Flagellar adhesion in Trypanosoma brucei relies on interactions between different skeletal structures in the flagellum and cell body.

The Trypanosoma brucei flagellum is an essential organelle anchored along the surface of the cell body through a specialized structure called the flagellum attachment zone (FAZ). Adhesion relies on the interaction of the extracellular portion of two transmembrane proteins, FLA1 and FLA1BP. Here, we identify FLAM3 as a novel large protein associated with the flagellum skeleton whose ablation inhibits flagellum attachment. FLAM3 does not contain transmembrane domains and its flagellar localization matches closely, but not exactly, that of the paraflagellar rod, an extra-axonemal structure present in the flagellum. Knockdown of FLA1 or FLAM3 triggers similar defects in motility and morphogenesis, characterized by the assembly of a drastically reduced FAZ filament. FLAM3 remains associated with the flagellum skeleton even in the absence of adhesion or a normal paraflagellar rod. However, the protein is dispersed in the cytoplasm when flagellum formation is inhibited. By contrast, FLA1 remains tightly associated with the FAZ filament even in the absence of a flagellum. In these conditions, the extracellular domain of FLA1 points to the cell surface. FLAM3 is essential for proper distribution of FLA1BP, which is restricted to the most proximal portion of the flagellum upon knockdown of FLAM3. We propose that FLAM3 is a key component of the FAZ connectors that link the axoneme to the adhesion zone, hence it acts in an equivalent manner to the FAZ filament complex, but on the side of the flagellum.