Premium
Knockdown of axonemal inner arm dynein protein IC138 Trypanosoma brucei causes flagellar detachment and defects in motility
Author(s) -
Marchese Anthony,
Wilson Corinne,
Springer Amy L.
Publication year - 2016
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.30.1_supplement.1067.2
Subject(s) - flagellum , axoneme , dynein , trypanosoma brucei , microbiology and biotechnology , biology , motility , microtubule , chlamydomonas , gene knockdown , biochemistry , gene , mutant
Trypanosoma brucei is a protozoan flagellate that causes African sleeping sickness. Motility is critical for its life cycle progression and pathogenesis. However, the regulation of flagellar function is not well understood. T. brucei utilizes a flagellum that attaches along the length of the cell, beating from tip‐to‐base to move the cell forward. Studies have found that the assembly and regulation of proteins within the axoneme, the microtubule‐based cytoskeleton of the flagellum, are well conserved. Within the axoneme are outer and inner arm dyneins, structures known to be involved in microtubule sliding which drives cell motility. Inner arm dynein I1/f has been found to play an important role in regulation of the flagellar beating. We are interested in the characterization of T. brucei IC138, an intermediate chain of dynein I1/f. This protein has been has been studied in green alga Chlamydomonas reinhardtii and shown to be a phosphoprotein important for the regulation of dynein I1/f. A T. brucei strain was created to inducibly target IC138 by RNA interference. By 72‐hours post‐induction, quantification of the knockdown revealed that IC138 protein levels were reduced by 87%. The knockdown cells were monitored for phenotypic changes over 108 hours following induction, and exhibited reduced doubling times, slowed or abnormal motility and partially or completely detached flagellum. Abnormal motility phenotypes included slow twitching or irregular beating that became more severe over time, such that by 72 hours post‐induction approximately 80% of the cells were immotile. Flagellar detachment increased over time, although less dramatically than immotility, affecting less than 60% of the population. The motility defect is seen early following induction and is evident in both detached and intact flagella and thus is consistent with the role of IC138 as a regulator of motility. Depletion of IC138 may disrupt the assembly of dynein I1 or other interacting proteins, possibly contributing to the phenotypes observed. Classification of other proteins missing in IC138 depleted cells will help to identify C. reinhardtii orthologs or lead to the discovery of novel IC138 binding partners. This identification could reveal additional components of T. brucei motility regulation and a provide basis for future research. Further knowledge of the protein interactions within dyneinI1/f may lead to a better understanding of the phenotypic changes resulting from IC138 depletion. Support or Funding Information NIH 7R15AI101941‐02