CMF22 Is a Broadly Conserved Axonemal Protein and Is Required for Propulsive Motility in Trypanosoma brucei

Abstract

bThe eukaryotic flagellum (or cilium) is a broadly conserved organelle that provides motility for many pathogenic protozoa and is critical for normal development and physiology in humans. Therefore, defining core components of motile axonemes enhances understanding of eukaryotic biology and provides insight into mechanisms of inherited and infectious diseases in humans. In this study, we show that component of motile flagella 22 (CMF22) is tightly associated with the flagellar axoneme and is likely to have been present in the last eukaryotic common ancestor. The CMF22 amino acid sequence contains predicted IQ and ATPase associated with a variety of cellular activities (AAA) motifs that are conserved among CMF22 orthologues in diverse organisms, hinting at the importance of these domains in CMF22 function. Knockdown by RNA interference (RNAi) and rescue with an RNAi-immune mRNA demonstrated that CMF22 is required for propulsive cell motility in Trypanosoma brucei. Loss of propulsive motility in CMF22-knockdown cells was due to altered flagellar beating patterns, rather than flagellar paralysis, indicating that CMF22 is essential for motility regulation and likely functions as a fundamental regulatory component of motile axonemes. CMF22 association with the axoneme is weakened in mutants that disrupt the nexin-dynein regulatory complex, suggesting potential interaction with this complex. Our results provide insight into the core machinery required for motility of eukaryotic flagella.

Insect Stage-Specific Adenylate Cyclases Regulate Social Motility in African Trypanosomes

“…To assess the specific role of AC6, we asked whether the hypersocial phenotype could be rescued by restoring expression of the wild-type protein in the knockdown cell line. To achieve this, we generated a knockdown line targeting the AC6 3= UTR, "AC6-uKD," because this enables one to test for rescue under RNAiinducing conditions by expressing an AC6 transgene with an alternate 3= UTR that is immune to RNAi (34,38). Tetracycline induction of AC6-uKD cells effectively reduced AC6 expression, without affecting growth or individual cell motility in suspension culture (see Fig.…”

“…AC6-Ri and AC6**-Ri cell lines were generated by expressing wild-type or mutant AC6 in the AC6 UTR knockdown line (AC6uKD) (34). To generate the AC6-Ri line, a 426-bp fragment corresponding to the 3= terminus of the AC6 open reading frame (ORF) was cloned immediately upstream of the ␣/␤-tubulin intergenic region in the pMOTag plasmid (39).…”

“…Clonal lines were generated by limiting dilution. Efficacy of knockdown was assessed via qRT-PCR as described previously (34) at 72 h postinduction. qRT-PCR results are reported as arithmetic means Ϯ standard deviations from at least two independent RNA preparations, each analyzed in duplicate and normalized against the housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Tb927.6.4280/Tb927.6.4300) and RPS23 (Tb10.70.7020/Tb10.70.7030).…”

Motility and more: the flagellum of Trypanosoma brucei

“…The NDRC is a large (>1 MDa) complex that is present in virtually all motile axonemes and contains conserved as well as organism-specific subunits 40–42 . In T. brucei the NDRC includes trypanin 30 and component of motile flagella 70 protein (CMF70) 40 , as well as candidate subunits, trypanin-related protein, CMF46, CMF40 and CMF22 30, 41, 43 . Pioneering studies in C. reinhardtii demonstrate the NDRC operates in concert with the radial spokes and central pair apparatus as a reversible inhibitor of dynein 44–46 .…”

“…Reverse beating has also been observed in T. brucei 50 and is correlated with the parasite encountering obstacles 51 . Notably, NDRC subunits contain domains predicted to function in Ca ++ signaling 41, 43 . In addition to the NDRC, the PFR has emerged as a potential player in beat regulation with the discovery that it houses proteins predicted to participate in Ca ++ and cAMP signaling pathways, such as EF-hand calcium binding proteins and cAMP-specific phosphodiesterase 34, 35 .…”