Authors

H Berti Gabriel¹; JD Sunter¹;  ¹ Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK

Discussion

Alongside the microtubule axoneme in many flagellated cells there are additional protein structures such as the paraflagellar rod (PFR) of T. brucei. The PFR has an intricate structure composed of three distinct domains (inner, middle and outer) that runs next to the axoneme within the flagellum and is important for flagellar beat regulation and cell motility. However, the specific contribution of each of the PFR domains to motility is unknown. The TrypTag project highlighted the complexity of the PFR with 146 proteins found in this structure. We determined by bioinformatics analysis, conservation patterns of these proteins across Euglenozoa and identified two major conserved sets of PFR proteins. The first set was conserved across Euglenozoa; whereas, the second set was conserved but not present in organisms with a reduced PFR, such as Angomonas deanei. A semi-automated analysis of TrypTag images, measuring the distance between the kinetoplast and the start of the PFR signal revealed a discontinuous start to the PFR and predicted the domain to which a PFR protein localised. These predictions were confirmed for a subset of 15 proteins by analysing the distance to known flagellum proteins. We analysed the function of these 15 proteins in cell motility and depletion of four of them disrupted motility, reduced cell growth and led to a ‘blob’ forming at the flagellum tip. The motility phenotypes were only observed in proteins present in the inner and very outer PFR domains. However, for three of these proteins despite changes in motility, there were no obvious changes to the PFR ultrastructure, suggesting these proteins are not required for PFR assembly. These results suggest that the motility function of the PFR can be separated from its assembly and that there are domains and sub-structures of the PFR with a specific role in motility. This is an important step in assigning specific functions to the individual PFR domains.