Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment

Motility in the protozoan parasite Trypanosoma brucei is conferred by a single flagellum, attached alongside the cell, which moves the cell forward using a beat that is generated from tip-to-base. We are interested in characterizing components that regulate flagellar beating, in this study we extend the characterization of TbIC138, the ortholog of a dynein intermediate chain that regulates axonemal inner arm dynein f/I1. TbIC138 was tagged In situ-and shown to fractionate with the inner arm components of the flagellum. RNAi knockdown of TbIC138 resulted in significantly reduced protein levels, mild growth defect and significant motility defects. These cells tended to cluster, exhibited slow and abnormal motility and some cells had partially or fully detached flagella. Slight but significant increases were observed in the incidence of mis-localized or missing kinetoplasts. To document development of the TbIC138 knockdown phenotype over time, we performed a detailed analysis of flagellar detachment and motility changes over 108 hours following induction of RNAi. Abnormal motility, such as slow twitching or irregular beating, was observed early, and became progressively more severe such that by 72 hours-post-induction, approximately 80% of the cells were immotile. Progressively more cells exhibited flagellar detachment over time, but this phenotype was not as prevalent as immotility, affecting less than 60% of the population. Detached flagella had abnormal beating, but abnormal beating was also observed in cells with no flagellar detachment, suggesting that TbIC138 has a direct, or primary, effect on the flagellar beat, whereas detachment is a secondary phenotype of TbIC138 knockdown. Our results are consistent with the role of TbIC138 as a regulator of motility, and has a phenotype amenable to more extensive structure-function analyses to further elucidate its role in the control of flagellar beat in T. brucei.


Introduction
Trypanosoma brucei is a protozoan parasite found in sub Saharan Africa, where it causes sleeping sickness in humans and a related disease in other mammals, including cattle and other livestock [1].Current drug treatments are not adequate with respect to effectiveness, safety, and expense [2,3].There is need for better understanding of parasite biology to inform drug design, facilitate diagnosis and circumvent drug resistance [1,2,4,5].One critical feature of T. brucei cells is motility, directed by a single flagellum that emerges from the posterior end of the cell and is attached alongside the cell body [6].All forms of the parasite throughout its complex life cycle have motile flagella, including multiple developmental forms in both the mammalian host and the tsetse fly vector [7][8][9].An unusual feature of flagellar motion of African trypanosomes is that the flagellar waveform initiates at the tip of the flagellum and propagates toward the base [10,11], this motion is thought to facilitate movement through the highly viscous extracellular environments of the mammalian host [12,13], driving the cell forwards at speeds of up to 30 μm/s [14].Flagellar beating may not be strong enough to direct swimming against the circulatory system [14,15], but is critical for clearance of surface bound antibodies for immune evasion and may be important for parasites to efficiently negotiate complex surfaces they encounter in the blood vessels [14,16,17] and successful tissue invasion [18].In the tsetse vector, flagellar motility is required for proper development [19].
Although the precise mechanism of motility is still being elucidated [11,13,16,20], this flagellar beat is complex and takes on a helical component as the attached flagellum rotates the cell body during swimming [11,16].The flagellar beat can also reverse, causing tumbling and backward movement [14,[21][22][23].Because of these characteristics and the availability of molecular genetics and biochemical approaches, T. brucei presents an appealing model system for studying mechanisms of flagellar motility [13,24,25].
The axoneme is the skeletal structure that confers motility in eukaryotic flagella; it is highly conserved in form and protein composition [26].The T. brucei flagellum includes an additional paracrystalline protein structure, the paraflagellar rod (PFR), that runs parallel to the axoneme along the length of the flagellum within the flagellar membrane [27] and is essential for normal flagellar motility and parasite viability in the bloodstream form [27][28][29][30]. Attachment of the flagellum to the cell body is required for proper forward motility in T. brucei [31,32].The flagellar attachment zone (FAZ) is a specialized region of cell cortex situated along the inner leaflet of the plasma membrane with protein complexes that form interactions in the extracellular space between the plasma and flagellar membranes [33][34][35].These structures are physically tethered to components of the axoneme: electron-dense filaments connect the PFR to outer arm dynein complexes on microtubule doublets 4-7 [20,33,36], and structures connecting the FAZ into the axoneme are also visible [31,37].
Aspects of axoneme structure and function have been characterized in many organisms, including the green alga Chlamydomonas reinhardtii, and appear to be conserved in T. brucei flagella.The structure consists of a "9+2" array: a cylindrical array of nine peripheral microtubule doublets surrounding a central pair of microtubules [38].The microtubule doublets consist of A and B tubules, the former of which has outer arm dyneins attached along the outer rim of the axoneme, and inner arm dyneins (IAD) facing toward the inside of the axoneme [39].The A-tubules are also connected to radial spokes that connect with the central pair complex, and nexin-dynein regulatory complexes coordinate sliding [40][41][42] and connect neighboring doublet pairs to one another [39,43].Dyneins are ATP-dependent motor protein complexes that promote sliding of the A-tubule against the B-tubule of the adjacent microtubule doublet in the axoneme [44,45].Dyneins are composed of heavy chains (HC) that contain ATPase and motor domains, as well as several intermediate chains (IC) and light chains (LC).Specific components of axonemal dynein complexes of C. reinhardtii and other organisms have been identified [26,46], and structural studies have elucidated the arrangement of dyneins along the C. reinhardtii axoneme [47][48][49].The outer arm dynein complexes are arrayed 24 nm apart along the length of the A-tubule [50].IAD complexes are more heterogeneous, repeating every 96 nm and consisting of multiple dynein species including up to seven different dynein HC [30,51].T. brucei axonemes have similar dynein arrangements, although there are fewer individual dynein HCs in both outer and inner arm complexes [36].The different dynein HCs in C. reinhardtii have distinct mechanical properties [26,52,53], so it is likely that they play distinct roles in coordinating proper flagellar beating [26,50,54,55].In particular the IAD complex known as dynein f/I1, the only two-HC dynein in the inner arm, has been suggested to be essential for formation of a normal flagellar waveform by down-regulating or inhibiting microtubule sliding to modulate flagellar bending [26].
In T. brucei, orthologs of some dynein f/I1 proteins have been identified, including the two HCs, IC138 and IC140 [51].We have previously reported that RNAi knockdown of the T. brucei ortholog, TbIC138, results in parasites with reduced motility, but no effect on overall axoneme structure [64].In this study we extend the analysis of the motility defect.We found that TbIC138 fractionated with inner arm components of axoneme, and characterized the phenotype of the TbIC138 knockdown in terms of protein levels, cell growth, morphological changes and the timing of onset of phenotypic changes such as flagellar detachment and immotility.The motility defect was observed early after induction, and progressively became more severe.Our results show that knockdown of TbIC138 results in rapid expression of flagellar beating defects in cells with or without detached flagella, suggesting that defective flagellar beating represents a primary effect of knockdown, while detachment is a secondary phenotype.

In situ tagging
The pMOTag system [68] was used to place a C-terminal 3xHA tag on a chromosomal copy of TbIC138 by double recombination.The 3' end of the gene and the sequence immediately beyond the stop codon (3'UTR) were PCR-amplified using Hot Star HiFidelity Polymerase (Qiagen.com) and the following primers (IC138 3' end: 5' ACA CGG GCC CTG TGG TCG CCT TTC GAA CTA 3' and 5' ACA CGG TAC CGC CCC ACA CAT GTT TCA TGG 3'; 3' UTR: 5' ACA CGC GGC GCT ACC GTG CGG TAT TTA GGC 3' and 5'ACA CTC TAG AGA TAC CCT TCC TGG TAT TGG CA 3').For cloning, restriction sites for ApaI and Kpn1 were added to the 3' end primer sets, and XbaI and NotI sites were added to the 3' UTR primer sets.The respective PCR products were cloned on either side of the 3xHA tag in pMOTag2H [68].Constructs were stably transfected into T. brucei strain 29-13 or into IC138 RNAi , and clonal lines were isolated as described previously [69] to produce, respectively IC138::3xHA and IC138 RNAi ::3xHA.

Quantification of knockdown
Uninduced and Tetracycline-induced IC138i::3x HA cultures were harvested at 48 hours-and 72 hours-post-induction (HPI), approximately 4 x 10 7 cells were washed with cytomix [24], pelleted and resuspended in PEME buffer [70] with 1X protease inhibitor cocktail (SigmaAldrich.com), and stored in Laemmli Sample Buffer [71] for a final concentration of 2.5 x 10 5 cells/mL.Approximately 2 x 10 6 cell equivalents were loaded per well onto a precast Any KD acrylamide gel (Bio-Rad.com),along with markers (Precision Plus Protein Kaleidoscope, Bio-Rad).Proteins were transferred to nitrocellulose using the Trans-blot turbo transfer system (Bio-Rad), transfer was confirmed using Ponceau stain before hybridization to anti-HAmouse monoclonal antibody, HA.11 Clone 16B12 (Covance.com)1:1000 and anti-β-tubulin mouse monoclonal antibody, E7 (DSHB.biology.Iowa.edu)1:5000, followed by Goat Antimouse horseradish peroxidase conjugate (EMDMillipore.com)1:2500, in 1X PBS/0.05%Tween/5% nonfat dry milk.Blots were visualized using West Pico Chemiluminescent substrate (Thermo Scientific) and imaged using a ChemiDoc XRS with Quantity One software (Bio-Rad).Density of protein bands was determined using the Gel analysis/Plot Lanes tool in ImageJ (http://rsb.info.nih.gov/ij),tubulin signal in each lane was used to normalize IC138::HA from different samples.Extent of knockdown was calculated by comparing normalized IC138::HA signals from induced cultures to those of uninduced, based on three separate experiments each comprising two independent 48-hour and two independent 72-hour inductions.

Microscopy
Slides were viewed in a Nikon Eclipse Ti-S inverted microscope (NikonUSA.com),60X objective for differential interference contrast (DIC) or epifluorescence, and a Nikon Eclipse E600 microscope, 100X objective lens for phase contrast optics.DAPI (4',6-diamidino-2-phenylindole)staining was performed as described [73], Immunofluorescence assay was described [74] for visualizing L13D6 antibody [75].Live cells were placed in a sliding chamber prepared using tape to create a 1-2 mm space between poly-L-glutamate-treated microscope slides and coverslips [24], and observed using DIC microscopy.Flagellar lengths (signal by L13D6 antibody) and cell lengths (posterior end to flagellum tip) were measured as described [76], using ImageJ.Phase contrast images were taken using a Spot RT camera (spotimaging.com),DIC images were taken using a FLASH 2.8 CMOS camera (Hamamatsu.com).For time course analysis, at each time point approximately 100 cells were scored for degree of flagellar detachment and extent of cell motility.In cells with detached flagella, the movement of flagella themselves was scored.A scoring sheet was designed to incorporate the full spectrum of possible characteristics for each cell (S1 Table ), scoring criteria and cells that could not be reliably scored are shown in S2 and S3 Tables.Statistical tests for significance were performed using MS Excel as described [77].

Characterization of TbIC138 RNAi knockdown
Tb927.2.4060 was previously identified by Bayesian analysis [51] to be the IC138 ortholog in T. brucei.This gene encodes a predicted protein of 864 amino acids, shorter than its C. reinhardtii homolog (CrIC138) of 1057 amino acids (Genbank accession: EDP00613).Sequence analysis identified five predicted WD domains [78] in the C-terminal third of the protein, the region that has the most significant identity (37% over 353 amino acids) to CrIC138, and to the human IC138 homolog (Swiss-Prot: Q5VTH9.1) of 848 amino acids (28% identity over 683 amino acids).
To study TbIC138 function, the gene was silenced using tetracycline-inducible RNAi as described previously [64].To determine extent of knockdown of TbIC138 protein in strain IC138 RNAi , we constructed an in situ 3x HA tag onto the C-terminal end of one chromosomal copy of the gene using the pMOtag system [68], insertion at the chromosomal copy in the IC138 RNAI strain was confirmed by PCR (not shown).Clonal IC138 RNAi ::3xHA was used to prepare lysates from cultures that were uninduced, or were 48 hours-or 72 hours-post-induction, respectively, using tetracycline.The resulting lysates were analyzed by western blot using anti-HA antibody to detect tagged TbIC138, and anti-tubulin antibody for normalization (Fig 1A).The predicted size of IC138::3xHA is 98 kD, and a band of this size was readily detectable.
Quantification of band intensities revealed IC138::3xHA protein levels were reduced to 15% of uninduced levels at 48 hours-post-induction, or 13% of uninduced at 72 hours-post-induction (Fig 1B).This suggests that the knockdown of TbIC138 protein is substantial.IC138::3xHA can also be used to confirm that this protein is actually localized in the IAD fraction in T. brucei.Clonal lines of IC138::3xHA were used to prepare lysates from various steps in flagellar preparation.The resulting fractions were analyzed by western blot using anti-HA antibody to detect tagged protein of the appropriate size in various fractions (Fig 1C).A band of the correct size was readily detectable in low-and high-salt insoluble flagellar fractions (P1, P2), indicating that TbIC138 is not extracted with high salt, as expected of an inner arm protein.Antibodies against two other proteins with known localization were used as controls [79], both exhibited fractionation patterns as expected: β-tubulin is a cytoskeletal protein and was found in cytoskeletal and insoluble flagellar fractions as well as in the S2 fraction [80].Trypanin localizes at the base of the inner arm and was found in low-and high-salt insoluble flagellar fractions but not in supernatant fractions [81].Control samples prepared from the untransfected host strain 29-13 yielded no anti-HA signal in any fractions (S5 Fig).

Effect on cell cycle
Microscopy was used to observe abnormalities associated with defects in cell growth and cell cycle progression in induced IC138 RNAi cells compared to uninduced cultures (Fig 3A ).By 48 hours-post-induction, several clusters of multiple cells were visible (Fig 3B) and these increased in frequency and number of cells through the course of induction, often the number of cells was difficult to classify (Fig 3C).In some cases flagella in these clusters appeared to be entangled, and some clusters seemed to have more one flagellum per cell body.Small clusters were occasionally seen in uninduced IC138 RNAi cultures, but much less frequently.The cell cycle in T. brucei includes replication of nuclear and kinetoplast DNA, and defects affecting cell cycle progression can result in abnormal nucleus:kinetoplast (N:K) ratios or kinetoplast localization.We used DAPI to stain nuclei and kinetoplasts in both uninduced and 48 hours-post-induction IC138 RNAi cultures.At 48 hours-post-induction, the mean number of nuclei per cell in IC138 R- NAi was 1.20 and the mean for uninduced cultures was 1.13 (S4 Table ), a difference that was not significant by single factor analysis of variance (P < 0.001).In IC138 RNAi cultures 48 hours post-induction kinetoplasts were more likely to be mis-localized (Fig 3D and 3E), generally by being located closer to the nucleus than expected (roughly one third or less of the average distance) or not discernable.Approximately 33% of the induced IC138 RNAi cells had mis-localized kinetoplasts at 48 hours-post-induction, as compared to 16% of uninduced ( significant.48 hours-post-induction cultures also showed a 13% incidence of cells with N:K ratios greater than 1, compared to 5% in uninduced cultures (Fig 3G and S2 Fig), this difference was significant by both Chi Squared and Fisher's exact tests for independence (P< 0.001).The frequency of cells with N:K ratios less than 1 was 14% for cultures and 13% for uninduced cultures, a difference that was not significant.

Morphological features
We used microscopy to examine individual IC138 RNAi induced cells for changes in flagellar structure and cell size.Depletion of flagellar proteins has been shown to affect flagellar morphology [6], including the attachment to the cell body.Partially detached flagella were defined as those in which the flagellum was attached to the cell body at the flagellar pocket and at the anterior end, but had a portion in between that is disconnected, forming a loop.Fully detached flagella were attached only at the flagellar pocket and were not in contact with any other part of the cell body.Cells with either partially or fully detached flagella were observed in IC138 RNAi induced cultures. .We also measured lengths of flagella, as well as total cell length in uninduced and induced IC138 RNAi cultures.Flagellar length was measured using anti-PFR antibody L13D6, and cell length was

Development of phenotype over time
To observe the time course of the knockdown phenotype, cells were scored for 108 hours at 12 hour time points following induction of RNAi.One of the aspects of this phenotype is that over time more and more cells were found in clusters of three or more cells, and by later time points some of these clusters contained upwards of ten cells.These clusters could be broken up to some extent by mechanical agitation (i.e shaking flasks by hand rapidly for 2-3 seconds), but then new clusters formed within 24 hours.This clustering caused some technical difficulties in obtaining reliable observations of characteristics of induced IC138 RNAi cells.Care was taken to vigorously agitate cultures before sampling to separate cells, and pipetting was used to further enrich for separation, although occasionally clusters were observed on microscope slides (S3 Table ).Some cells that appeared to be stuck to the surface of the slide were eliminated from scoring; if too many cells appeared to be stuck a new slide was prepared.For consistency, only individual cells for which motility and flagellar attachment could be definitively scored were counted.
The results presenting development of abnormal motility and flagellar detachment are shown in Fig 5 .By 24 hours-post-induction, some abnormal motility was observed (Fig 5A).Cells scored as slow or abnormal included those with slowed rotation or pulsating body movements, as well as cells that twitched irregularly (these behaviors were combined into one category: "slow and abnormal motility").Abnormal motility was generally initiated tip-to-base, even in cells that were twitching or very slow.However, we did not specifically score the direction of beat in each cell in this study, so we cannot rule out the occasional occurrence of a reverse beat within this phenotype.Cells scored as immotile had no body movement but usually still had some flagellar beating, either at the flagellar tip or along detached portions of the flagellum.Cells that showed no movement at all of any part of the body or flagellum were very rare, and could not be distinguished from non-living cells.Motility became steadily more defective over the time course of the study.Cells exhibiting slow and abnormal movement increased as a proportion of the population for 36 hours-post-induction, then decreased and the percentage of immotile cells increased.By 108 hours-post-induction more than 80% of cells were immotile.Abnormal movement was rarely observed in uninduced cultures (S3 Fig).
Flagellar detachment was first observed somewhat later than abnormal motility, by the 36 hours-post-induction time point, and partial detachment was generally more prevalent than complete detachment.The percentage of affected cells increased over time and by 108 hourspost-induction, cells with partially or fully detached flagella comprised nearly 60% of the population (Fig 5B).Flagella that were detached, either partially or fully, usually showed defective beating (Fig 5C and S4 Fig) although generally less than 50% of detached flagella showed complete paralysis (no detectable movement).Detached flagella were very rare in the uninduced cultures (S3 Fig), but were occasionally observed.Since we made a point of using uninduced IC138 RNAi as our reference control instead of a wild type strain, our controls may show a low frequency of phenotypes associated with flagellar defects if repression of dsRNA transcription was leaky in the absence of tetracycline.

Discussion
In this study we have examined TbIC138, ortholog of an IAD intermediate chain protein believed to be a regulator of flagellar motility [49,56,58].We confirmed that RNAi knockdown of TbIC138 results in substantially reduced protein levels using an in situ 3xHA tag at the 3' end of a chromosomal copy of the gene.Western analyses confirmed that IC138::3xHA is found in flagellar fractions (Fig 1C) and in particular, in the insoluble fraction following high salt extraction (P2).In this fraction the outer arm dyneins are missing and central pair proteins are significantly reduced, but IADs largely remain [24].The localization of TbIC138 is consistent with that of IAD structures [81], as would be expected of an IC138 ortholog.
Knockdown of TbIC138 results in defective motility [64]; in this study we have extended the characterization of the motility defect using sedimentation and microscopic analyses to show that IC138 RNAi induced cells sedimented at a faster rate than uninduced cells ( To assess whether the knockdown affected other aspects of cell cycle function, nuclear and kinetoplast characteristics were measured, such as multinucleate cells, high N:K ratios and missing or mis-localized kinetoplasts.All of these parameters increase in incidence upon knockdown of another component of dynein f/I1, the alpha HC TbDNAH10 [74].In IC138 RNAi 48 hourspost-induction, the number of multinucleate cells was not significantly increased relative to uninduced cultures (S2 Table ), but there were slight yet significant increases in cells with N:K > 1 and in cells with mis-localized kinetoplasts (Fig 3F and 3G).The increase in N:K ratio can result from kinetoplasts that overlapped the nucleus in the image and so were blocked from view, which may be a result of mis-localization.Disruption of cell cycle events can lead to changes in cell and flagellar length [31,[82][83][84][85], but we found no differences in these lengths following TbIC138 knockdown.Taken together, these results may suggest a mild effect on timing of kinetoplast division and/or kinetoplast segregation.Previous reviews have discussed the occurrence of cell cycle defects associated with depletion of flagellar proteins in T. brucei [6,86], and suggest motility may play a role in the completion of cell division.The phenotypes we report here, with respect to growth rate and N:K ratio were milder defects than those seen after knockdown of TbDNAH10, even though extent of knockdown was similar [74].It is perhaps not surprising that depletion of this protein, thought to modulate dynein function, does not have as drastic an impact on cell growth and movement as depletion of a dynein HC [58,74].
To document the time course of development of the IC138 RNAi phenotype, we monitored cells at 12 hour intervals following induction of RNAi.We focused on features of flagellar detachment and motility that could be easily scored in live cells.While some features were limited to only a small number of cells, the study does reveal some consistent trends: discernable changes in cell motion were recorded as early as 24 hours-post-induction (Fig 5A ), and motility became more defective over time until nearly all cells were immotile.It remains to be determined if the increase in immotility over time was related to the dynamics of assembly and recycling of axonemal complexes following knockdown, or resulted from loss of proper coordination between axonemal dyneins.
Incidence of flagellar detachment was first observed a little bit later (36 hours-post-induction), partially detached flagella were more common than completely detached flagella, but both populations increased in frequency over time (Fig 5B).By 108 hours nearly 60% of cells showed some form of flagellar detachment.Detachment of flagella from along the cell is not unique to the TbIC138 knockdown, it has been reported in studies of other flagellar and cell cycle factors, [21,[86][87][88][89], and there may be many ways to compromise attachment.
Attachment of the flagellum to the cell body occurs at the FAZ, a structure demarcated by an electron-dense filament that includes multi-domain protein assemblies [6,35,36,90], including FLAM3 that attaches into the axoneme itself [31].Proper positioning of a new flagellum is important for the progression of cell division, including kinetoplast localization [82,[91][92][93][94]. Failure to properly attach results in shorter cells and significant morphological changes [31,83,85,90], unlike the phenotype seen upon knockdown of TbIC138.Depletion of TbIC138 was previously shown to not affect axoneme structure by transmission electron microscopic analysis [64] that shows the major IAD structures are still intact despite the detachment phenotype.
The mechanism by which TbIC138 depletion leads to flagellar detachment is unknown, but it is possible that irregular beating of flagella pulls apart the attachment anchoring complexes, resulting in progressively more flagellar detachment over time, but we were not able to observe individual cells long enough to determine this.Alternatively, there may be components that interact with anchoring complexes whose assembly into the flagellum is compromised after TbIC138 knockdown [63,95], resulting in weaker flagellum-cell body attachment.
Flagella with partial or complete detachment nearly always had abnormal beating (Fig 5C ), that appeared as a slow and often irregular beat, or as complete paralysis.Because the numbers of cells with detached flagella were low, it was not possible to determine by the end of this study if all detached flagella would eventually become paralyzed, although it seems likely since immotility increases in the population overall.By characterizing cells with respect to flagellar attachment and separately to body movement, we are able to make distinctions between behavior of cells with attached and detached flagella.Clearly, depletion of TbIC138 can affect flagellar beating in the absence of flagellar detachment, indeed when development of abnormal motility over time is considered in only those cells with completely attached flagella, the pattern is essentially the same (S4 Fig) as for the total cell population (Fig 5A).Since we are interested in regulation of flagellar beating in T. brucei, monitoring the course of phenotype development allowed us to determine time points that might be useful for analyses of flagellar beating specifically.Since the earliest patterns of abnormal motility were seen by 24 hours-post-induction, detailed aspects of flagellar beating patterns can be analyzed shortly after induction without concern that secondary phenotypes associated with cell cycle timing will interfere with the analysis.Alternatively, later time points in which individual cells with fully detached flagella can be observed could be studied to observe variations in beating patterns of free flagella, comparing patterns between time points, or relative to a strain such as the FLAM3 knockdown strain that has detached flagella with normal motility [31].
In genome-wide studies, depletion of any of several IAD proteins are reported to result in loss of fitness in bloodstream form parasites [30,96], suggesting that components of IAD play important roles in parasite survival in mammalian hosts.Among IAD proteins, IC138 is an important regulator [49,56], recent studies have suggested that IC138 functions to position the dynein f/I1 complex properly in C. reinhardtii [26,97].The phenotype observed for knockdown of TbIC138 represents a stronger motility defect than seen in C. reinhardtii, in which loss of CrIC38 does not cause immotility [26], but a less severe motility defect than that of knockdown of a dynein f/I1 HC, consistent with its role as a regulator, as stated above.Differences in flagellar motility between the two organisms may result in slightly different roles for dynein f/I1 and thus regulators like IC138.The TbIC138 knockdown resulted in a distinct defect in flagellar beating, suggesting this strain is amenable for more extensive structure -function analyses of the role of this important protein in T. brucei.Flagellar motility is important not only in protozoan pathogens [12], but in many eukaryotic organisms, where it plays a role in cell movement but also in the sensory and transduction functions of this organelle [25].

Fig 1 .
Fig 1. Tagged TbIC138 protein: extent of knockdown and localization.A) Western blot showing detection of IC138::3xHA (98 kD) and Tubulin (50 kD) in representative examples of IC138 RNAi cultures 0, 48 hours-and 72 hours-post-induction. B) Quantification of Western blot showing extent of knockdown of IC138 protein levels.The level of protein in uninduced cells (uninduced) was set as 1.0, and the relative expression in 48 hours-or 72 hours-post-induction cells is indicated.Data represent averages from three independent sets of uninduced and induced cultures, with standard deviation indicated by error bars.C) Western blot

Localization
RNAi knockdown of TbIC138 resulted in a slight reduction in growth rate (Fig 2A) observable within 48 hours-post-induction. Individual cells became noticeably less motile and clusters of three or more cells were visible.Cell growth did not appear to stop even after inductions of as many as 8 days (S1 Fig), but as cell clusters became larger and more frequent, it is possible that clumped cells could cause an underestimate of cell titers at later times post-induction.Mild agitation of cultures for the duration of the growth curve analysis did not seem to affect the growth rate difference (S1 Fig) and did not eliminate clustering behavior.Sedimentation analysis of IC138 RNAi showed that induced cells settled out of solution more rapidly than uninduced cells, representing defective cell motility (Fig 2B).

Fig 2 .Fig 3 .
Fig 2. Growth and sedimentation analyses of TbIC138 knockdown.A) A representative cumulative growth curve of IC138 RNAi uninduced (dashed line) or tetracycline-induced (solid line).Cell titer = cells/mL.B) Fig 4 shows examples of a cell with completely attached flagellum (Fig 4A), a partially detached flagellum (Fig 4B) and with fully detached flagellum (Fig 4C)

Fig 4 .
Fig 4. Morphological features of TbIC138 knockdown.DIC images showing examples of attached (A), partially detached (B) and fully detached (C) flagella.Scale bar represents 5 μm.D) Graph showing flagellar lengths and total cell lengths of uninduced, 48 hours-and 72 hours-post-induction IC138 RNAi cells.Error bars show standard deviation, number of samples (n) is indicated below each point.doi:10.1371/journal.pone.0139579.g004

Fig 5 .
Fig 5. Occurrence of phenotypes over time following knockdown of TbIC138.A) Frequency of cells with normal, slow and abnormal motility or immotile.See S2 Table for motility classification.B) Frequency of cells with attached, partially detached or fully detached flagella.C) Frequency of detached flagella showing normal movement, aberrant movement or paralysis.Error bars represent standard deviation between two independent experiments, keys are at left of each graph.doi:10.1371/journal.pone.0139579.g005 Fig 2B) and that cell movement was progressively more impaired over time following induction, such that nearly 80% of cells were immotile by 72 hours-post-induction (Fig 5).Cell growth showed only a slight reduction in rate upon induction of RNAi (Fig 2A), and cells did not stop growing even after 8 days (S1 Fig).TbIC138 knockdown resulted in formation of clusters of cells, noticeable within 48 hours-post-induction (Fig 3B and 3C); mechanical agitation could be used to separate cells but the clustering behavior continued upon subsequent culturing.Clustering is a feature that has been observed following RNAi against other flagellar proteins [21,42,74], and may indicate that the final stages of cytokinesis do not function properly in these strains.