A roadmap for assembly of a eukaryotic flagellum

Cilia and flagella (terms used interchangeably) are hair-like appendages that carry out critical motility and/or sensory functions for eukaryotic cells. These complex structures are built from hundreds of distinct proteins and their assembly is a significant challenge, a task made even more difficult as assembly occurs in a cellular compartment distinct from the site of protein synthesis within the main body of the cell. In order to form flagella, eukaryotic cells must therefore target and transport all component proteins to this environmentally distinct region, and subsequently ensure the coordinated assembly of these proteins into the flagellum structure. Previous studies have shown that proteins destined for the flagellum are targeted first to the basal body; a pivotal structure that both templates formation of the axoneme (main structural component of flagella) and acts as a docking platform for recruitment of flagellum proteins. Flagellum assembly thus involves a pathway that allows regulated transport of proteins to the basal body, and hence into the flagellum compartment and their subsequent coordinated assembly into a complex 3-dimensional structure. The obvious dangers for ciliated/flagellated cells are (i) flagellum proteins may be incorrectly targeted (and thus never reach the basal body and/or flagellum); (ii) proteins are correctly targeted but are incapable of flagellum incorporation; (iii) abnormal proteins are incorporated into the flagellum resulting in structural defects. To better understand flagellum assembly this project aims to generate a 'roadmap' by studying how proteins are targeted to the basal body and/or the flagellum and also exploring the role of protein processing (i.e. quality control) in ensuring the functional integrity of flagellum structures. As part of this study we will specifically investigate the biochemical function of TbRP2, a critical basal body located protein which, according to our published data and previous work on human RP2 protein by others, is proposed to carry out essential 'quality control' checks on tubulin (the major protein building block of the axoneme) prior to its import into the flagellum. In addition to investigating the role of TbRP2 in protein processing we will also use the specific intracellular localisation of TbRP2 to identify specific basal body targeting signals and investigate the co-ordination of basal body protein recruitment and protein transport into (and along) the flagellum. In the second interrelated part of this project we will investigate the role of one type of motif which targets proteins beyond the basal body into the flagellum. We have previously shown that this motif is sufficient to target proteins to the flagellum but that subsequent tethering of proteins into flagellum structures can be dictated by relative positioning of the motif within the protein. By investigating properties of this targeting determinant we can learn a great deal about the specific targeting and tethering of flagellum proteins. The project will provide critical new insight into processes widely conserved in ciliated/flagellated eukaryotic cells and are thus of wide biological interest. These studies could also have practical implications, since defects in cilium/flagellum structure and function underpin a wide range of human diseases; including infertility, chronic respiratory disease, cancer, diabetes and obesity. Greater knowledge of fundamental mechanisms required for formation of eukaryotic cilia/flagella could lead to increased understanding of human health and disease. Conversely, since the model to be used in our studies is the important microbial pathogen Trypanosoma brucei (in which flagellum function is critical for virulence) identification of parasite specific differences to the normal flagellum assembly process could lead to the generation of new and urgently needed drug therapies for the sleeping sickness this parasite causes.

Our study of protein targeting and flagellum assembly in trypanosomes will be informed by complementary and overlapping approaches. 1. To define amino acid sequences necessary and sufficient to (a) target TbRP2 protein to flagellar basal bodies (BB) and (b) anchor TbRP2 to the transitional fibres of the BB, N- and C-terminal truncated variants of TbRP2 will be fused to GFP. Protein expression will be controlled by use of either a tetracycline-inducible gene expression system or endogenous TbRP2 cis-regulatory elements. Live cell GFP imaging or immunofluorescence of fixed cells will readily identify targeting determinants; biochemical and immunofluorescence analyses of detergent-extracted cytoskeletons/isolated flagella will distinguish BB anchoring determinants from mere targeting determinants. 2. Our study of adenylate kinase targeting to the trypanosome paraflagellar rod (PFR) and axoneme will also utilise a tractable domain swapping approach coupled to immunofluorescence analysis of protein chimeras in whole cells, detergent-extracted cytoskeletons and isolated flagella. Essential reagents, such as antibodies recognising marker antigens in the axoneme or PFR, respectively are at our disposal. 3. TbRP2 biochemistry will be informed by yeast-2-hybrid (Y2H) screens to identify candidate interacting proteins. All interacting candidates (including additional molecules identified by literature searching) will be localised using either an epitope-tagging approach or polyclonal antibodies raised against recombinant protein. These experiments will determine whether interacting candidature is supported by an appropriate BB-localisation. Putative function of interacting candidates in flagellum assembly will be informed using gene-specific RNA interference and by reference to the published TbRP2 RNAi phenotype. 4. Fine-mapping of TbRP2 targeting/tethering motifs will be conducted by pair-wise Y2H against newly identified and validated TbRP2 interacting proteins.