Abstract
Eukaryotic flagella are complex microtubule-based organelles that, in many organisms, contain extra-axonemal structures, such as the outer dense fibres of mammalian sperm and the paraflagellar rod (PFR) of trypanosomes. Flagellum assembly is a complex process occurring across three main compartments, the cytoplasm, the transition zone and the flagellum itself. The process begins with the translation of protein components followed by their sorting and trafficking into the flagellum, transport to the assembly site and incorporation. Flagella are formed from over 500 proteins and the principles governing assembly of the axonemal components are relatively clear. However, the coordination and location of assembly of extra-axonemal structures are less clear. We have discovered two cytoplasmic proteins in Trypanosoma brucei that are required for PFR formation, PFR assembly factors 1 and 2 (PFR-AF1 and PFR-AF2, respectively). Deletion of either PFR-AF1 or PFR-AF2 dramatically disrupted PFR formation and caused a reduction in the amount of major PFR proteins. The existence of cytoplasmic factors required for PFR formation aligns with the concept that processes facilitating axoneme assembly occur across multiple compartments, and this is likely a common theme for extra-axonemal structure assembly.