The paraflagellar rod of kinetoplastid parasites: from structure to components and function.
Bottom Line: 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.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.
Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford OX13RE, UK.Show MeSH
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Mentions: Our increasing knowledge of the molecular complexity of the PFR suggests a role for this structure in a variety of functions within the flagellum. A fuller understanding of these roles will require an expansion of our knowledge of the protein components contributing to these functions. To this end we expanded on our earlier proteomic analysis (Pullen et al., 2004) of the snl2 mutant cell line and used cutting edge comparative proteomics techniques (difference gel electrophoresis – DIGE (Fig. 2); and isobaric tags for relative and absolute quantitation – iTRAQ) to produce a PFR proteome consisting of 30 proteins (Portman et al., 2009). The proteins in this cohort include PFR1, PFR2, PAR1, PFR5, ADKA, ADKB, Tb5.20 and calmodulin which have all previously been proposed as PFR components. Interestingly, several other components discussed above, including some with strong evidence for a PFR localisation, were not identified in this study. There are many likely reasons for the absence of these components from the PFR2-dependent cohort: low abundance, presence in the inner sub-domain of the PFR that is still assembled in the absence of PFR2, or protein/peptide characteristics that effect detection in gel-based and/or mass spectrometric analyses. In addition to the previously identified components, 20 proteins in the PFR2-dependent cohort (paraflagellar rod proteome components – PFCs) were proteins of unknown function that have not previously been associated with the PFR. Amongst these were further examples of potential structural, regulatory and calcium interacting proteins as well as several for which a predicted function remains elusive. By following the logic loop and repeating our RNAi/comparative proteomic analysis with newly identified PFR components, we were able to show a number of dependency relationships between proteins within the PFR2-dependent cohort (Portman et al., 2009; Lacomble et al., 2009). One of these dependency networks involved the two novel PFR proteins PFC1 and PFC15 and the previously identified PFR adenylate kinases TbADKA and TbADKB. RNAi ablation of either PFC1 or PFC15 resulted in a loss of both of these proteins from purified flagellar fractions as well as decreases in the volume of spots corresponding to TbADKA and TbADKB. Most intriguingly, our bioinformatic analyses of PFC1 and PFC15 predicted that both contain Pfam domains associated with calcium signalling and sensing. PFC1 had a weakly predicted EF-Hand calcium binding domain and PFC15 was predicted to contain an IQ-calmodulin binding domain. This potential association of calcium signalling and adenine nucleotide homeostasis provides tantalising insights into the regulatory and functional networks operating within the PFR and implicates this structure in the wider regulation of flagellar function.
Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford OX13RE, UK.