title: Knockdown of Inner Arm Protein IC138 in Trypanosoma brucei Causes Defective Motility and Flagellar Detachment.
abstract: 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.
authors.affiliation: Department of Biology, Siena College, Loudonville, New York, United States of America.
authors.forename: Corinne S
authors.initials: CS
authors.lastname: Wilson
authors.affiliation: Department of Biology, Amherst College, Amherst, Massachusetts, United States of America.
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Chang
authors.affiliation: Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, United States of America.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Greene
authors.affiliation: Department of Biology, Amherst College, Amherst, Massachusetts, United States of America.
authors.forename: Sulynn
authors.initials: S
authors.lastname: Machado
authors.affiliation: Department of Biology, Amherst College, Amherst, Massachusetts, United States of America.
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Parsons
authors.affiliation: Department of Biology, Siena College, Loudonville, New York, United States of America.
authors.forename: Taylor A
authors.initials: TA
authors.lastname: Takats
authors.affiliation: Department of Biology, Amherst College, Amherst, Massachusetts, United States of America.
authors.forename: Luke J
authors.initials: LJ
authors.lastname: Zambetti
authors.affiliation: Department of Biology, Siena College, Loudonville, New York, United States of America.
authors.forename: Amy L
authors.initials: AL
authors.lastname: Springer
mesh_terms: D054468:Axoneme / Q000502:physiology; D002453:Cell Cycle; D002467:Cell Nucleus / Q000648:ultrastructure; D004398:Dyneins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D005407:Flagella / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D008928:Mitochondria / Q000648:ultrastructure; D009068:Movement; D010641:Phenotype; D015800:Protozoan Proteins / Q000235:genetics / Q000502:physiology*; D034622:RNA Interference; D014346:Trypanosoma brucei brucei / Q000502:physiology* / Q000648:ultrastructure
chemical_list: D015800:Protozoan Proteins; D004398:Dyneins