Sue Vaughan

Molecular Evolution of FtsZ Protein Sequences Encoded Within the Genomes of Archaea, Bacteria, and Eukaryota

The FtsZ protein is a polymer-forming GTPase which drives bacterial cell division and is structurally and functionally related to eukaryotic tubulins. We have searched for FtsZ-related sequences in all freely accessible databases, then used strict criteria based on the tertiary structure of FtsZ and its well-characterized invitro and invivo properties to determine which sequences represent genuine homologues of FtsZ. We have identified 225 full-length FtsZ homologues, which we have used to document, phylum by phylum, the primary sequence characteristics of FtsZ homologues from the Bacteria, Archaea, and Eukaryota. We provide evidence for at least five independent ftsZ gene-duplication events in the bacterial kingdom and suggest the existence of three ancestoral euryarchaeal FtsZ paralogues. In addition, we identify “FtsZ-like” sequences from Bacteria and Archaea that, while showing significant sequence similarity to FtsZs, are unlikely to bind and hydrolyze GTP.

γ-Tubulin functions in the nucleation of a discrete subset of microtubules in the eukaryotic flagellum

gamma-tubulin is an essential part of a multiprotein complex that nucleates the minus end of microtubules. Although the function of gamma-tubulin in nucleating cytoplasmic and mitotic microtubules from organizing centers such as the centrosome and spindle pole body is well documented, its role in microtubule nucleation in the eukaryotic flagellum is unclear. Here, we have used Trypanosoma brucei to investigate possible functions of gamma-tubulin in the formation of the 9 + 2 flagellum axoneme. T. brucei possesses a single flagellum and forms a new flagellum during each cell cycle. We have used an inducible RNA interference (RNAi) approach to ablate expression of gamma-tubulin, and, after induction, we observe that the new flagellum is still formed but is paralyzed, while the old flagellum is unaffected. Electron microscopy reveals that the paralyzed flagellum lacks central pair microtubules but that the outer doublet microtubules are formed correctly. These differences in microtubule nucleation mechanisms during flagellum growth provide insights into spatial and temporal regulation of gamma-tubulin-dependent processes within cells and explanations for the organization and evolution of axonemal structures such as the 9 + 0 axonemes of sensory cells and primary cilia.

The flagella connector of Trypanosoma brucei: an unusual mobile transmembrane junction.

Throughout its elongation, the new flagellum of the procyclic form of the African trypanosome Trypanosoma brucei is tethered at its tip to the lateral aspect of the old flagellum. This phenomenon provides a cytotactic mechanism for influencing inheritance of cellular pattern. Here, we show that this tethering is produced via a discrete, mobile transmembrane junction – the flagella connector. Light and electron microscopy reveal that the flagella connector links the extending microtubules at the tip of the new flagellum to the lateral aspect of three of the doublet microtubules in the old flagellar axoneme. Two sets of filaments connect the microtubules to three plates on the inner faces of the old and new flagellar membranes. Three differentiated areas of old and new flagellar membranes are then juxtaposed and connected by a central interstitial core of electron-dense material. The flagella connector is formed early in flagellum extension and is removed at the end of cytokinesis, but the exact timing of the latter event is slightly variable. The flagella connector represents a novel form of cellular junction that is both dynamic and mobile.

An Essential Quality Control Mechanism at the Eukaryotic Basal Body Prior to Intraflagellar Transport

Constructing a eukaryotic cilium/flagellum is a demanding task requiring the transport of proteins from their cytoplasmic synthesis site into a spatially and environmentally distinct cellular compartment. The clear potential hazard is that import of aberrant proteins could seriously disable cilia/flagella assembly or turnover processes. Here, we reveal that tubulin protein destined for incorporation into axonemal microtubules interacts with a tubulin cofactor C (TBCC) domain‐containing protein that is specifically located at the mature basal body transitional fibres. RNA interference‐mediated ablation of this protein results in axonemal microtubule defects but no effect on other microtubule populations within the cell. Bioinformatics analysis indicates that this protein belongs to a clade of flagellum‐specific TBCC‐like proteins that includes the human protein, XRP2, mutations which lead to certain forms of the hereditary eye disease retinitis pigmentosa. Taken with other observations regarding the role of transitional fibres in cilium/flagellum assembly, we suggest that a localized protein processing capacity embedded at transitional fibres ensures the ‘quality’ of tubulin imported into the cilium/flagellum, and further, that loss of a ciliary/flagellar quality control capability may underpin a number of human genetic disorders.

Basal body movements orchestrate membrane organelle division and cell morphogenesis in Trypanosoma brucei

The defined shape and single-copy organelles of Trypanosoma brucei mean that it provides an excellent model in which to study how duplication and segregation of organelles is interfaced with morphogenesis of overall cell shape and form. The centriole or basal body of eukaryotic cells is often seen to be at the centre of such processes. We have used a combination of electron microscopy and electron tomography techniques to provide a detailed three-dimensional view of duplication of the basal body in trypanosomes. We show that the basal body duplication and maturation cycle exerts an influence on the intimately associated flagellar pocket membrane system that is the portal for secretion and uptake from this cell. At the start of the cell cycle, a probasal body is positioned anterior to the basal body of the existing flagellum. At the G1–S transition, the probasal body matures, elongates and invades the pre-existing flagellar pocket to form the new flagellar axoneme. The new basal body undergoes a spectacular anti-clockwise rotation around the old flagellum, while its short new axoneme is associated with the pre-existing flagellar pocket. This rotation and subsequent posterior movements results in division of the flagellar pocket and ultimately sets parameters for subsequent daughter cell morphogenesis.

Serial block face scanning electron microscopy—the future of cell ultrastructure imaging

One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

Assembly of the flagellum and its role in cell morphogenesis in Trypanosoma brucei

Eukaryotic flagella are microtubule-based structures required for a variety of functions including cell motility and sensory perception. Most eukaryotic flagella grow out from a cell into the surrounding medium, but when the flagellum of the protozoan parasite Trypanosoma brucei exits the cell via the flagellar pocket, it is attached along the length of the cell body by a cytoskeletal structure called the flagellum attachment zone (FAZ). The exact reasons for flagellum attachment have remained elusive, but evidence is emerging that the attached flagellum plays a major role in cell morphogenesis in this organism. In this review we discuss evidence published in the past four years that is unravelling the role of the flagellum in organelle segregation, inheritance of cell shape and cytokinesis.

Cytokinesis in Trypanosoma brucei differs between bloodstream and tsetse trypomastigote forms: implications for microtubule-based morphogenesis and mutant analysis.

Trypanosomes use a microtubule‐focused mechanism for cell morphogenesis and cytokinesis. We used scanning electron and video microscopy of living cells to provide the first detailed description of cell morphogenesis and cytokinesis in the early‐branching eukaryote Trypanosoma brucei. We outline four distinct stages of cytokinesis and show that an asymmetric division fold bisects the two daughter cells, with a cytoplasmic bridge‐like structure connecting the two daughters immediately prior to abscission. Using detection of tyrosinated α‐tubulin as a marker for new or growing microtubules and expression of XMAP215, a plus end binding protein, as a marker for microtubule plus ends we demonstrate spatial asymmetry in the underlying microtubule cytoskeleton throughout the cell division cycle. This leads to inheritance of different microtubule cytoskeletal patterns and demonstrates the major role of microtubules in achieving cytokinesis. RNA interference techniques have led to a large set of mutants, often with variations in phenotype between procyclic and bloodstream life cycle forms. Here, we show morphogenetic differences between these two life cycle forms of this parasite during new flagellum growth and cytokinesis. These discoveries are important tools to explain differences between bloodstream and procyclic form RNAi phenotypes involving organelle mis‐positioning during cell division and cytokinesis defects.

Assessing mechanisms of GPIHBP1 and lipoprotein lipase movement across endothelial cells

Lipoprotein lipase (LPL) is secreted into the interstitial spaces by adipocytes and myocytes but then must be transported to the capillary lumen by GPIHBP1, a glycosylphosphatidylinositol-anchored protein of capillary endothelial cells. The mechanism by which GPIHBP1 and LPL move across endothelial cells remains unclear. We asked whether the transport of GPIHBP1 and LPL across endothelial cells was uni- or bidirectional. We also asked whether GPIHBP1 and LPL are transported across cells in vesicles and whether this transport process requires caveolin-1. The movement of GPIHBP1 and LPL across cultured endothelial cells was bidirectional. Also, GPIHBP1 moved bidirectionally across capillary endothelial cells in live mice. The transport of LPL across endothelial cells was inhibited by dynasore and genistein, consistent with a vesicular transport process. Also, transmission electron microscopy (EM) and dual-axis EM tomography revealed GPIHBP1 and LPL in invaginations of the plasma membrane and in vesicles. The movement of GPIHBP1 across capillary endothelial cells was efficient in the absence of caveolin-1, and there was no defect in the internalization of LPL by caveolin-1-deficient endothelial cells in culture. Our studies show that GPIHBP1 and LPL move bidirectionally across endothelial cells in vesicles and that transport is efficient even when caveolin-1 is absent.

Cell morphogenesis of Trypanosoma brucei requires the paralogous, differentially expressed calpain-related proteins CAP5.5 and CAP5.5V.

Proteins from the calpain super-family are involved in developmentally- and environmentally-regulated re-modelling of the eukaryotic cytoskeleton and the dynamic organisation of signal transduction cascades. In trypanosomatid parasites, calpain-related gene families are unusually large, but we have little insight into the functional roles played by these molecules during trypanosomatid lifecycles. Here we report that CAP5.5, a cytoskeletal calpain-related protein subject to strict stage-specific expression in the sleeping sickness parasite Trypanosoma brucei, is essential and required for correct cell morphogenesis of procyclic (tsetse mid-gut stage) T. brucei. Striking consequences of CAP5.5 RNA interference are the loss of protein from the posterior cell-end, organelle mis-positioning giving rise to aberrant cytokinesis, and disorganisation of the sub-pellicular microtubules that define trypanosome cell shape. We further report that the stage-specificity of CAP5.5 expression can be explained by the presence of a paralogue, CAP5.5V, which is required for cell morphogenesis in bloodstream T. brucei; RNAi against this paralogous protein results in a qualitatively similar phenotype to that described for procyclic CAP5.5 RNAi mutants. By comparison to recently described phenotypes for other procyclic trypanosome RNAi mutants, likely functions for CAP5.5 and CAP5.5V are discussed.