CrLC1, including conservation of all residuesproposed to bind �HC and p45, the only other protein knownto interact with LC1 (Horvath et al., 2005; Sakato and King,2004). Additionally, both of the basic residues believed tomake ionic contact within the ATP-hydrolyzing site of themotor domain are conserved in TbLC1 (Sakato and King,2004). Notably, neither of these residues is conserved in thehuman protein, suggesting potential differences in mechanismsof dynein regulation. The T. cruzi protein exhibits a unique N-terminal extension. Whether this reflects unique aspects ofLC1 function in that organism, or an error in genomeannotation is not presently known. Beyond primary sequencesimilarity, structure modeling of TbLC1 on the coordinates ofthe CrLC1 structure (Wu et al., 2003; Wu et al., 1999; Wu etal., 2000) revealed a similar topology, including placement ofthe �HC-interacting sites within the �-sheet face of the proteinand positioning of the �-helical regions that interact with p45(Fig. 1C). The identity of p45 is not known, but the T. bruceigenome does encode a single �HC homologue (GenBankAccession number XP_838596). Together, this suggests thatTbLC1 is a bona fide LC1 homologue.

To determine whether TbLC1 was localized to the flagellum,we constructed a tetracycline (Tet)-inducible GFP fusion.TbLC1-GFP was localized to the flagellum in live cells andwas stably associated with the axoneme after detergentextraction in a manner consistent with outer arm localization(Fig. 2A). TbLC1-GFP fluorescence was observed along theaxoneme, but did not extend all the way to the basal body, asevidenced by the gap between TbLC1-GFP and the kinetoplast.Biochemical fractionation revealed that some TbLC1-GFP wassolubilized with detergent (Fig. 2B, lane S1+), although asignificant amount remained associated with detergent-extracted cytoskeletons (Fig. 2B, lane P1+), corroborating theGFP fluorescence data. When cytoskeletons were furtherextracted with 500 mM NaCl, which removes the majority ofouter dynein arms (K.H., unpublished observation), TbLC1-GFP was distributed between the solubilized material and theextracted axoneme (Fig. 2B, lane S2+ and P2+, respectively).This is consistent with TbLC1 being a true homologue,because CrLC1 can also be solubilized in a complex with outerdynein arms by salt extraction (Wu et al., 2000), yet asignificant amount can associate with the axonemeindependently of the outer dynein arms (DiBella et al., 2005).

Tet-inducible RNAi was used to ablate TbLC1 expression inorder to ascertain whether TbLC1 is required for dyneinassembly and/or function. Clear and effective knockdown wasobtained within 24 hours of Tet induction (Fig. 2C). Three

independent assays demonstrated that TbLC1 knockdownresults in a dramatic motility defect. First, induction with Tetover a 5-day period resulted in multicellular clusters that beganto appear 48 hours post induction (hpi) (Fig. 2D). The mutantsremained viable, although growth was reduced (Fig. 2E).Trypanosomes divide through binary fission and flagellarmotility is required for efficient completion of cytokinesis(Ralston et al., 2006). In motility mutants, multiple rounds ofcytokinesis initiate, but are not completed, resulting inmulticellular clusters (Baron et al., 2007; Branche et al., 2006;Ralston et al., 2006). Our results are therefore consistent witha primary defect in flagellar motility that leads to faulty celldivision. To test this, TbLC1 knockdowns were next examinedfor motility defects using a sedimentation assay (Baron et al.,2007; Bastin et al., 1999; Ralston et al., 2006) at 24 hpi, whenthe mRNA was clearly knocked down (Fig. 2C) but no clusterswere present (Fig. 2D). TbLC1 mutants sedimented similarly topreviously reported moderate motility mutants (Baron et al.,2007), whereas uninduced cultures did not (Fig. 2F). Therefore,a primary motility defect preceded the cytokinesis defect.

To directly investigate motility, TbLC1 mutants wereexamined using high-resolution DIC microscopy as described(Baron et al., 2007). A unique and hallmark feature oftrypanosome flagellar motility is a tractile beat that initiates atthe tip of the flagellum and propagates toward the base, drivingcell movement with the flagellum tip leading (Hill, 2003;Walker, 1961; Walker and Walker, 1963). Occasionally, wild-type trypanosomes will stop moving forward and tumble,before continuing again in the forward direction (Hill, 2003;Hutchings et al., 2002). Uninduced TbLC1 cells werevigorously motile, moving with an average forward velocity of2.50±0.839 �m/second (Fig. 3A, –Tet). In stark contrast, mostTbLC1 mutants moved steadily backward at an averagevelocity of 0.738±0.250 �m/second (see supplementarymaterial Movie 1), with the flagellum tip trailing (Fig. 3A,+Tet). Occasionally, we observed cells that remained in oneposition, but they never moved forward. This is the first everreport of sustained backward motility in any trypanosomatid.Motility traces demonstrated that the majority of TbLC1mutant cells exhibited aberrant motility (Fig. 3B). The smallpercentage of cells labeled as runners in the motility trace ofinduced cells probably represents cells not fully affected by theknockdown at the time of the experiment.

Careful examination of TbLC1 mutant flagella revealedanother striking phenotype. Flagellar beat in these mutants wasreversed, i.e. initiated at the base of the flagellum andpropagated toward the tip (Fig. 3C). Wild-type trypanosomeflagella beat tip-to-base and will intermittently reverse to a base-to-tip direction (Branche et al., 2006; Holwill, 1974; Holwill,1965; Walker and Walker, 1963), but reverse beat is notsustained. In TbLC1 mutants, every cell examined at 30 hpi and49 hpi had a sustained base-to-tip beat and most of these movedbackward (30 hpi cells are shown in supplementary materialMovie 1). Significant cytokinesis problems precluded detailedmotility analysis at later time points, but at 49 hpi mutantscontinued to exhibit reverse beat and backward motility (notshown). The reverse beat is mostly symmetrical, although itbecomes erratic at the flagellum tip. There is some slow rotationof the cell around its long axis, in the counter-clockwisedirection when looking toward the cell posterior. However,TbLC1 mutants did not exhibit the tumbling behavior that

Jour

nal o

f Cel

l Sci

ence

Jour

nal o

f Cel

l Sci

ence

1516 Journal of Cell Science 120 (9)

Fig. 2. TbLC1 localizes along theflagellum and is required formotility. (A) Fluorescencemicroscopy of live cells andcytoskeletons from TbLC1-GFPstrains shows that TbLC1-GFP islocalized to the flagellum ininduced cells (+Tet). There isbackground autofluorescence inthe cell body of induced anduninduced (–Tet) live cell samplesthat is variable (Baron et al.,2007). Cytoskeletons were stainedwith DAPI (blue) to visualize thenucleus and kinetoplast relative toTbLC1-GFP (green). (B) Westernblot analysis with anti-GFPantibody of TbLC1-GFP cellularfractions from induced (+Tet, 24hpi) and uninduced (–Tet) cellsconfirms that TbLC1 is stablyassociated with the flagellum. L,lysates; S1, detergent-solubleproteins; P1, cell cytoskeletons;S2, NaCl soluble proteins; P2,flagellar cytoskeletons. The samefractions were blotted with anti-trypanin (TPN) monoclonalantibody as a loading control(bottom panel). (C) Northern blotsof RNA from an uninduced (–Tet)and induced (+Tet, 24 hpi) TbLC1RNAi knockdown strain probedwith TbLC1 and TbCMF46(control) DNA fragments.(D) Images of whole cultures ofuninduced (–Tet) and induced(+Tet) TbLC1 knockdown cellsover a 5-day induction. At 48 hpimulticellular clusters appear in theculture containing 5-10 cells each,and increase in size and numberover time. (E) Growth curve ofuninduced (–Tet) and induced(+Tet) TbLC1 cells over a 5-dayperiod. (F) Sedimentation curves(Baron et al., 2007; Bastin et al.,1999; Ralston et al., 2006) for theuninduced (–TET) and induced(+TET, 24 hpi) cells. Error barsshow the s.d. for threeexperiments.

Jour

nal o

f Cel

l Sci

ence

Jour

nal o

f Cel

l Sci

ence

1517Functional analysis of LC1 in T. brucei

occurs periodically in wild-type cells and continuously in DRCmutants (Hill, 2003; Hutchings et al., 2002). Therefore, loss ofTbLC1 expression results in the complete loss of the dominanttractile beat that is a hallmark of trypanosome flagella and theconcomitant emergence of a sustained reverse beat thatpropagates base-to-tip and drives cell movement in reverse.

In Chlamydomonas, all of the outer arm IC and LC mutantspreviously studied save one, LC6, have been shown to berequired for stable assembly of the outer arm (King, 2003;Pazour and Witman, 2000; Sakato and King, 2004). Toascertain the extent to which the loss of TbLC1 affectsaxoneme structure in T. brucei, we used transmission electronmicroscopy. Compared with flagella from control cells, TbLC1mutant flagella show normal outer doublets, central pair andradial spoke structures (Fig. 4, +Tet). However, outer dyneinarms were missing or reduced in most axonemes of TbLC1

mutants (Fig. 4A, +Tet). Cross-sections of mutant flagellaexamined at 30, 49 and 72 hpi showed that this effect wasconstant at all time points, indicating that motility phenotypesobserved at early time points accurately reflect theconsequence of outer arm deficiency that is due to loss ofTbLC1. Branche and co-workers (Branche et al., 2006),recently demonstrated that loss of the dynein intermediatechain DNAI1 in T. brucei results in loss of outer dynein arms,loss of tip-to-base flagellar beat and loss of forward motility.Our data are consistent with these findings, supporting the ideathat tip-to-base flagellar beat propagation and forward motilityin T. brucei requires outer dynein arms, since outer dyneindeficiency caused by two independent mechanisms(knockdown of DNAI1 or LC1) blocks tip-to-base beat.Interestingly, the reverse base-to-tip beat of DNAI1knockdown mutants did not drive reverse cell motility.

Fig. 3. TbLC1 knockdown causesreverse cell motility. (A) Time-lapseseries taken from video clips ofinduced (+Tet, 26 hpi) or uninduced(–Tet) TbLC1 knockdown cells. Thedashed line marks the cell posterior(P) at the start of the time-lapseseries. The black arrows in the firstpanel represent the direction expectedfor wild-type movement, with theanterior end (A) leading. Red arrowsat the bottom show the actualdirection of cellular movement.TbLC1 mutants move backward, withthe posterior end leading. Significantcytokinesis problems preventeddetailed motility analysis at later timepoints, but single cells observed at 49hpi continued to exhibit reverse beatand reverse motility (not shown).Bars, 10 �m. (B) Cell traces (Baronet al., 2007) of uninduced (–Tet) andinduced (+Tet, 26 hpi) TbLC1knockdown cells. Lines show thedistance traveled by cells over a 30-second interval. n=47 (–Tet) or 52(+Tet) cells. Bars, 50 �m. Cellclassifications are described inMaterials and Methods. Inset piecharts display the percentage of eachcell type observed in each strain. (C) Time-lapse series shows beatdirection in two flagellar mutants thatare stuck to slides. TbLC1 mutants(24 hpi) display a flagellar beat thatoriginates at the base of the flagellumand is propagated to the tip(supplementary material Movie 3).Trypanin mutants (5 days postinfection) display a tumblingphenotype (Hutchings et al., 2002)and maintain the wild-type tip-to-base flagellar waveform movement.Black arrows show the waveformposition at the beginning of theseries. White arrows show theposition of the waveform at each timepoint.

Jour

nal o

f Cel

l Sci

ence

Jour

nal o

f Cel

l Sci

ence

1518

Whether this reflects differences in the requirement for DNAI1and TbLC1 in outer dynein assembly and/or function orexperimental differences is not presently clear. Orientation ofcentral pair microtubules is restricted within a small range inT. brucei (Branche et al., 2006; Ralston et al., 2006). TbLC1mutants exhibited a defect in central pair orientation (Fig. 4B),

as observed for DNAI1 mutants (Branche etal., 2006). This defect increased withinduction time in TbLC1 mutants, eventhough the outer arm defect did not (Fig. 4),suggesting that central pair misorientationmight be due to pleiotropic effectscontributing to the phenotype at later timepoints.

Branche and co-workers also suggested aninteresting model in which reverse wavepropagation in T. brucei causes cellularreorientation without backward motility. Wetherefore examined beat direction in trypaninmutants, which undergo continuous cellularreorientation without net directional motility(Hutchings et al., 2002). In cases where beatdirection could clearly be determined,flagellar beat progressed primarily from tip-to-base, with occasional bursts of base-to-tipbeat (Fig. 3C and supplementary materialMovie 2) as seen for wild-type T. brucei(Branche et al., 2006; Walker and Walker,1963). These results, together with thefinding that TbLC1 mutants exhibit acontinuous reverse beat and maintain steadyreverse cell movement, indicate that reversebeat alone is not sufficient to drive cellreorientation. Perhaps concurrent andcompeting reverse and forward beats in thesame flagellum are what cause cellreorientation. Transient bursts of base-to-tipbeat propagation have previously beendemonstrated to drive short periods of reversecell motion in T. cruzi (Jahn and Fonseca,1963) and Crithidia oncopelti (Holwill,1964; Holwill, 1965). Therefore, the abilityof reverse flagellar beat to drive backwardcell movement is conserved among thesetrypanosomatids. Tip-to-base beating oftrypanosome flagella, as well as the capacityto reverse flagellar beat implies the presenceof specialized regulatory systems forcontrolling flagellar beat in these organisms.

The ultrastructural deficit of TbLC1mutants was specific to the outer dyneinarms, as inner arms, radial spokes and centralpair structures were still present. Likewise,loss of outer arm dyneins is not a commondefect in other T. brucei motility mutants(Baron, 2007; Bastin et al., 1998; Branche etal., 2006; Maga et al., 1999). Therefore, ourdata indicate a specific requirement for LC1in the assembly and/or stability of outer armdyneins in T. brucei. Defects in outer armdynein assembly are often seen in mutants

lacking ICs or LCs associated with the HC coiled-coil domainthat mediates cargo attachment and oligomerization in bothChlamydomonas (King, 2003; Sakato and King, 2004) and T.brucei (Branche et al., 2006). However, LC1 is bound to themotor domain of the �HC rather than the coiled-coil domain(Benashski et al., 1999). As such, the requirement for TbLC1

Journal of Cell Science 120 (9)

Fig. 4. TbLC1 is required for stable outer arm dynein assembly. (A) Electronmicrographs of uninduced (–Tet) and induced (+Tet, 30 hpi) whole cell TbLC1 mutantflagellar cross-sections. Arrows indicate examples of positions on doublets where outerdynein arms are present (– Tet) or absent (+ Tet). The table shows the breakdown of howmany arms were missing from uninduced and induced cells at the indicated time points.Increasing the time of induction does not affect the extent of outer arm loss. (B) Analysis of central pair orientation in TbLC1 knockdown cells. Central pairorientation was determined as described previously (Ralston et al., 2006). n=22-37sections (–Tet) or 30-40 sections (+Tet). The percentage of sections having central pairorientation outside the range observed in associated –Tet samples are indicated.

Jour

nal o

f Cel

l Sci

ence

Jour

nal o

f Cel

l Sci

ence

1519Functional analysis of LC1 in T. brucei

in outer dynein assembly is somewhat surprising, because C.reinhardtii mutants lacking the entire �HC or the �HC motordomain still assemble outer arm dyneins (Sakakibara et al.,1991; Sakakibara et al., 1993). It is possible that LC1 mightnot be specific to the �HC motor domain in T. brucei. However,all of the predicted �HC binding sites are conserved in theprimary structure and in the modeled tertiary structure ofTbLC1, as are the C-terminal amino acids predicted to contactthe ATP-hydrolyzing site of the AAA+ domain. This suggeststhat TbLC1 does bind the cognate domain of the T. brucei �HCand indicates that outer arm assembly in trypanosomes issensitive to perturbations of the �HC. This might reflectchanges specifically at the motor domain where LC1 binds.Alternatively, loss of TbLC1 might directly affect stability ofthe entire �HC, similarly to the influence of Oda7p – a leucine-rich repeat (LRR) protein like LC1 (Freshour et al., 2007) – on�HC stability in C. reinhardtii (Fowkes and Mitchell, 1998).Loss of the �HC might then lead to a block in outer dyneinassembly.

Our results and those of Branche et al. (Branche et al., 2006)highlight an interesting feature of outer dynein contributions toflagellar beat in Chlamydomonas versus trypanosomes. C.reinhardtii outer dynein arm mutants cannot generate flagellar-type beating, in which a sinusoidal beat propagates base-to-tipand drives cell movement with the flagellum tip trailing(Kamiya and Okamoto, 1985). Conversely, this is the only typeof beat that occurs in T. brucei outer dynein arm mutants. Tip-to-base beating is a distinguishing feature of trypanosomeflagella and does not occur in C. reinhardtii. Our data and thoseof Branche and co-workers indicate that outer dyneins arerequired for tip-to-base beating. Hence, outer dynein armsappear to be a target of trypanosome-specific regulatorymechanisms that enable this distinctive beat, indicating thatouter dyneins in T. brucei have specialized functionality that isnot observed in C. reinhardtii. This emphasizes the need forcontinued analysis of flagellar motility in diverse organisms.Recent work strongly suggests that flagellar motility isessential in bloodstream-form T. brucei (Broadhead et al.,2006; Ralston and Hill, 2006). Hence, identifying andunderstanding unique features of trypanosome flagellarmotility will enhance the potential of the flagellum as a targetfor chemotherapeutic intervention in African sleeping sickness.

Materials and MethodsBioinformatic analysisA previous genomic screen (Baron et al., 2007) identified a T. brucei protein (GeneDB ID number Tb11.02.3390, NCBI accession XP_828643) that was similar toCrLC1 (E-34). Protein sequence alignments between the bona fide CrLC1, otherLC1 homologues and TbLC1 were performed using the Clustal W algorithm(Thompson et al., 1994). NCBI accession numbers of other LC1 homologues: C.reinhardtii (AAD41040), L. major (CAJ04675), T. cruzi (XP_815756), H. sapiens(AAQ11377). Structural homology between CrLC1 (PDB accession 1DS9) andTbLC1 was determined using Deep View/Swiss-PDB Viewer (http://www.expasy.org/spdbv/).

Cloning of TbLC1 RNAi and GFP-tagged constructsRNAiThe RNAi construct was created as described previously (Baron et al., 2007) usinga 485 bp fragment corresponding to nucleotides 121 to 605 identified by theTrypanofan RNAit algorithm (Redmond et al., 2003; http://trypanofan.path.cam.ac.uk/cgi-bin/rnait.org). Primers used for amplification were: forward, 5�-aggaggctcgtgttttgcta-3� and reverse, 5�-ctctttccggatccactggta-3�. Inserts wereverified by sequencing at the UCLA genomics center.

GFP-taggingFull-length TbLC1 ORF was amplified from 29-13 (Wirtz et al., 1999) genomic

DNA using primers containing HindIII and XbaI sites and ligated into pKH12 atthe N-terminus of GFP as described previously (Baron et al., 2007). Althoughexpression levels and epitope tag might influence protein localization, this plasmidwas selected because it drives expression of other axonemal proteins at nearendogenous levels and accurately reports the location of flagellar proteins havingexpression levels similar to that expected for TbLC1, i.e. one or a few copies peraxonemal repeat unit (Baron et al., 2007). Primers used for amplification were:forward, 5�-cccaagcttgcatgtcaggaacaacttcca-3� and reverse, 5�-gctctagaccggccg -cgctccgcctcttc-3�. All sequences were verified by DNA sequencing at the UCLAgenomics center.

Trypanosome transfection and cell maintenanceProcyclic 29-13 cells (Wirtz et al., 1999) were used to generate RNAi and GFP-tagged TbLC1 strains. Cells were maintained and transfected as describedpreviously (Hill et al., 1999; Hutchings et al., 2002) and clonal lines were obtainedby limiting dilution. Knockdown mutants and expression of TbLC1-GFP wasinduced with 1 �g/ml of tetracycline. At 24 hpi, TbLC1-GFP fusion protein cellswere viewed on a Zeiss Axioskop II compound fluorescent microscope using a 63�oil objective.

Northern blotsRNA was isolated from induced (24 hpi) and uninduced TbLC1 cells using a QiagenRNeasy Miniprep kit according to the manufacturer’s instructions. Northern blots(Hill et al., 1991) using 5 �g of total RNA were probed with 32P-labeled DNAfragments corresponding to the TbLC1 region used for RNAi knockdown, or toTbCMF 46 nucleotides 129 to 608.

Motility assays and ultrastructural analysisSedimentation assays, whole culture observations, cell tracking assays and DICmicroscopy were performed as described previously (Baron et al., 2007).Transmission electron microscopy was performed as described previously(Hutchings et al., 2002). Central pair orientation analysis was performed asdescribed previously (Ralston et al., 2006). In order to minimize the influence ofpleiotropic effects, cells were examined at 24-30 hpi, a time point where mRNA isclearly knocked down and motility defects are clearly evident, but before clusteringbecame apparent in the culture. Ultrastructural analyses were also done at 49 and72 hpi as described in the text. For motility traces, ‘runners’ were classified asvigorously motile cells that progressed forward along a curvilinear path for 5seconds or more. ‘Crawlers’ were cells with slow movement that progressed awayfrom their point of origin, whereas ‘immotiles’ were cells that remained at theirpoint of origin during the time interval.

Trypanosome cellular fractionation and western blottingTrypanosome lysates, detergent-soluble proteins and cell cytoskeletons wereprepared as described previously (Hill et al., 2000) and western blotted as described(Hill et al., 1999). Primary antibody dilutions were: monoclonal anti-GFP antibody(Clontech no. 632380) 1:500 and monoclonal anti-trypanin antibody 1:4000(Ralston et al., 2006).

We would like to thank Randy Nessler (University of Iowa) for hisassistance with electron microscopy. We would also like to thankGeorge Cross (Rockefeller University) for the 29-13 cell line andKatherine Ralston for assistance with fluorescence microscopy. Thiswork was supported by grants from the National Institute of Health(R01AI52348), Ellison Medical Foundation (ID-NS-0148-03) andBeckman Young Investigator Program to K.L.H. D.M.B. is therecipient of a USPHS National Research Service Award in MicrobialPathogenesis (2-T32-AI-07323) and the UCLA Graduate DissertationYear Fellowship for 2006-2007.

ReferencesBaron, D., Ralston, K. S., Kabututu, Z. P. and Hill, K. L. (2007). Functional genomics

in Trypanosoma brucei identifies evolutionarily conserved components of motileflagella. J. Cell Sci. 120, 478-491.

Bastin, P., Sherwin, T. and Gull, K. (1998). Paraflagellar rod is vital for trypanosomemotility. Nature 391, 548.

Bastin, P., Pullen, T. J., Sherwin, T. and Gull, K. (1999). Protein transport and flagellumassembly dynamics revealed by analysis of the paralysed trypanosome mutant snl-1.J. Cell Sci. 112, 3769-3777.

Benashski, S. E., Patel-King, R. S. and King, S. M. (1999). Light chain 1 from theChlamydomonas outer dynein arm is a leucine-rich repeat protein associated with themotor domain of the gamma heavy chain. Biochemistry 38, 7253-7264.

Branche, C., Kohl, L., Toutirais, G., Buisson, J., Cosson, J. and Bastin, P. (2006).Conserved and specific functions of axoneme components in trypanosome motility. J.Cell Sci. 119, 3443-3455.

Broadhead, R., Dawe, H. R., Farr, H., Griffiths, S., Hart, S. R., Portman, N., Shaw,

Jour

nal o

f Cel

l Sci

ence

Jour

nal o

f Cel

l Sci

ence

1520

M. K., Ginger, M. L., Gaskell, S. J., McKean, P. G. et al. (2006). Flagellar motilityis required for the viability of the bloodstream trypanosome. Nature 440, 224-227.

DiBella, L. M. and King, S. M. (2001). Dynein motors of the Chlamydomonas flagellum.Int. Rev. Cytol. 210, 227-268.

DiBella, L. M., Gorbatyuk, O., Sakato, M., Wakabayashi, K., Patel-King, R. S.,Pazour, G. J., Witman, G. B. and King, S. M. (2005). Differential light chainassembly influences outer arm dynein motor function. Mol. Biol. Cell 16, 5661-5674.

Fowkes, M. E. and Mitchell, D. R. (1998). The role of preassembled cytoplasmiccomplexes in assembly of flagellar dynein subunits. Mol. Biol. Cell 9, 2337-2347.

Freshour, J., Yokoyama, R. and Mitchell, D. R. (2007). Chlamydomonas flagellar outerrow dynein assembly protein Oda7 interacts with both outer row and I1 inner rowdyneins. J. Biol. Chem. 282, 5404-5412.

Hill, K. L. (2003). Mechanism and biology of trypanosome cell motility. Eukaryotic Cell2, 200-208.

Hill, K. L., Li, H. H., Singer, J. and Merchant, S. (1991). Isolation and structuralcharacterization of the Chlamydomonas reinhardtii gene for cytochrome c6. Analysisof the kinetics and metal specificity of its copper-responsive expression. J. Biol. Chem.266, 15060-15067.

Hill, K. L., Hutchings, N. R., Russell, D. G. and Donelson, J. E. (1999). A novel proteintargeting domain directs proteins to the anterior cytoplasmic face of the flagellar pocketin African trypanosomes. J. Cell Sci. 112, 3091-3101.

Hill, K. L., Hutchings, N. R., Grandgenett, P. M. and Donelson, J. E. (2000). TLymphocyte triggering factor of African trypanosomes is associated with the flagellarfraction of the cytoskeleton and represents a new family of proteins that are present inseveral divergent eukaryotes. J. Biol. Chem. 275, 39369-39378.

Holwill, M. E. J. (1964). High-speed cine-photography of certain of theTrypanosomatidae. J. Protozool. 11 suppl. 40, 122.

Holwill, M. E. J. (1965). The motion of Strigomona Oncopelti. J. Exp. Biol. 42, 125-137.Holwill, M. E. (1974). Some physical aspects of the motility of ciliated and flagellated

microorganisms. Sci. Prog. 61, 63-80.Horvath, J., Fliegauf, M., Olbrich, H., Kispert, A., King, S. M., Mitchison, H.,

Zariwala, M. A., Knowles, M. R., Sudbrak, R., Fekete, G. et al. (2005).Identification and analysis of axonemal dynein light chain 1 in primary ciliarydyskinesia patients. Am. J. Respir. Cell Mol. Biol. 33, 41-47.

Hutchings, N. R., Donelson, J. E. and Hill, K. L. (2002). Trypanin is a cytoskeletallinker 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 ofcilia in development and disease. Hum. Mol. Genet. 12, R27-R35.

Jahn, T. L. and Fonseca, J. R. (1963). Mechanisms of locomotion of flagellates: V.Trypanosoma lewisi and T. cruzi. J. Protozool. 10, 11.

Kamiya, R. and Okamoto, M. (1985). A mutant of Chlamydomonas reinhardtii thatlacks the flagellar outer dynein arm but can swim. J. Cell Sci. 74, 181-191.

King, S. M. (2000). The dynein microtubule motor. Biochim. Biophys. Acta 1496, 60-75.King, S. M. (2003). Organization and regulation of the dynein microtubule motor. Cell

Biol. Int. 27, 213-215.King, S. M. and Witman, G. B. (1990). Localization of an intermediate chain of outer

arm dynein by immunoelectron microscopy. J. Biol. Chem. 265, 19807-19811.King, S. M., Patel-King, R. S., Wilkerson, C. G. and Witman, G. B. (1995). The

78,000-M(r) intermediate chain of Chlamydomonas outer arm dynein is a microtubule-binding protein. J. Cell Biol. 131, 399-409.

Kohl, L., Robinson, D. and Bastin, P. (2003). Novel roles for the flagellum in cellmorphogenesis and cytokinesis of trypanosomes. EMBO J. 22, 5336-5346.

Luck, D., Piperno, G., Ramanis, Z. and Huang, B. (1977). Flagellar mutants ofChlamydomonas: studies of radial spoke-defective strains by dikaryon and revertantanalysis. Proc. Natl. Acad. Sci. USA 74, 3456-3460.

Maga, J. A., Sherwin, T., Francis, S., Gull, K. and LeBowitz, J. H. (1999). Genetic

dissection of the Leishmania paraflagellar rod, a unique flagellar cytoskeletonstructure. J. Cell Sci. 112, 2753-2763.

Neuwald, A. F., Aravind, L., Spouge, J. L. and Koonin, E. V. (1999). AAA+: A classof chaperone-like ATPases associated with the assembly, operation, and disassemblyof protein complexes. Genome Res. 9, 27-43.

Ogawa, K. (1991). Four ATP-binding sites in the midregion of the beta heavy chain ofdynein. Nature 352, 643-645.

Pan, J., Wang, Q. and Snell, W. J. (2005). Cilium-generated signaling and cilia-relateddisorders. Lab. Invest. 85, 452-463.

Pazour, G. J. and Witman, G. B. (2000). Forward and reverse genetic analysis ofmicrotubule motors in Chlamydomonas. Methods 22, 285-298.

Pazour, G. J. and Witman, G. B. (2003). The vertebrate primary cilium is a sensoryorganelle. Curr. Opin. Cell Biol. 15, 105-110.

Pazour, G. J., Agrin, N., Leszyk, J. and Witman, G. B. (2005). Proteomic analysis ofa eukaryotic cilium. J. Cell Biol. 170, 103-113.

Ralston, K. S. and Hill, K. L. (2006). Trypanin, a component of the flagellar Dyneinregulatory complex, is essential in bloodstream form African trypanosomes. PLoSPathog. 2, e101.

Ralston, K. S., Lerner, A. G., Diener, D. R. and Hill, K. L. (2006). Flagellar motilitycontributes to cytokinesis in Trypanosoma brucei and is modulated by an evolutionarilyconserved dynein regulatory system. Eukaryotic Cell 5, 696-711.

Redmond, S., Vadivelu, J. and Field, M. C. (2003). RNAit: an automated web-basedtool for the selection of RNAi targets in Trypanosoma brucei. Mol. Biochem. Parasitol.128, 115-118.

Sakakibara, H., Mitchell, D. R. and Kamiya, R. (1991). A Chlamydomonas outer armdynein mutant missing the alpha heavy chain. J. Cell Biol. 113, 615-622.

Sakakibara, H., Takada, S., King, S. M., Witman, G. B. and Kamiya, R. (1993). AChlamydomonas outer arm dynein mutant with a truncated beta heavy chain. J. CellBiol. 122, 653-661.

Sakato, M. and King, S. M. (2004). Design and regulation of the AAA+ microtubulemotor dynein. J. Struct. Biol. 146, 58-71.

Solter, K. M. and Gibor, A. (1977). Evidence for role of flagella as sensory transducersin mating of Chlamydomonas reinhardi. Nature 265, 444-445.

Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994). CLUSTAL W: improvingthe sensitivity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680.

Vickerman, K., Tetley, L., Hendry, K. A. and Turner, C. M. (1988). Biology of Africantrypanosomes in the tsetse fly. Biol. Cell 64, 109-119.

Walker, P. J. (1961). Organization of function in trypanosome flagella. Nature 189, 1017-1018.

Walker, P. J. and Walker, J. C. (1963). Movement of trypanosome flagella. J. Protozool.10, suppl. 32.

Wilkerson, C. G., King, S. M., Koutoulis, A., Pazour, G. J. and Witman, G. B. (1995).The 78,000 M(r) intermediate chain of Chlamydomonas outer arm dynein is a WD-repeat protein required for arm assembly. J. Cell Biol. 129, 169-178.

Wirtz, E., Leal, S., Ochatt, C. and Cross, G. A. (1999). A tightly regulated inducibleexpression system for conditional gene knock-outs and dominant-negative genetics inTrypanosoma brucei. Mol. Biochem. Parasitol. 99, 89-101.

Wu, H., Maciejewski, M. W., Benashski, S. E., Mullen, G. P. and King, S. M. (1999).1H, 15N and 13C resonance assignments for the 22 kDa LC1 light chain fromChlamydomonas outer arm dynein. J. Biomol. NMR 13, 309-310.

Wu, H., Maciejewski, M. W., Marintchev, A., Benashski, S. E., Mullen, G. P. andKing, S. M. (2000). Solution structure of a dynein motor domain associated light chain.Nat. Struct. Biol. 7, 575-579.

Wu, H., Blackledge, M., Maciejewski, M. W., Mullen, G. P. and King, S. M. (2003).Relaxation-based structure refinement and backbone molecular dynamics of the dyneinmotor domain-associated light chain. Biochemistry 42, 57-71.

Journal of Cell Science 120 (9)

Jour

nal o

f Cel

l Sci

ence

Jour

nal o

f Cel

l Sci

ence