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Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei

Katherine S. Ralston, Neville K. Kisalu, Kent L. Hill
Katherine S. Ralston
1Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095
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Neville K. Kisalu
1Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095
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Kent L. Hill
1Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, California 90095
2Molecular Biology Institute, University of California, Los Angeles, California 90095
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  • For correspondence: kenthill@mednet.ucla.edu
DOI: 10.1128/EC.00298-10
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ABSTRACT

The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei. Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.

FOOTNOTES

    • Received 30 November 2010.
    • Accepted 23 February 2011.
    • Accepted manuscript posted online 4 March 2011.
  • ↵† Supplemental material for this article may be found at http://dx.doi.org/10.1128/EC.00298-10.

  • Copyright © 2011, American Society for Microbiology
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Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei
Katherine S. Ralston, Neville K. Kisalu, Kent L. Hill
Eukaryotic Cell Jul 2011, 10 (7) 884-894; DOI: 10.1128/EC.00298-10

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Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei
Katherine S. Ralston, Neville K. Kisalu, Kent L. Hill
Eukaryotic Cell Jul 2011, 10 (7) 884-894; DOI: 10.1128/EC.00298-10
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