The 912 microtubule axoneme of flagella and cilia represents one of the most iconic structures built by eukaryotic cells and organisms. Both unity and diversity are present among cilia and flagella on the evolutionary as well as the developmental scale. Some cilia are motile, whereas others function as sensory organelles and can variously possess 912 and 910 axonemes and other associated structures 1 . How such unity and diversity are reflected in molecular repertoires is unclear. The flagellated protozoan parasite Trypanosoma brucei is endemic in sub-Saharan Africa, causing devastating disease in humans and other animals 2 . There is little hope of a vaccine for African sleeping sickness and a desperate need for modern drug therapies 3 . Here we present a detailed proteomic analysis of the trypanosome flagellum. RNA interference (RNAi)-based interrogation of this proteome provides functional insights into human ciliary diseases and establishes that flagellar function is essential to the bloodstream-form trypanosome. We show that RNAi-mediated ablation of various proteins identified in the trypanosome flagellar proteome leads to a rapid and marked failure of cytokinesis in bloodstream-form (but not procyclic insect-form) trypanosomes, suggesting that impairment of flagellar function may provide a method of disease control. A postgenomic meta-analysis, comparing the evolutionarily ancient trypanosome with other eukaryotes including humans, identifies numerous trypanosome-specific flagellar proteins, suggesting new avenues for selective intervention.Flagellum-mediated migration between the gut and salivary glands of its tsetse fly vector is essential for progression of the trypanosome life cycle. However, the necessity for motility in an extracellular bloodstream-form trypanosome is unclear. In both forms, a single attached flagellum emerges from a posterior flagellar pocket ( Fig. 1a) and comprises a membrane-bound axoneme and associated paraflagellar rod (PFR; Fig 1b). We isolated the structural axoneme and associated PFR and basal body from procyclic trypanosomes by a well-characterized procedure of detergent and high-salt treatment 4 . Bands and spots were cut from one- (Fig. 1c) and twodimensional gels and digested with trypsin, and the resulting peptides analysed by reverse-phase high-performance liquid chromatography coupled with tandem mass spectrometry. Bands and spots excised from ten representative gels were analysed, resulting in the identification of 522 nonredundant proteins.Further manual mass spectrometric validation confirmed 380 proteins (see Supplementary Methods and Supplementary Fig. 1 for peptide numbers and coverage). Highly basic proteins are known to contaminate microtubule preparations owing to charge interactions 4 , and we noted some highly basic ribosomal proteins as recognizable contaminants. To limit contamination, we filtered the data by using an isolectric point (pI) value of 10.2 as a cut-off (the pI of the most acidic ribosomal protein), and placed 49 proteins (30 of which were r...

The role of the eukaryotic flagellum in cell motility is well established but its importance in many other aspects of cell biology, from cell signalling to developmental regulation, is becoming increasingly apparent. In addition to this diversity of function the core structure of the flagellum, which has been inherited from the earliest ancestor of all eukaryotes, is embellished with a range of extra-axonemal structures in many organisms. One of the best studied of these structures is the paraflagellar rod of kinetoplastid protozoa in which the morphological characteristics have been well defined and some of the major protein constituents have been identified. Here we discuss recent advances in the identification of further molecular components of the paraflagellar rod, how these impact on our understanding of its function and regulation and the implications for therapeutic intervention in a number of devastating human pathologies.

Three inhibitors of CDK4/6 kinases were recently FDA approved for use in combination with endocrine therapy, and they significantly increase the progression-free survival of patients with advanced estrogen receptor-positive (ER+) breast cancer in the first-line treatment setting. As the new standard of care in some countries, there is the clinical emergence of patients with breast cancer that is both CDK4/6 inhibitor and endocrine therapy resistant. The strategies to combat these cancers with resistance to multiple treatments are not yet defined and represent the next major clinical challenge in ER+ breast cancer. In this review, we discuss how the molecular landscape of endocrine therapy resistance may affect the response to CDK4/6 inhibitors, and how this intersects with biomarkers of intrinsic insensitivity. We identify the handful of pre-clinical models of acquired resistance to CDK4/6 inhibitors and discuss whether the molecular changes in these models are likely to be relevant or modified in the context of endocrine therapy resistance. Finally, we consider the crucial question of how some of these changes are potentially amenable to therapy.