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Motility and sensory functions of the Trypanosoma brucei flagellum

  • Author(s): Langousis, Gerasimos
  • Advisor(s): Hill, Kent L
  • et al.
Abstract

Eukaryotic flagella, also known as cilia, are emblematic features of most extant eukaryotic cells. Cilia have important motility and sensory roles for cellular and organismal physiology. A prime example is the flagellum of Trypanosoma brucei, the unicellular causative agent of sleeping sickness in Africa. The T.brucei flagellum harbors a canonical eukaryotic axoneme and provides cell propulsion, dictates cell morphogenesis as well as constitutes a critical host pathogen interface.

Flagellar motility results from the coordinated activity of axonemal dyneins. While the mechanism, by which thousands of dynein motors are spatiotemporally controlled, is enigmatic, motility requires the Nexin Dynein Regulatory Complex (NDRC). The NDRC is a massive axonemal protein complex, present in virtually all flagellated eukaryotes, that reversibly inhibits dyneins. NDRC has important roles in human and trypanosome biology yet its exact composition and mode of action are unknown. We employed quantitative proteomics as well as a candidate approach to illuminate the T. brucei NDRC subunit composition. We discovered 14 proteins that are consistently reduced in axonemes of NDRC mutants and are thus prime NDRC subunit candidates. Of these candidates, only two were specific to the trypanosome lineage while the remainder had clear orthologs in diverse eukaryotes. Our results support the existence of an NDRC core consisting of phylogenetically conserved subunits that operate in unison with lineage specific subunits. Moreover, NDRC is likely a calcium-regulated hub given the extensive representation of cognate domains among its subunits.

The trypanosome flagellum remains attached to the cell body for most of its length at the flagellum attachment zone (FAZ). FAZ has important structural and morphogenetic roles that dictate the shape and size of the trypanosome cell. Nevertheless, the protein repertoire of the FAZ has remained elusive. We focused on FS179, an uncharacterized membrane protein recently detected in our T.brucei flagellar membrane proteome. We showed that this putative calcium channel localizes to a subdomain of the flagellar membrane along the FAZ. Interestingly, FS179 mediates flagellum attachment and is required for cell shape and parasite viability. Our findings thus reveal a novel component of the trypanosome FAZ and shed light on flagellum roles in cell morphogenesis.

The trypanosome flagellar membrane is the interface with the extracellular milieu and its proteins have vital roles in host-pathogen interactions. This is exemplified by the flagellar pocket, a membrane invagination at the base of the flagellum where all endocytosis and uptake of host factors takes place. During efforts to understand flagellar membrane functions, we studied BBS proteins, subunits of the BBSome complex that controls cilium localization for certain membrane proteins in other eukaryotes. We show that T.brucei BBS proteins assemble into a BBSome-like complex and localize to the flagellar pocket membrane and vesicles therein. Genetic knockouts of BBS genes further reveal that BBSome is dispensable for flagellum biogenesis yet is essential for proper uptake of host transferrin. Our results support a model whereby T. brucei BBSome functions as a clathrin adaptor facilitating endocytosis of a subset of flagellar pocket membrane proteins such as the transferrin receptor. In summary, our results highlight the importance of T.brucei as a model system to study conserved and parasite specific flagellar functions, illuminate novel regulators of axonemal motility and expand the roles of a cilium gatekeeper to endocytosis.

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