A novel function for the atypical small G protein Rab-like 5 in the assembly of the trypanosome flagellum

Abstract

The atypical small G protein Rab-like 5 has been shown to traffic in sensory cilia of Caenorhabditis elegans, where it participates in signalling processes but not in cilia construction. In this report, we demonstrate that RABL5 colocalises with intraflagellar transport (IFT) proteins at the basal body and in the flagellum matrix of the protist Trypanosoma brucei. RABL5 fused to GFP exhibits anterograde movement in the flagellum of live trypanosomes, suggesting it could be associated with IFT. Accordingly, RABL5 accumulates in the short flagella of the retrograde IFT140RNAi mutant and is restricted to the basal body region in the IFT88RNAi anterograde mutant, a behaviour that is identical to other IFT proteins. Strikingly, RNAi silencing reveals an essential role for RABL5 in trypanosome flagellum construction. RNAi knock-down produces a phenotype similar to inactivation of retrograde IFT with formation of short flagella that are filled with a high amount of IFT proteins. These data reveal for the first time a functional difference for a conserved flagellar matrix protein between two different ciliated species and raise questions related to cilia diversity.

Intraflagellar transport is required for the maintenance of the trypanosome flagellum composition but not its length

“…Each p2675 construct was linearized with a specific enzyme allowing recombination within the PKAR (SgrdI), RSP9 (BbsI) or AKB (NsiI) gene. The eGFP::Hydin fusion contains 1-1200 bp of the hydin gene in frame downstream of the eGFP gene in the vector pPCPFReGFP (Adhiambo et al, 2009). In this vector, the fusion gene is flanked by PFR1 and PFR2 intergenic sequences.…”

“…IFT has been imaged by tagging multiple IFT-B or dynein subunits and is active during both construction and maintenance. The expression of various components of the IFT-A and IFT-B complexes has been individually silenced using inducible RNA interference (RNAi), resulting in failure of flagellum construction (Absalon et al, 2008;Adhiambo et al, 2009;Blisnick et al, 2014;Davidge et al, 2006;Franklin and Ullu, 2010;Huet et al, 2014;Kohl et al, 2003). Usually, inhibition of IFT-B proteins (for example IFT88) blocks axoneme construction in agreement with a default in anterograde transport, whereas absence of IFT-A proteins (such as IFT140) leads to formation of short flagella filled with IFT material, indicative of defects in retrograde transport (Absalon et al, 2008).…”

“…However, efficient tools to investigate IFT were not available at the time of these studies. We have since produced a monoclonal antibody against the IFT-B protein IFT172 (Adhiambo et al, 2009) and set up conditions to reliably visualize IFT in trypanosomes (Buisson et al, 2013). Moreover, the development of vectors for in situ tagging now allows expression of fluorescent fusion proteins at their endogenous level, an essential condition to monitor IFT in mutant situations (Kelly et al, 2007).…”

Biology of Cilia and Ciliopathies

“…The null IFTA-2 (IFT22) mutant had intact sensory cilia, effectively suggesting IFTA-2 is not essential to ciliogenesis. However, RNAi knockdown experiments of Trypanosome RABL5 lead to the buildup of IFT particles in the flagella compartment and subsequent shortening of the flagella [60]. This phenotype is similar to mutants with defective retrograde IFT, suggesting RABL5 is important for ciliogenesis.…”

Forward motility is essential for trypanosome infection in the tsetse fly

“…Subclones of double mutant cells were transformed with a rescue copy of the DNAI1 gene tagged to EGFP and integrated in the PFR intergenic region of the trypanosome nuclear genome to be used as an add-back control strain (AB). For this purpose, the full DNAI1 coding sequence (1968 bp) was cloned with NheI and EcoRI at the 3′ end of the EGFP coding sequence in the pPCPFR-EGFP vector (Absalon et al, 2008;Adhiambo et al, 2009).…”