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.

Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins

“…The rapid and efficient knockout achieved with RNP complexes allowed us to also investigate the function of genes suspected or known to be essential and, thus, not amenable to previous knockout protocols that require an extended drug-selection period ( 17 , 19 , 20 ). Among such genes are those essential to the flagellar structure, as demonstrated using RNAi knockdown in T. brucei ( 21 ). Electroporation of RNP complexes targeting the FAZ1 ( 22 ) and KMP11 ( 23 ) genes results in a substantial population of epimastigotes with clear flagellar and cytokinesis defects within a few days after transfection ( Fig.…”

Targeted copy number screening highlights an intragenic deletion of WDR63 as the likely cause of human occipital encephalocele and abnormal CNS development in zebrafish

“…IC140 forms a complex with IC138 and binds via WDR domains to the tubulin axoneme via WDR domains. Conversely, knockout of IC140 disturbs the proper function of the cilium (Hendrickson, Goss, Seaton, & Rohrs, ; Hendrickson et al., ; Wilson et al., ; Perrone, Yang, O'Toole, Sale, & Porter, ). Our morpholino knockdown experiments in zebrafish (Supp.…”

“…Conversely, knockout of IC140 disturbs the proper function of the cilium (Hendrickson, Goss, Seaton, & Rohrs, 2013;Hendrickson et al, 2004;Wilson et al, 2015;Perrone, Yang, O'Toole, Sale, & Porter, 1998).…”