Direction of flagellum beat propagation is controlled by proximal/distal outer dynein arm asymmetry.

The 9 + 2 axoneme structure of the motile flagellum/cilium is an iconic, apparently symmetrical cellular structure. Recently, asymmetries along the length of motile flagella have been identified in a number of organisms, typically in the inner and outer dynein arms. Flagellum-beat waveforms are adapted for different functions. They may start either near the flagellar tip or near its base and may be symmetrical or asymmetrical. We hypothesized that proximal/distal asymmetry in the molecular composition of the axoneme may control the site of waveform initiation and the direction of waveform propagation. The unicellular eukaryotic pathogens Trypanosoma brucei and Leishmania mexicana often switch between tip-to-base and base-to-tip waveforms, making them ideal for analysis of this phenomenon. We show here that the proximal and distal portions of the flagellum contain distinct outer dynein arm docking-complex heterodimers. This proximal/distal asymmetry is produced and maintained through growth by a concentration gradient of the proximal docking complex, generated by intraflagellar transport. Furthermore, this asymmetry is involved in regulating whether a tip-to-base or base-to-tip beat occurs, which is linked to a calcium-dependent switch. Our data show that the mechanism for generating proximal/distal flagellar asymmetry can control waveform initiation and propagation direction.

Genome-wide subcellular protein map for the flagellate parasite Trypanosoma brucei.

Trypanosoma brucei is a model trypanosomatid, an important group of human, animal and plant unicellular parasites. Understanding their complex cell architecture and life cycle is challenging because, as with most eukaryotic microbes, ~50% of genome-encoded proteins have completely unknown functions. Here, using fluorescence microscopy and cell lines expressing endogenously tagged proteins, we mapped the subcellular localization of 89% of the T. brucei proteome, a resource we call TrypTag. We provide clues to function and define lineage-specific organelle adaptations for parasitism, mapping the ultraconserved cellular architecture of eukaryotes, including the first comprehensive 'cartographic' analysis of the eukaryotic flagellum, which is vital for morphogenesis and pathology. To demonstrate the power of this resource, we identify novel organelle subdomains and changes in molecular composition through the cell cycle. TrypTag is a transformative resource, important for hypothesis generation for both eukaryotic evolutionary molecular cell biology and fundamental parasite cell biology.

A Trypanosoma brucei protein required for maintenance of the flagellum attachment zone and flagellar pocket ER domains.

Trypanosomes and Leishmanias are important human parasites whose cellular architecture is centred on the single flagellum. In trypanosomes, this flagellum is attached to the cell along a complex flagellum attachment zone (FAZ), comprising flagellar and cytoplasmic components, the integrity of which is required for correct cell morphogenesis and division. The cytoplasmic FAZ cytoskeleton is conspicuously associated with a sheet of endoplasmic reticulum termed the 'FAZ ER'. In the present work, 3D electron tomography of bloodstream form trypanosomes was used to clarify the nature of the FAZ ER. We also identified TbVAP, a T. brucei protein whose knockdown by RNAi in procyclic form cells leads to a dramatic reduction in the FAZ ER, and in the ER associated with the flagellar pocket. TbVAP is an orthologue of VAMP-associated proteins (VAPs), integral ER membrane proteins whose mutation in humans has been linked to familial motor neuron disease. The localisation of tagged TbVAP and the phenotype of TbVAP RNAi in procyclic form trypanosomes are consistent with a function for TbVAP in the maintenance of sub-populations of the ER associated with the FAZ and the flagellar pocket. Nevertheless, depletion of TbVAP did not affect cell viability or cell cycle progression.