Structure-Function Analysis of Dynein Light Chain 1 Identifies Viable Motility Mutants in Bloodstream-Form Trypanosoma brucei

Abstract

The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that is receiving increasing attention as a potential drug target and as a system for studying flagellum biology. RNA interference (RNAi) knockdown is widely used to test the requirement for a protein in flagellar motility and has suggested that normal flagellar motility is essential for viability in bloodstream-form trypanosomes. However, RNAi knockdown alone provides limited functional information because the consequence is often loss of a multiprotein complex. We therefore developed an inducible system that allows functional analysis of point mutations in flagellar proteins in T. brucei. Using this system, we identified point mutations in the outer dynein light chain 1 (LC1) that allow stable assembly of outer dynein motors but do not support propulsive motility. In procyclic-form trypanosomes, the phenotype of LC1 mutants with point mutations differs from the motility and structural defects of LC1 knockdowns, which lack the outer-arm dynein motor. Thus, our results distinguish LC1-specific functions from broader functions of outer-arm dynein. In bloodstream-form trypanosomes, LC1 knockdown blocks cell division and is lethal. In contrast, LC1 point mutations cause severe motility defects without affecting viability, indicating that the lethal phenotype of LC1 RNAi knockdown is not due to defective motility. Our results demonstrate for the first time that normal motility is not essential in bloodstream-form T. brucei and that the presumed connection between motility and viability is more complex than might be interpreted from knockdown studies alone. These findings open new avenues for dissecting mechanisms of flagellar protein function and provide an important step in efforts to exploit the potential of the flagellum as a therapeutic target in African sleeping sickness.

Forward motility is essential for trypanosome infection in the tsetse fly

“…RNAi mutants (Baron et al, 2007;Ralston et al, 2011). In agreement with these observations of monomorphic strains in vitro, the pleomorphic DNAI1 mutant parasites were still able to invade the posterior midgut and to proliferate.…”

Functional Architecture of the Outer Arm Dynein Conformational Switch

“…Similar mutations in Trypanosoma also lead to defective motility (20), whereas RNAi-driven knockdowns caused failure of outer arm dynein assembly (28). In the context of a ciliated epithelium, reduction of LC1 levels in the planarian Schmidtea mediterranea resulted in lower beat frequency and disruption of the hydrodynamic coupling necessary for generating metachronal synchrony but did not appear to affect dynein assembly (29).…”

“…Introduction of different mutant forms of LC1 produced graded effects: for example, mutation of Met 182 to Ala was much less disruptive than alterations to either Gly or Pro, which were predicted to change the dynamic behavior. Mutation of the equivalent residues in Trypanosoma brucei LC1 also caused major motility defects (20).…”

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

“…A 405-bp fragment of the CMF22 3= UTR was selected using parameters described previously (43,44) and designed using the RNAit algorithm (45) as described previously (44,46). This region was amplified with forward (5=-ATGGATCCAGCCCGGAATGTTCCTCAC-3=) and reverse (5=-ATAAGCTTGCTTCGCACCTGTACAACG-3=) primers containing a BamHI site (underlined) and a HindIII site (underlined), respectively.…”

“…The rescue UTRknockdown RNAi-immune (UKD-Ri) cell line was generated by stably transfecting the CMF22 C-terminal in situ tagging cassette (see above) into the CMF22-UKD line. The same CMF22 C-terminal in situ tagging cassette (see above) was stably transfected into the trypanin-UKD (46) and CKF70-knockdown (CMF70-KD) (25) cell lines to generate trypanin and CMF70 knockdowns that express tagged CMF22. These cell lines were grown in medium without puromycin for a week prior to tetracycline induction.…”