Axonemal dyneins are multisubunit molecular motors that provide the driving force for flagellar motility. Dynein light chain 1 (LC1) has been well studied in Chlamydomonas reinhardtii and is unique among all dynein components as the only protein known to bind directly to the catalytic motor domain of the dynein heavy chain. However, the role of LC1 in dynein assembly and/or function is unknown because no mutants have previously been available. We identified an LC1 homologue (TbLC1) in Trypanosoma brucei and have investigated its role in trypanosome flagellar motility using epitope tagging and RNAi studies. TbLC1 is localized along the length of the flagellum and partitions between the axoneme and soluble fractions following detergent and salt extraction. RNAi silencing of TbLC1 gene expression results in the complete loss of the dominant tip-to-base beat that is a hallmark of trypanosome flagellar motility and the concomitant emergence 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 in TbLC1 mutants. Therefore LC1 is required for stable dynein assembly and forward motility in T. brucei. Our work provides the first functional analysis of LC1 in any organism. Together with the recent findings in T. brucei DNAI1 mutants [Branche et al. (2006). Conserved and specific functions of axoneme components in trypanosome motility. J. Cell Sci. 119, 3443-3455], our data indicate functionally specialized roles for outer arm dyneins in T. brucei and C. reinhardtii. Understanding these differences will provide a more robust description of the fundamental mechanisms underlying flagellar motility and will aid efforts to exploit the trypanosome flagellum as a drug target.

Introduction

Flagella and cilia (eukaryotic cilia and flagella are structurally and functionally analogous and we will use these terms interchangeably) are dynamic organelles involved in several biological processes including sensing the surrounding environment, cellular adhesion, mating (Pazour and Witman, 2003; Solter and Gibor, 1977; Vickerman et al., 1988) and most notably, motility. For example, in mammals, motile cilia are required for proper reproduction, left-right axis determination, brain and respiratory function. As such, ciliary defects can lead to serious diseases including infertility, situs inversus, hydrocephalus and respiratory malfunction (Ibanez-Tallon et al., 2003; Pan et al., 2005). Flagella are also the primary mode of locomotion for many pathogenic protozoa, such as Trypanosoma brucei, Giardia lamblia and Trichomonas vaginalis. For many pathogens, motility is believed to be vital for disease pathogenesis and in some cases, flagella serve additional, 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). Therefore flagellar motility impacts infectious and heritable diseases in humans. At the heart of motile flagella and cilia is the conserved 9+2 axoneme, which is composed of approximately 250 proteins (Luck et al., 1977; Pazour et al., 2005). It is the coordinated activation of dynein molecular motors on adjacent outer doublets of the axoneme that is the driving force behind the motility of this structure. The axoneme contains several discrete populations of dyneins having distinct subunit compositions and specialized roles in flagellar beat. Determining the contribution of these subunits to dynein assembly and function is critical for understanding the mechanism and regulation of flagellar motility in normal and diseased states.

Dyneins are ATP-driven, microtubule-based molecular motors. These massive, multi-subunit complexes contain one or more catalytic heavy chains associated with several light and intermediate chains (Fig. 1) (King, 2000). The heavy chain (HC) is divided into three regions: the narrow neck, where cargo binding occurs, the globular head, which corresponds to the motor domain, and the stalk, which extends from the globular head and is responsible for ATP-dependent microtubule 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 two or more HCs, a complex at the base of the HC, composed of intermediate chains (IC) and light chains (LC), is responsible for matching the appropriate cargo to the motor enzyme (DiBella and King, 2001; King et al., 1995; King and Witman, 1990; Wilkerson et al., 1995). Most of these accessory proteins are also required for dynein assembly and are suspected to play regulatory roles (King, 2003; Sakato and King, 2004). The extensively studied outer dynein arms of the Chlamydomonas flagellar axoneme are comprised of three HCs (α, β, γ), two intermediate chains (IC1 and IC2) and nine light chains (LC1-LC9). LC2-LC9 are associated with the intermediate chains bound to the neck region (Fig. 1A) (King, 2003; Sakato and King, 2004). LC1, which binds to the γHC, is unique in that it is the only protein known to bind directly to a HC catalytic motor domain, rather than the cargo binding neck (Benashski et al., 1999). This interaction is mediated by a hydrophobic patch on the β-sheet face of LC1, which binds at or near the P1 AAA+ loop (Wu et al., 2000). Two basic residues at the carboxyl end of the protein are thought to make ionic contacts within the ATP-hydrolyzing site of the P1 AAA+ domain, suggesting that LC1 is directly involved in regulating motor activity (Benashski et al., 1999). Despite the unique and interesting aspects of LC1-dynein interactions, the role of this protein in dynein function and/or assembly is unknown, because no mutants have been available for analysis.

Fig. 1.

TbLC1 has sequence and structural homology to CrLC1. (A) Schematic diagram of the Chlamydomonas outer arm dynein complex [adapted from King (King, 2003) with permission]. It is composed of three heavy chains (HC), each associated with at least one light chain (LC). HCs have three regions, a globular head motor domain, a microtubule-binding stalk and a neck domain that contributes to correct cargo binding. (B) Sequence alignment between TbLC1 and LC1 homologues (accession numbers listed in Materials and Methods). There is strong conservation, particularly in the LRR region (underlined residues are 40.5% identical, 94.6% similar). Key residues are conserved including those predicted to bind the γHC (#), p45 (*) and two C-terminal basic residues (x) thought to contact the ATP-hydrolyzing site in the motor domain. Yellow and blue highlighted amino acids are identical between all and most organisms, respectively. Green highlighted amino acids represent conservative substitutions. (C) Space filling model of CrLC1 compared with TbLC1. The TbLC1 structure was predicted by sequence comparison to CrLC1 and modeling on the confirmed CrLC1 structure (Wu et al., 2000) as described in the Materials and Methods. CrLC1 residues predicted to bind the γHC (green) as well as the basic residues in the C-terminus (blue) are conserved in the T. brucei protein. Red, α-helices; yellow, β-sheets.

Fig. 1.

TbLC1 has sequence and structural homology to CrLC1. (A) Schematic diagram of the Chlamydomonas outer arm dynein complex [adapted from King (King, 2003) with permission]. It is composed of three heavy chains (HC), each associated with at least one light chain (LC). HCs have three regions, a globular head motor domain, a microtubule-binding stalk and a neck domain that contributes to correct cargo binding. (B) Sequence alignment between TbLC1 and LC1 homologues (accession numbers listed in Materials and Methods). There is strong conservation, particularly in the LRR region (underlined residues are 40.5% identical, 94.6% similar). Key residues are conserved including those predicted to bind the γHC (#), p45 (*) and two C-terminal basic residues (x) thought to contact the ATP-hydrolyzing site in the motor domain. Yellow and blue highlighted amino acids are identical between all and most organisms, respectively. Green highlighted amino acids represent conservative substitutions. (C) Space filling model of CrLC1 compared with TbLC1. The TbLC1 structure was predicted by sequence comparison to CrLC1 and modeling on the confirmed CrLC1 structure (Wu et al., 2000) as described in the Materials and Methods. CrLC1 residues predicted to bind the γHC (green) as well as the basic residues in the C-terminus (blue) are conserved in the T. brucei protein. Red, α-helices; yellow, β-sheets.

Previously, we identified a candidate T. brucei LC1 homologue (TbLC1) in a screen for conserved components of motile flagella (Baron et al., 2007). Taking advantage of the tools available for reverse genetics in T. brucei, we created an LC1 knockdown mutant by RNAi. Data from direct and indirect assays demonstrate that TbLC1 is necessary for proper forward flagellar motility. The TbLC1 mutant presents a novel motility phenotype with nearly all of the mutant cells displaying a slow backward propulsion and a reverse flagellar beat. Additionally, loss of TbLC1 destabilizes the outer arm dyneins, which is remarkable given its distal position in the dynein motor complex. Our data are therefore consistent with the findings of Branche and co-workers (Branche et al., 2006) supporting the idea that tip-to-base wave propagation requires outer arm dyneins. By understanding how each of the subcomponents of the dynein motor complex contribute to overall dynein function, a better comprehension of the role of dynein in axonemal motility can be achieved.

Results and Discussion

Alignment of TbLC1 with CrLC1 and homologous sequences from other kinetoplastids and humans demonstrates strong sequence conservation (Fig. 1B). Overall, TbLC1 is 46.1% identical to CrLC1, including conservation of all residues proposed to bind γHC and p45, the only other protein known to interact with LC1 (Horvath et al., 2005; Sakato and King, 2004). Additionally, both of the basic residues believed to make ionic contact within the ATP-hydrolyzing site of the motor domain are conserved in TbLC1 (Sakato and King, 2004). Notably, neither of these residues is conserved in the human protein, suggesting potential differences in mechanisms of dynein regulation. The T. cruzi protein exhibits a unique N-terminal extension. Whether this reflects unique aspects of LC1 function in that organism, or an error in genome annotation is not presently known. Beyond primary sequence similarity, structure modeling of TbLC1 on the coordinates of the CrLC1 structure (Wu et al., 2003; Wu et al., 1999; Wu et al., 2000) revealed a similar topology, including placement of the γHC-interacting sites within the β-sheet face of the protein and positioning of the α-helical regions that interact with p45 (Fig. 1C). The identity of p45 is not known, but the T. brucei genome does encode a single γHC homologue (GenBank Accession number XP_838596). Together, this suggests that TbLC1 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 and was stably associated with the axoneme after detergent extraction in a manner consistent with outer arm localization (Fig. 2A). TbLC1-GFP fluorescence was observed along the axoneme, but did not extend all the way to the basal body, as evidenced by the gap between TbLC1-GFP and the kinetoplast. Biochemical fractionation revealed that some TbLC1-GFP was solubilized with detergent (Fig. 2B, lane S1+), although a significant amount remained associated with detergent-extracted cytoskeletons (Fig. 2B, lane P1+), corroborating the GFP fluorescence data. When cytoskeletons were further extracted with 500 mM NaCl, which removes the majority of outer dynein arms (K.H., unpublished observation), TbLC1-GFP was distributed between the solubilized material and the extracted 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 outer dynein arms by salt extraction (Wu et al., 2000), yet a significant amount can associate with the axoneme independently of the outer dynein arms (DiBella et al., 2005).

Tet-inducible RNAi was used to ablate TbLC1 expression in order to ascertain whether TbLC1 is required for dynein assembly and/or function. Clear and effective knockdown was obtained within 24 hours of Tet induction (Fig. 2C). Three independent assays demonstrated that TbLC1 knockdown results in a dramatic motility defect. First, induction with Tet over a 5-day period resulted in multicellular clusters that began to appear 48 hours post induction (hpi) (Fig. 2D). The mutants remained viable, although growth was reduced (Fig. 2E). Trypanosomes divide through binary fission and flagellar motility is required for efficient completion of cytokinesis (Ralston et al., 2006). In motility mutants, multiple rounds of cytokinesis initiate, but are not completed, resulting in multicellular clusters (Baron et al., 2007; Branche et al., 2006; Ralston et al., 2006). Our results are therefore consistent with a primary defect in flagellar motility that leads to faulty cell division. To test this, TbLC1 knockdowns were next examined for motility defects using a sedimentation assay (Baron et al., 2007; Bastin et al., 1999; Ralston et al., 2006) at 24 hpi, when the mRNA was clearly knocked down (Fig. 2C) but no clusters were present (Fig. 2D). TbLC1 mutants sedimented similarly to previously 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 were examined using high-resolution DIC microscopy as described (Baron et al., 2007). A unique and hallmark feature of trypanosome flagellar motility is a tractile beat that initiates at the tip of the flagellum and propagates toward the base, driving cell 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 were vigorously motile, moving with an average forward velocity of 2.50±0.839 μm/second (Fig. 3A, –Tet). In stark contrast, most TbLC1 mutants moved steadily backward at an average velocity of 0.738±0.250 μm/second (see supplementary material Movie 1), with the flagellum tip trailing (Fig. 3A, +Tet). Occasionally, we observed cells that remained in one position, but they never moved forward. This is the first ever report of sustained backward motility in any trypanosomatid. Motility traces demonstrated that the majority of TbLC1 mutant cells exhibited aberrant motility (Fig. 3B). The small percentage of cells labeled as runners in the motility trace of induced cells probably represents cells not fully affected by the knockdown at the time of the experiment.

Careful examination of TbLC1 mutant flagella revealed another striking phenotype. Flagellar beat in these mutants was reversed, i.e. initiated at the base of the flagellum and propagated toward the tip (Fig. 3C). Wild-type trypanosome flagella 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 not sustained. In TbLC1 mutants, every cell examined at 30 hpi and 49 hpi had a sustained base-to-tip beat and most of these moved backward (30 hpi cells are shown in supplementary material Movie 1). Significant cytokinesis problems precluded detailed motility analysis at later time points, but at 49 hpi mutants continued to exhibit reverse beat and backward motility (not shown). The reverse beat is mostly symmetrical, although it becomes erratic at the flagellum tip. There is some slow rotation of the cell around its long axis, in the counter-clockwise direction when looking toward the cell posterior. However, TbLC1 mutants did not exhibit the tumbling behavior that occurs periodically in wild-type cells and continuously in DRC mutants (Hill, 2003; Hutchings et al., 2002). Therefore, loss of TbLC1 expression results in the complete loss of the dominant tractile beat that is a hallmark of trypanosome flagella and the concomitant emergence of a sustained reverse beat that propagates base-to-tip and drives cell movement in reverse.

Fig. 2.

TbLC1 localizes along the flagellum and is required for motility. (A) Fluorescence microscopy of live cells and cytoskeletons from TbLC1-GFP strains shows that TbLC1-GFP is localized to the flagellum in induced cells (+Tet). There is background autofluorescence in the cell body of induced and uninduced (–Tet) live cell samples that is variable (Baron et al., 2007). Cytoskeletons were stained with DAPI (blue) to visualize the nucleus and kinetoplast relative to TbLC1-GFP (green). (B) Western blot analysis with anti-GFP antibody of TbLC1-GFP cellular fractions from induced (+Tet, 24 hpi) and uninduced (–Tet) cells confirms that TbLC1 is stably associated with the flagellum. L, lysates; S1, detergent-soluble proteins; P1, cell cytoskeletons; S2, NaCl soluble proteins; P2, flagellar cytoskeletons. The same fractions were blotted with anti-trypanin (TPN) monoclonal antibody as a loading control (bottom panel). (C) Northern blots of RNA from an uninduced (–Tet) and induced (+Tet, 24 hpi) TbLC1 RNAi knockdown strain probed with TbLC1 and TbCMF46 (control) DNA fragments. (D) Images of whole cultures of uninduced (–Tet) and induced (+Tet) TbLC1 knockdown cells over a 5-day induction. At 48 hpi multicellular clusters appear in the culture containing 5-10 cells each, and increase in size and number over time. (E) Growth curve of uninduced (–Tet) and induced (+Tet) TbLC1 cells over a 5-day period. (F) Sedimentation curves (Baron et al., 2007; Bastin et al., 1999; Ralston et al., 2006) for the uninduced (–TET) and induced (+TET, 24 hpi) cells. Error bars show the s.d. for three experiments.

Fig. 2.

TbLC1 localizes along the flagellum and is required for motility. (A) Fluorescence microscopy of live cells and cytoskeletons from TbLC1-GFP strains shows that TbLC1-GFP is localized to the flagellum in induced cells (+Tet). There is background autofluorescence in the cell body of induced and uninduced (–Tet) live cell samples that is variable (Baron et al., 2007). Cytoskeletons were stained with DAPI (blue) to visualize the nucleus and kinetoplast relative to TbLC1-GFP (green). (B) Western blot analysis with anti-GFP antibody of TbLC1-GFP cellular fractions from induced (+Tet, 24 hpi) and uninduced (–Tet) cells confirms that TbLC1 is stably associated with the flagellum. L, lysates; S1, detergent-soluble proteins; P1, cell cytoskeletons; S2, NaCl soluble proteins; P2, flagellar cytoskeletons. The same fractions were blotted with anti-trypanin (TPN) monoclonal antibody as a loading control (bottom panel). (C) Northern blots of RNA from an uninduced (–Tet) and induced (+Tet, 24 hpi) TbLC1 RNAi knockdown strain probed with TbLC1 and TbCMF46 (control) DNA fragments. (D) Images of whole cultures of uninduced (–Tet) and induced (+Tet) TbLC1 knockdown cells over a 5-day induction. At 48 hpi multicellular clusters appear in the culture containing 5-10 cells each, and increase in size and number over time. (E) Growth curve of uninduced (–Tet) and induced (+Tet) TbLC1 cells over a 5-day period. (F) Sedimentation curves (Baron et al., 2007; Bastin et al., 1999; Ralston et al., 2006) for the uninduced (–TET) and induced (+TET, 24 hpi) cells. Error bars show the s.d. for three experiments.

Fig. 3.

TbLC1 knockdown causes reverse cell motility. (A) Time-lapse series taken from video clips of induced (+Tet, 26 hpi) or uninduced (–Tet) TbLC1 knockdown cells. The dashed line marks the cell posterior (P) at the start of the time-lapse series. The black arrows in the first panel represent the direction expected for wild-type movement, with the anterior end (A) leading. Red arrows at the bottom show the actual direction of cellular movement. TbLC1 mutants move backward, with the posterior end leading. Significant cytokinesis problems prevented detailed motility analysis at later time points, but single cells observed at 49 hpi continued to exhibit reverse beat and reverse motility (not shown). Bars, 10 μm. (B) Cell traces (Baron et al., 2007) of uninduced (–Tet) and induced (+Tet, 26 hpi) TbLC1 knockdown cells. Lines show the distance traveled by cells over a 30-second interval. n=47 (–Tet) or 52 (+Tet) cells. Bars, 50 μm. Cell classifications are described in Materials and Methods. Inset pie charts display the percentage of each cell type observed in each strain. (C) Time-lapse series shows beat direction in two flagellar mutants that are stuck to slides. TbLC1 mutants (24 hpi) display a flagellar beat that originates at the base of the flagellum and is propagated to the tip (supplementary material Movie 3). Trypanin mutants (5 days post infection) display a tumbling phenotype (Hutchings et al., 2002) and maintain the wild-type tip-to-base flagellar waveform movement. Black arrows show the waveform position at the beginning of the series. White arrows show the position of the waveform at each time point.

Fig. 3.

TbLC1 knockdown causes reverse cell motility. (A) Time-lapse series taken from video clips of induced (+Tet, 26 hpi) or uninduced (–Tet) TbLC1 knockdown cells. The dashed line marks the cell posterior (P) at the start of the time-lapse series. The black arrows in the first panel represent the direction expected for wild-type movement, with the anterior end (A) leading. Red arrows at the bottom show the actual direction of cellular movement. TbLC1 mutants move backward, with the posterior end leading. Significant cytokinesis problems prevented detailed motility analysis at later time points, but single cells observed at 49 hpi continued to exhibit reverse beat and reverse motility (not shown). Bars, 10 μm. (B) Cell traces (Baron et al., 2007) of uninduced (–Tet) and induced (+Tet, 26 hpi) TbLC1 knockdown cells. Lines show the distance traveled by cells over a 30-second interval. n=47 (–Tet) or 52 (+Tet) cells. Bars, 50 μm. Cell classifications are described in Materials and Methods. Inset pie charts display the percentage of each cell type observed in each strain. (C) Time-lapse series shows beat direction in two flagellar mutants that are stuck to slides. TbLC1 mutants (24 hpi) display a flagellar beat that originates at the base of the flagellum and is propagated to the tip (supplementary material Movie 3). Trypanin mutants (5 days post infection) display a tumbling phenotype (Hutchings et al., 2002) and maintain the wild-type tip-to-base flagellar waveform movement. Black arrows show the waveform position at the beginning of the series. White arrows show the position of the waveform at each time point.

In Chlamydomonas, all of the outer arm IC and LC mutants previously studied save one, LC6, have been shown to be required for stable assembly of the outer arm (King, 2003; Pazour and Witman, 2000; Sakato and King, 2004). To ascertain the extent to which the loss of TbLC1 affects axoneme structure in T. brucei, we used transmission electron microscopy. Compared with flagella from control cells, TbLC1 mutant flagella show normal outer doublets, central pair and radial spoke structures (Fig. 4, +Tet). However, outer dynein arms were missing or reduced in most axonemes of TbLC1 mutants (Fig. 4A, +Tet). Cross-sections of mutant flagella examined at 30, 49 and 72 hpi showed that this effect was constant at all time points, indicating that motility phenotypes observed at early time points accurately reflect the consequence of outer arm deficiency that is due to loss of TbLC1. Branche and co-workers (Branche et al., 2006), recently demonstrated that loss of the dynein intermediate chain 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 idea that tip-to-base flagellar beat propagation and forward motility in T. brucei requires outer dynein arms, since outer dynein deficiency caused by two independent mechanisms (knockdown of DNAI1 or LC1) blocks tip-to-base beat. Interestingly, the reverse base-to-tip beat of DNAI1 knockdown mutants did not drive reverse cell motility. Whether this reflects differences in the requirement for DNAI1 and TbLC1 in outer dynein assembly and/or function or experimental differences is not presently clear. Orientation of central pair microtubules is restricted within a small range in T. brucei (Branche et al., 2006; Ralston et al., 2006). TbLC1 mutants exhibited a defect in central pair orientation (Fig. 4B), as observed for DNAI1 mutants (Branche et al., 2006). This defect increased with induction time in TbLC1 mutants, even though the outer arm defect did not (Fig. 4), suggesting that central pair misorientation might be due to pleiotropic effects contributing to the phenotype at later time points.

Fig. 4.

TbLC1 is required for stable outer arm dynein assembly. (A) Electron micrographs of uninduced (–Tet) and induced (+Tet, 30 hpi) whole cell TbLC1 mutant flagellar cross-sections. Arrows indicate examples of positions on doublets where outer dynein arms are present (– Tet) or absent (+ Tet). The table shows the breakdown of how many 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 pair orientation was determined as described previously (Ralston et al., 2006). n=22-37 sections (–Tet) or 30-40 sections (+Tet). The percentage of sections having central pair orientation outside the range observed in associated –Tet samples are indicated.

Fig. 4.

TbLC1 is required for stable outer arm dynein assembly. (A) Electron micrographs of uninduced (–Tet) and induced (+Tet, 30 hpi) whole cell TbLC1 mutant flagellar cross-sections. Arrows indicate examples of positions on doublets where outer dynein arms are present (– Tet) or absent (+ Tet). The table shows the breakdown of how many 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 pair orientation was determined as described previously (Ralston et al., 2006). n=22-37 sections (–Tet) or 30-40 sections (+Tet). The percentage of sections having central pair orientation outside the range observed in associated –Tet samples are indicated.

Branche and co-workers also suggested an interesting model in which reverse wave propagation in T. brucei causes cellular reorientation without backward motility. We therefore examined beat direction in trypanin mutants, which undergo continuous cellular reorientation without net directional motility (Hutchings et al., 2002). In cases where beat direction could clearly be determined, flagellar beat progressed primarily from tip-to-base, with occasional bursts of base-to-tip beat (Fig. 3C and supplementary material Movie 2) as seen for wild-type T. brucei (Branche et al., 2006; Walker and Walker, 1963). These results, together with the finding that TbLC1 mutants exhibit a continuous reverse beat and maintain steady reverse cell movement, indicate that reverse beat alone is not sufficient to drive cell reorientation. Perhaps concurrent and competing reverse and forward beats in the same flagellum are what cause cell reorientation. Transient bursts of base-to-tip beat propagation have previously been demonstrated to drive short periods of reverse cell motion in T. cruzi (Jahn and Fonseca, 1963) and Crithidia oncopelti (Holwill, 1964; Holwill, 1965). Therefore, the ability of reverse flagellar beat to drive backward cell movement is conserved among these trypanosomatids. Tip-to-base beating of trypanosome flagella, as well as the capacity to reverse flagellar beat implies the presence of specialized regulatory systems for controlling flagellar beat in these organisms.

The ultrastructural deficit of TbLC1 mutants was specific to the outer dynein arms, as inner arms, radial spokes and central pair structures were still present. Likewise, loss of outer arm dyneins is not a common defect in other T. brucei motility mutants (Baron, 2007; Bastin et al., 1998; Branche et al., 2006; Maga et al., 1999). Therefore, our data indicate a specific requirement for LC1 in the assembly and/or stability of outer arm dyneins in T. brucei. Defects in outer arm dynein assembly are often seen in mutants lacking ICs or LCs associated with the HC coiled-coil domain that mediates cargo attachment and oligomerization in both Chlamydomonas (King, 2003; Sakato and King, 2004) and T. brucei (Branche et al., 2006). However, LC1 is bound to the motor domain of the γHC rather than the coiled-coil domain (Benashski et al., 1999). As such, the requirement for TbLC1 in outer dynein assembly is somewhat surprising, because C. reinhardtii mutants lacking the entire αHC or the βHC motor domain still assemble outer arm dyneins (Sakakibara et al., 1991; Sakakibara et al., 1993). It is possible that LC1 might not be specific to the γHC motor domain in T. brucei. However, all of the predicted γHC binding sites are conserved in the primary structure and in the modeled tertiary structure of TbLC1, as are the C-terminal amino acids predicted to contact the ATP-hydrolyzing site of the AAA+ domain. This suggests that TbLC1 does bind the cognate domain of the T. brucei γHC and indicates that outer arm assembly in trypanosomes is sensitive to perturbations of the γHC. This might reflect changes specifically at the motor domain where LC1 binds. Alternatively, loss of TbLC1 might directly affect stability of the entire γHC, similarly to the influence of Oda7p – a leucine-rich repeat (LRR) protein like LC1 (Freshour et al., 2007) – on αHC stability in C. reinhardtii (Fowkes and Mitchell, 1998). Loss of the γHC might then lead to a block in outer dynein assembly.

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

Materials and Methods

Bioinformatic analysis

A previous genomic screen (Baron et al., 2007) identified a T. brucei protein (Gene DB ID number Tb11.02.3390, NCBI accession XP_828643) that was similar to CrLC1 (E-34). Protein sequence alignments between the bona fide CrLC1, other LC1 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) and TbLC1 was determined using Deep View/Swiss-PDB Viewer (http://www.expasy.org/spdbv/).

Cloning of TbLC1 RNAi and GFP-tagged constructs RNAi

The RNAi construct was created as described previously (Baron et al., 2007) using a 485 bp fragment corresponding to nucleotides 121 to 605 identified by the Trypanofan 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 were verified by sequencing at the UCLA genomics center.

GFP-tagging

Full-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 at the N-terminus of GFP as described previously (Baron et al., 2007). Although expression levels and epitope tag might influence protein localization, this plasmid was selected because it drives expression of other axonemal proteins at near endogenous levels and accurately reports the location of flagellar proteins having expression levels similar to that expected for TbLC1, i.e. one or a few copies per axonemal repeat unit (Baron et al., 2007). Primers used for amplification were: forward, 5′-cccaagcttgcatgtcaggaacaacttcca-3′ and reverse, 5′-gctctagaccggccgcgctccgcctcttc-3′. All sequences were verified by DNA sequencing at the UCLA genomics center.

Trypanosome transfection and cell maintenance

Procyclic 29-13 cells (Wirtz et al., 1999) were used to generate RNAi and GFP-tagged TbLC1 strains. Cells were maintained and transfected as described previously (Hill et al., 1999; Hutchings et al., 2002) and clonal lines were obtained by limiting dilution. Knockdown mutants and expression of TbLC1-GFP was induced with 1 μg/ml of tetracycline. At 24 hpi, TbLC1-GFP fusion protein cells were viewed on a Zeiss Axioskop II compound fluorescent microscope using a 63× oil objective.

Northern blots

RNA was isolated from induced (24 hpi) and uninduced TbLC1 cells using a Qiagen RNeasy Miniprep kit according to the manufacturer's instructions. Northern blots (Hill et al., 1991) using 5 μg of total RNA were probed with 32P-labeled DNA fragments corresponding to the TbLC1 region used for RNAi knockdown, or to TbCMF 46 nucleotides 129 to 608.

Motility assays and ultrastructural analysis

Sedimentation assays, whole culture observations, cell tracking assays and DIC microscopy 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 as described previously (Ralston et al., 2006). In order to minimize the influence of pleiotropic effects, cells were examined at 24-30 hpi, a time point where mRNA is clearly knocked down and motility defects are clearly evident, but before clustering became apparent in the culture. Ultrastructural analyses were also done at 49 and 72 hpi as described in the text. For motility traces, `runners' were classified as vigorously motile cells that progressed forward along a curvilinear path for 5 seconds or more. `Crawlers' were cells with slow movement that progressed away from their point of origin, whereas `immotiles' were cells that remained at their point of origin during the time interval.

Trypanosome cellular fractionation and western blotting

Trypanosome lysates, detergent-soluble proteins and cell cytoskeletons were prepared 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).

Acknowledgements

We would like to thank Randy Nessler (University of Iowa) for his assistance with electron microscopy. We would also like to thank George Cross (Rockefeller University) for the 29-13 cell line and Katherine Ralston for assistance with fluorescence microscopy. This work was supported by grants from the National Institute of Health (R01AI52348), Ellison Medical Foundation (ID-NS-0148-03) and Beckman Young Investigator Program to K.L.H. D.M.B. is the recipient of a USPHS National Research Service Award in Microbial Pathogenesis (2-T32-AI-07323) and the UCLA Graduate Dissertation Year Fellowship for 2006-2007.

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