Silencing of a putative inner arm dynein heavy chain results in flagellar immotility in Trypanosoma brucei

Abstract

The Trypanosoma brucei flagellum controls motility and is crucial for cell polarity and division. Unique features of trypanosome motility suggest that flagellar beat regulation in this organism is unusual and worthy of study. The flagellar axoneme, required for motility, has a structure that is highly conserved among eukaryotes. Of the several dyneins in the axonemal inner arm complex, dynein f is thought to control flagellar waveform shape. A T. brucei gene predicted to encode the dynein f alpha heavy chain, TbDNAH10, was silenced using RNA interference in procyclic T. brucei cells. This resulted in immotile flagella, showing no movement except for occasional slight twitches at the tips. Cell growth slowed dramatically and cells were found in large clusters. Microscopic analysis of silenced cultures showed many cells with detached flagella, sometimes entangled between multiple cells. DAPI staining showed an increased frequency of mis-positioned kinetoplasts and multinucleate cells, suggesting that these cells experience disruption at an early cell cycle stage, probably secondary to the motility defect. TEM images showed apparently normal axonemes and no discernable defects in inner arm structure. This study demonstrates the use of RNAi as an effective method to study very large genes such as dynein heavy chains (HCs), and the immotility phenotype of these dynein knockdowns suggests that an intact inner arm is necessary for flagellar beating in T. brucei. Since analogous mutants in Chlamydomonas reinhardtii retain motility, this phenotype likely reflects differences in requirements for motility and/or dynein assembly between the two organisms and these comparative studies will help elucidate the mechanisms of flagellar beat regulation.

Figure 1

Axoneme structure. A. Transmission electron micrograph of a T.brucei flagellum cross-section with accompanying schematic representation of the axoneme showing the ring of nine outer doublet microtubules and central pair of single microtubules (9+2). Dynein complexes of the outer arm (OA, in blue) and inner arm (IA, in ocher) are indicated, as well as the radial spoke (RS, in green) structures that extend between the central pair and outer doublet microtubules. The micrograph image also shows the electron-dense paraflagellar rod structure underneath the axoneme and the flagellar membrane. Scale bar is 100nm. B. Schematic diagram showing a longitudinal view model for the structure outer arm (OA) and inner arm (IA) dynein complexes of C. reinhardtii, adapted from [9,48]. In this view, the microtubule is represented as a horizontal tube with base- and tip-proximal ends indicated, OA, IA and RS are indicated. Each of the eight major inner arm dynein HCs is shown. DNAH10 encodes the dynein f alpha heavy chain, part of the two-headed dynein f, and is shown in red. Scale bar is indicated. Although no model has been reported for the T. brucei axoneme, the genome sequence includes only two outer arm dynein HC genes (equivalent to β and γ shown here) and seven inner arm dynein HC genes including those of dynein f [44].

Figure 2

Analyses of DNAH10 ^RNAi knockdown cells. A. Relative transcript levels of DNAH10 RNA by real time RT-PCR analysis. Gene expression was normalized to the housekeeping genes GAPDH and RPS23. The level of gene expression in uninduced cells (-Tet) was set as 1.0, and the relative expression in tetracycline-induced DNAH10^RNAi cells (+Tet) is indicated. Data represent averages from two independent sets of uninduced and induced cultures, with standard deviation indicated by error bars. B. A representative cumulative growth curve of DNAH10^RNAi uninduced (solid line) or tetracycline-induced (dashed line), averages of cell densities are shown with standard deviation indicated by error bars. C, D, E. Phase contrast images of live DNAH10^RNAi cultures viewed at 10× magnification, showing clusters in tetracycline-induced cultures: C. Uninduced (-Tet). D. 48 hours post induction (+ Tet). E. 96 hours post-induction (+ Tet). Scale bar is 40 μm.

Figure 3

Motility is disrupted in DNAH10 ^RNAi knockdown cells. A. Sedimentation assay showing the motility of DNAH10^RNAi cells in the absence (closed diamonds) or presence (open squares) of tetracycline (48 hours post induction). B. Summaries of traces from time-lapse motility assays on DNAH10^RNAi. Pie charts with percentages of cells scored as runners (white), tumblers (gray) or immotile (black) in cultures that were uninduced, 24 hours, or 48 hours post-induction with tetracycline. Raw numbers of cells scored for each sample indicated in parenthesis.

Figure 4

Microscopic analyses of DNAH10 ^RNAi cultures. A-C. Paired DIC and fluorescent images of DNAH10RNAi that were uninduced (A), or induced for 48 hours with tetracycline (B-C). Multinucleate cells and cells with detached flagella are visible in the induced cultures. Representative examples of multinucleate cell (white arrowhead) and detached flagellum (white arrow) are indicated. Scale bar is 10 μm. D. Enlarged image of individual cells from white square in image B, shown to highlight the flagellar detail. Scale bar is 10 μm. E. Bar graph showing percentages of different morphological classes scored from uninduced, 24 hours post-induction, and 48 hours post-induction DNAH10^RNAi cells. Inset shows key to shading: Black = cells with detached or partially detached flagella. Gray = cells with two or more nuclei (the percent of cells with greater than two nuclei is indicated by the darker gray boxes within the gray bar). Hatched = cells with kinetoplast: nucleus ratio <1. F-H. SEM images of DNAH10^RNAi cells that were uninduced (F) or 48 hours post induction (G-H). Scale bar is 5μm.

Figure 5

TEM analysis of DNAH10 ^RNAi cultures. Representative axonemes prepared from high salt extraction of flagella from DNAH10 ^RNAi uninduced (A) or induced (B-C) cultures. This extraction protocol removes outer arm dyneins as well as some internal axonemal structures, but yields very good visualization of inner arm structures. Representative inner arm complexes scored as “large” (arrow) or “small” (arrowhead) are indicated, see text for details. Scale bar is 100 nm.