Modulation of flagellum attachment zone protein FLAM3 and regulation of the cell shape in Trypanosoma brucei life cycle transitions.

The cell shape of Trypanosoma brucei is influenced by flagellum-to-cell-body attachment through a specialised structure - the flagellum attachment zone (FAZ). T. brucei exhibits numerous morphological forms during its life cycle and, at each stage, the FAZ length varies. We have analysed FLAM3, a large protein that localises to the FAZ region within the old and new flagellum. Ablation of FLAM3 expression causes a reduction in FAZ length; however, this has remarkably different consequences in the tsetse procyclic form versus the mammalian bloodstream form. In procyclic form cells FLAM3 RNAi results in the transition to an epimastigote-like shape, whereas in bloodstream form cells a severe cytokinesis defect associated with flagellum detachment is observed. Moreover, we demonstrate that the amount of FLAM3 and its localisation is dependent on ClpGM6 expression and vice versa. This evidence demonstrates that FAZ is a key regulator of trypanosome shape, with experimental perturbations being life cycle form dependent. An evolutionary cell biology explanation suggests that these differences are a reflection of the division process, the cytoskeleton and intrinsic structural plasticity of particular life cycle forms.

Platinum trimethyl bipyridyl thiolates--new, tunable, red- to near IR emitting luminophores for bioimaging applications.

Synthetic, spectroscopic, computational and biological imaging studies of platinum trimethyl bipyridyl thiolate complexes of the general formula [PtMe3(bpy)SR] reveal these to be easily accessed, tunable bioimaging agents which feature an unusual σ-π* Inter-Ligand Charge Transfer (ILCT) transition, and in some cases emit into the Near infra-red (NIR).

An alternative model for the role of RP2 protein in flagellum assembly in the African trypanosome.

The tubulin cofactor C domain-containing protein TbRP2 is a basal body (centriolar) protein essential for axoneme formation in the flagellate protist Trypanosoma brucei, the causal agent of African sleeping sickness. Here, we show how TbRP2 is targeted and tethered at mature basal bodies and provide novel insight into TbRP2 function. Regarding targeting, understanding how several hundred proteins combine to build a microtubule axoneme is a fundamental challenge in eukaryotic cell biology. We show that basal body localization of TbRP2 is mediated by twinned, N-terminal TOF (TON1, OFD1, and FOP) and LisH motifs, motifs that otherwise facilitate localization of only a few conserved proteins at microtubule-organizing centers in animals, plants, and flagellate protists. Regarding TbRP2 function, there is a debate as to whether the flagellar assembly function of specialized, centriolar tubulin cofactor C domain-containing proteins is processing tubulin, the major component of axonemes, or general vesicular trafficking in a flagellum assembly context. Here we report that TbRP2 is required for the recruitment of T. brucei orthologs of MKS1 and MKS6, proteins that, in animal cells, are part of a complex that assembles at the base of the flagellum to regulate protein composition and cilium function. We also identify that TbRP2 is detected by YL1/2, an antibody classically used to detect α-tubulin. Together, these data suggest a general processing role for TbRP2 in trypanosome flagellum assembly and challenge the notion that TbRP2 functions solely in assessing tubulin "quality" prior to tubulin incorporation into the elongating axoneme.

The tubulin cofactor C family member TBCCD1 orchestrates cytoskeletal filament formation.

TBCCD1 is an enigmatic member of the tubulin-binding cofactor C (TBCC) family of proteins required for mother-daughter centriole linkage in the green alga Chlamydomonas reinhardtii and nucleus-centrosome-Golgi linkage in mammalian cells. Loss of these linkages has severe morphogenetic consequences, but the mechanism(s) through which TBCCD1 contributes to cell organisation is unknown. In the African sleeping sickness parasite Trypanosoma brucei a microtubule-dominant cytoskeleton dictates cell shape, influencing strongly the positioning and inheritance patterns of key intracellular organelles. Here, we show the trypanosome orthologue of TBCCD1 is found at multiple locations: centrioles, the centriole-associated Golgi 'bi-lobe', and the anterior end of the cell body. Loss of Trypanosoma brucei TBCCD1 results in disorganisation of the structurally complex bi-lobe architecture and loss of centriole linkage to the single unit-copy mitochondrial genome (or kinetoplast) of the parasite. We therefore identify TBCCD1 as an essential protein associated with at least two filament-based structures in the trypanosome cytoskeleton. The last common ancestor of trypanosomes, animals and green algae was arguably the last common ancestor of all eukaryotes. On the basis of our observations, and interpretation of published data, we argue for an unexpected co-option of the TBCC domain for an essential non-tubulin-related function at an early point during evolution of the eukaryotic cytoskeleton.

Towards an integrative soil health assessment strategy: a three tier (integrative biomarker response) approach with Eisenia fetida applied to soils subjected to chronic metal pollution.

This is a pilot study for assessing soil ecosystem health in chronically polluted sites on the basis of a 3-tier approach (screening+scoring+understanding) designed to be cost-effective and scientifically based, and to provide straightforward advice and support to managers and stakeholders involved in environmental protection. For the initial screening (Tier 1), the use of a highly sensitive, low-cost biomarker such as neutral red uptake (NRU) in earthworm coelomocytes is proposed. In sites where an alteration in NRU has been established, the stress level may be further assessed by utilising a suite of low-cost and rapid biomarkers of effect integrated in an integrative biological response (IBR) index to obtain an objective (scored) assessment of the induced stress syndrome (Tier 2). The IBR/n index is based on the integration of biomarkers at different levels of biological organisation. Acyl-CoA oxidase activity (AOX), catalase activity (CAT), lipofuscin optical density (LOD%), NRU and the mean epithelial thickness (MET) have been used to calculate the IBR/n index. Biomarkers are determined in earthworms, Eisenia fetida, exposed ex situ to real soils (three mining sites and a reference) for 3, 10 and 17d. The 3d NRU (Tier 1) provided signal of stress. After 3d, PCA, based on the suite of biomarkers (Tier 2), discriminated reference and polluted sites according to toxicity profiles and at 17d, the most polluted site is segregated from less polluted and reference sites. Soils were classified as harmful, unhealthy (not apparently toxic) or healthy. Soils were investigated by microarray transcriptomics (Tier 3), to understand the causes (aetiology) and consequences (prognosis) of health impairment. Tier 3 discriminates, according to stress syndrome traits, soils that did not fall into the category of highly stressed and revealed the main agent causing toxicity at each site by identifying the toxicity mechanisms and biological responses.

Accumulated metal speciation in earthworm populations with multigenerational exposure to metalliferous soils: cell fractionation and high-energy synchrotron analyses.

Predicting metal bioaccumulation and toxicity in soil organisms is complicated by site-specific biotic and abiotic parameters. In this study we exploited tissue fractionation and digestion techniques, combined with X-ray absorption spectroscopy (XAS), to investigate the whole-body and subcellular distributions, ligand affinities, and coordination chemistry of accumulated Pb and Zn in field populations of the epigeic earthworm Lumbricus rubellus inhabiting three contrasting metalliferous and two unpolluted soils. Our main findings were (i) earthworms were resident in soils with concentrations of Pb and Zn ranging from 1200 to 27,000 mg kg(-1) and 200 to 34,000 mg kg(-1), respectively; (ii) Pb and Zn primarily accumulated in the posterior alimentary canal in nonsoluble subcellular fractions of earthworms; (iii) site-specific differences in the tissue and subcellular partitioning profiles of populations were observed, with earthworms from a calcareous site partitioning proportionally more Pb to their anterior body segments and Zn to the chloragosome-rich subcellular fraction than their acidic-soil inhabiting counterparts; (iv) XAS indicated that the interpopulation differences in metal partitioning between organs were not accompanied by qualitative differences in ligand-binding speciation, because crystalline phosphate-containing pyromorphite was a predominant chemical species in the whole-worm tissues of all mine soil residents. Differences in metal (Pb, Zn) partitioning at both organ and cellular levels displayed by field populations with protracted histories of metal exposures may reflect theirinnate ecophysiological responses to essential edaphic variables, such as Ca2+ status. These observations are highly significant in the challenging exercise of interpreting holistic biomarker data delivered by "omic" technologies.

Riboflavin content of coelomocytes in earthworm (Dendrodrilus rubidus) field populations as a molecular biomarker of soil metal pollution.

The effect of Pb + Zn on coelomocyte riboflavin content in the epigeic earthworm Dendrodrilus rubidus inhabiting three metalliferous soils and one reference soil was measured by flow cytometry and spectrofluorimetry. A reciprocal polluted<-->unpolluted worm transfer experiment (4-week exposure) was also performed. High proportions of autofluorescent eleocytes were counted in worms from all localities, but intense riboflavin-derived autofluorescence was detectable only in reference worm eleocytes. Other findings were: (i) fluorophore(s) other than riboflavin is/are responsible for eleocyte autofluorescence in residents of metalliferous soils; (ii) riboflavin content was reduced in the eleocytes of worms transferred from unpolluted to metal-polluted soil; (iii) the riboflavin content of D. rubidus eleocytes is a promising biomarker of exposure; (iv) COII mitochondrial genotyping revealed that the reference population is genetically distinct from the three mine populations; (v) metal exposure rather than genotype is probably the main determinant of inter-population differences in eleocyte riboflavin status.