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First published online 16 January 2007
doi: 10.1242/jcs.03352

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Functional genomics in Trypanosoma brucei identifies evolutionarily conserved components of motile flagella

¹ Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA
² Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA

View larger version (56K): [in this window] [in a new window]   Fig. 1. Comparative genomics identifies T. brucei components of motile flagella. (A) Overview of the strategy used to identify and characterize TbCMF genes. Genomes of organisms with motile cilia or flagella (H. sapiens, M. musculus, D. melanogaster, L. major, T. cruzi, C. reinhardtii and C. intestinalis) were compared to the T. brucei genome for common genes, then those genes found in organisms that have non-motile flagella or lack flagella (C. elegans and A. thaliana, respectively) were subtracted. Homology was confirmed by protein alignments and reciprocal BLAST analysis. To determine whether the subset of 41 functionally uncharacterized proteins have a role in motility, RNAi mutants were analyzed (see Materials and Methods for details). (B) Amino acid sequence alignment of TbCMF 9 with top BLAST hits from humans (Hs, accession number AAI09127) and C. reinhardtii (Cr, protein ID C_230118). Amino acids highlighted in yellow and blue are identical in all or most sequences, respectively; green highlighting represents conservative substitutions. The RNAi region is underlined and the borders of the SMC domain in TbCMF 9 are denoted by red arrowheads.

View larger version (94K): [in this window] [in a new window]   Fig. 2. TbCMF knockdown is specific and mutants can be placed into four phenotypic classes. (A) Classification of TbCMF knockdown mutants. The majority of TbCMF mutants exhibit cytokinesis defects that result in the formation of multicellular clusters (white arrows). Mutants were placed into one of four classes based on the size and time of formation of these clusters. A representative example of each mutant class is shown. Each panel shows a phase-contrast image of the indicated TbCMF knockdown strain grown in the absence (uninduced) or presence (induced) of 0.3 µg/ml tetracycline for five days. (B) Northern blots of RNA from two sample TbCMF mutants from each mutant class. RNA samples were probed with the gene targeted by RNAi and with a non-target gene as a loading control. *For TbCMF 2, equal loading was confirmed using rRNA visualized by UV.

View larger version (63K): [in this window] [in a new window]   Fig. 3. Class 3 and 4 TbCMF mutants have clear motility defects. The indicated mutants were assessed for cell motility defects using a sedimentation assay (A) (Bastin et al., 1999; Ralston et al., 2006) and by direct observation using DIC microscopy (B). In both cases, cells were assayed before significant clustering was evident to avoid secondary effects on motility. (A) Sedimentation curves. OD is the difference between the average OD600 readings for sedimented (S) and resuspended (R) samples. Error bars show a standard deviation of three independent experiments. A trypanin knockdown mutant (Hutchings et al., 2002) is included as a control for samples without (-tet) and with (+tet) motility defects. (B) Time-lapse series taken from a video clip of TbCMF 46 (a Class 3 mutant) grown in the absence (-tet) or presence (+tet) of tetracycline for 30 hours. The black arrow marks the point of origin at the start of the time-lapse series while the white arrow follows the cell. Bar, 10 µm. A video of TbCMF 46 motility is provided as supplementary material Movie 2. Additional movies of WT (Movie 5), class 2 (TbCMF 3, Movie 1) and class 4 (TbCMF 5, Movie 4; TbCMF 9, Movie 3) are provided as supplementary material.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Quantitative analysis of TbCMF cell motility. (A) Cell traces of uninduced and induced (30 hpi) TbCMF 46 cells. Analysis was done as described in Materials and Methods. Running cells (R) were classified as having sustained propulsive movement for at least 5 seconds. Tumbling cells (T) had visible movement, but did not sustain a run for 5 seconds or more. Immotile cells (I) were classified as cells where movement was visible at the cellular level, but the cell did not move from the point of origin. (B) Graphical representation of motility differences between uninduced and induced mutant cells. Class 2, TbCMF 2 and TbCMF 3; Class 3, TbCMF 46 and TbCMF 76b; Class 4, TbCMF 5 and TbCMF 9. n=49-53 cells; 30-second interval. Blue, immotile cells; red, tumbler cells; yellow, runner cells. Bars, 50 µm.

View larger version (108K): [in this window] [in a new window]   Fig. 5. TbCMF 9 and TbCMF 76b are part of a novel family of axonemal stabilizing proteins. Transmission EM images of flagella from the indicated TbCMF mutants. (A-C) Knockdown of TbCMF 5 does not significantly perturb the axoneme or PFR structure including the orientation of central pair microtubules. (D-F) TbCMF 9 knockdown causes a range of defects in outer-doublet arrangement. Axonemes ranged from outer doublets falling away from the axoneme (D, arrow), to groups of doublets that are completely separated from the axoneme (E, arrow), to split `hemi-axonemes', in which half of the axoneme has twisted around to a piggyback arrangement on the other half (F). This range of structural defects was observed in 44% of sections, 68% displayed doublets in disarray and 32% displayed a piggyback configuration. A total of 100 sections were examined. (G-I) Knockdown of TbCMF 76b, a protein related to TbCMF 9, results in similar ultrastructural defects. These are observed in 40% of sections, 81% of which show aberrant outer-doublet arrangement and 19% show the hemi-axoneme arrangement. A total of 75 sections were examined. (J) Longitudinal sections of TbCMF 9 and 76b flagella. Doublets that have lost their linkages with the rest of the doublets bow out from the axoneme (arrows). Longitudinal images were taken at the same time point as the cross-sections shown in A-I.

View larger version (62K): [in this window] [in a new window]   Fig. 6. TbCMF proteins localize along, and are stably associated with, the flagellum. (A) Anti-TPN western blot on whole-cell lysates (L), detergent-solubilized proteins (S1) and detergent-extracted cytoskeletons prepared (Hill et al., 2000) from a trypanosome cell line harboring a Tet-inducible TPN-GFP fusion protein. The TPN-GFP fusion protein is expressed at levels equivalent to endogenous TPN and is quantitatively associated with the cytoskeleton. (B-F) Fluorescence microscopy shows that TPN-GFP is correctly localized to the flagellum (arrows) in live cells (C-D) and in cytoskeletons (E-F). (B) Live cells exhibit background autofluorescence that does not overlap with the flagellum and is also evident in the 29-13 parent cell line. As a negative control, GFP alone was used. Although fluorescence was evident in the cell body of live cells, there was no fluorescence in the flagellum of detergent-extracted cytoskeletons (supplementary material Fig. S2). (G) Fluorescence microscopy of detergent-extracted cytoskeletons prepared from cell lines harboring the indicated Tet-inducible TbCMF-GFP fusion proteins. In all cases, the GFP fusions are localized along the flagellum (arrows). (H) Anti-GFP western blots of whole cell lysates (L), detergent-solubilized proteins (S1) and detergent-extracted cytoskeletons (P1) prepared from induced (+) and uninduced (-) TbCMF-GFP strains. The asterisk indicates a secondary band at 100 kDa that reacts with the anti-GFP primary antibody. Bars, 5 µm (B); 10 µm (G).

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