Assembly of the paraflagellar rod and the flagellum attachment zone complex during the Trypanosoma brucei cell cycle

Abstract

Trypanosomes possess a single flagellum that is attached to their cell body via the flagellum attachment zone (FAZ). The FAZ is composed of two structures: a cytoplasmic filament complex and four microtubules situated next to it. There is a complex transmembrane crosslinking of this FAZ to the paraflagellar rod (PFR) and axoneme within the flagellum. We have partially purified the FAZ complex and have produced monoclonal antibodies both against the FAZ and the paraflagellar rod. The two antibodies against the FAZ (L3B2 and L6B3) recognise the cytoplasmic filament in immunofluorescence and in immunoelectron microscopy. On western blot, they detect a doublet of high molecular weight (M(r) 200,000). Two anti-PFR antibodies (L13D6 and L8C4) recognise the paraflagellar rod in immunofluorescence, but show a difference on Western blot: L13D6 recognises both major PFR proteins, whereas L8C4 is specific for only one of them. Using these new antibodies we have shown that although the growth of both cytoplasmic FAZ filament and external PFR are related, their growth initiates at different time points during the cell cycle and the two structures elongate at distinct rates.