Abstract
Flagellar attachment is a visibly striking morphological feature of African trypanosomes but little is known about the requirements for attachment at a molecular level. This study characterizes a previously undescribed membrane protein, FLA3, which plays an essential role in flagellar attachment in Trypanosoma brucei. FLA3 is heavily N-glycosylated, locates to the flagellar attachment zone and appears to be a bloodstream stage specific protein. Ablation of the FLA3 mRNA rapidly led to flagellar detachment and a concomitant failure of cytokinesis in the long slender bloodstream form but had no effect on the procyclic form. Flagellar detachment was obvious shortly after induction of the dsRNA and the newly synthesized flagellum was often completely detached after it emerged from the flagellar pocket. Within 12 h most cells possessed detached flagella alongside the existing attached flagellum. These results suggest that proteins involved in attachment are not shared between the new and old attachment zones. In other respects the detached flagella appear normal, they beat rapidly although directional motion was lost, and they possess an apparently normal axoneme and paraflagellar rod structure. The flagellar attachment zone appeared to be disrupted when FLA3 was depleted. Thus, while flagellar attachment is a constitutive feature of the life cycle of trypanosomes, attachment requires stage specific elements at the protein level.
Highlights
African trypanosomes are flagellated protozoan parasites that cause sleeping sickness in humans and trypanosomiasis in cattle
This study describes a previously uncharacterized membrane glycoprotein, FLA3, and demonstrates that it locates to the FAZ and plays an essential role in flagellar attachment
FLA3 appears to be a bloodstream stage specific protein, which distinguishes it from other FAZ proteins, such as FLA1 and FAZ1 that appear to be involved in attachment throughout the life cycle
Summary
African trypanosomes are flagellated protozoan parasites that cause sleeping sickness in humans and trypanosomiasis in cattle. The FAZ contains an electron dense filament, positioned in a gap in the subpellicular array of microtubules that are located immediately beneath the surface membrane of the cell body. This filament appears as a regularly spaced structure that runs the length of the attachment zone [1,13]. A second feature of the attachment zone is the presence of a special quartet of microtubules located immediately to the left of the FAZ filament when the cell is viewed towards the anterior end These microtubules originate at the end of the flagellum closest to the basal bodies and run the length of the attachment zone alongside the FAZ filament. This quartet is intimately associated with a sheet of endoplasmic reticulum, which interdigitates between the four microtubules [13]
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