Protein transport and flagellum assembly dynamics revealed by analysis of the paralysed trypanosome mutant snl-1

The paraflagellar rod (PFR) of Trypanosoma brucei is a large, complex, intr aflagellar structure that represents an excellent system in which to study flagellum assembly. Molecular ablation of one of its major constituents, th e PFRA protein, in the snl-1 mutant causes considerable alteration of the P FR structure, leading to cell paralysis. Mutant trypanosomes sedimented to the bottom of the flask rather than staying in suspension but divided at a rate close to that of wild-type cells, This phenotype was complemented by t ransformation of snl-1 with a plasmid overexpressing an epitope-tagged copy of the PFRA gene. In the snl-1 mutant, other PFR proteins such as the seco nd major constituent, PFRC, accumulated at the distal tip of the growing fl agellum, forming a large dilation or 'blob', This was not assembled as fila ments and was removed by detergent-extraction. Axonemal growth and structur e was unmodified in the snl-1 mutant and the blob was present only at the t ip of the new flagellum, Strikingly, the blob of unassembled material was s hifted towards the base of the flagellum after cell division and was not de tectable when the daughter cell started to produce a new flagellum in the n ext cell cycle. The dynamics of blob formation and regression are likely in dicators of anterograde and retrograde transport systems operating in the f lagellum, In this respect, the accumulation of unassembled PFR precursors i n the flagellum shows interesting similarities with axonemal mutants in oth er systems, illustrating transport of components of a flagellar structure d uring both flagellum assembly and maintenance, Observation of PFR component s indicate that these are likely to be regulated and modulated throughout t he cell cycle.