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Mutations at Proposed Phosphorylation Sites in Flagellar Inner Arm Dynein Protein IC138 of Trypanosoma brucei
Author(s) -
Rios Kelly,
Takats Taylor,
Fuhrman Melissa,
Volpe Alexis,
Springer Amy
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.884.44
Subject(s) - trypanosoma brucei , flagellum , biology , microbiology and biotechnology , motility , mutant , dynein , gene knockdown , rna interference , phosphorylation , genetics , gene , microtubule , rna
Trypanosoma brucei are uniflagellated protozoan parasites that are responsible for African sleeping sickness. The flagellum of T. brucei is important for pathogenesis and proper cell division. We are interested in regulation of flagellar motility, which is not well understood, even though the structure of the flagellar axoneme is highly conserved among eukaryotes. We are studying a component of axonemal inner arm dynein protein IC138. Studies in Chlamydomonas reinhardtii suggest that IC138 is required for the regulation of flagellar motility and is controlled by phosphorylation. Since a phosphoproteomic study of T. brucei identified two possible serine phosphorylation sites in IC138, we constructed point mutations changing these residues, S46 and S158, into alanines to determine if they are important for motility. Two single mutants and one double mutant of IC138 were transfected into wild type trypanosomes to determine if they showed dominant effects. In order to observe mutant expression, endogenous IC138 was knocked down using RNAi. We describe the targeting of UTR regions to specifically knockdown endogenous IC138 without affecting the engineered mutants, and use of RT‐qPCR to assess the degree of knockdown effectiveness. If these sites are important for IC138 function, then the mutants should affect normal motility, which we evaluate using sedimentation assays, video microscopy and biochemical analyses. Discovering more about the regulation of motility in T. brucei is important for studying the pathogenicity of these parasites, and it may allow for the future development of treatments or preventative measures against the spread of African sleeping sickness.