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First published online 3 April 2007
doi: 10.1242/jcs.004846

Journal of Cell Science 120, 1513-1520 (2007)
Published by The Company of Biologists 2007
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Stuck in reverse: loss of LC1 in Trypanosoma brucei disrupts outer dynein arms and leads to reverse flagellar beat and backward movement

Desiree M. Baron1, Zakayi P. Kabututu1 and Kent L. Hill1,2,*

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


Figure 1
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  		Fig. 1. TbLC1 has sequence and structural homology to CrLC1. (A) Schematic diagram of the Chlamydomonas outer arm dynein complex [adapted from King (King, 2003Go) 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 {gamma}HC (#), 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., 2000Go) as described in the Materials and Methods. CrLC1 residues predicted to bind the {gamma}HC (green) as well as the basic residues in the C-terminus (blue) are conserved in the T. brucei protein. Red, {alpha}-helices; yellow, beta-sheets.

 

Figure 2
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  		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., 2007Go). 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., 2007Go; Bastin et al., 1999Go; Ralston et al., 2006Go) for the uninduced (–TET) and induced (+TET, 24 hpi) cells. Error bars show the s.d. for three experiments.

 

Figure 3
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  		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., 2007Go) 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., 2002Go) 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.

 

Figure 4
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  		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., 2006Go). 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.

 

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© The Company of Biologists Ltd 2007