Gareth W. Morgan

The kinetoplastida endocytic apparatus. Part I: a dynamic system for nutrition and evasion of host defences

The endocytic system of kinetoplastid parasites is a highly polarized membrane network focused on the flagellar pocket localized at one end of the cell. When first characterized, the endosomal network was envisioned as a simple system for uptake of extracellular material by fluid-phase or receptor-mediated mechanisms. Subsequently, it has become clear that the kinetoplastid endosomal system has an active and vital role in avoiding the host immune system and virulence, as well as providing the basic functions to fulfil cellular nutritional requirements. In two reviews, recent advances in the definition and comprehension of kinetoplastida endocytosis are discussed and, in Trypanosoma brucei in particular as the more developed experimental system. In Part 1, the endocytic system is considered in context of the surface molecules and their potential roles in virulence.

The endocytic apparatus of the kinetoplastida. Part II: machinery and components of the system.

Endocytic systems within eukaryotic cells are a diverse set of intracellular transport pathways responsible for uptake, recycling, interaction with the exocytic system and degradation of molecules. Each of these pathways requires the interaction of distinct protein components that function in macromolecule sorting, control of transport rates and in membrane biogenesis. In the second of two articles on kinetoplastida endocytosis, the endocytic system in Trypanosoma brucei is considered as a model, and the molecules that control this system and the protein components of the endocytic pathway are discussed. We also consider novel mechanisms for sorting that have been proposed to operate in trypanosomes.

New Approaches to the Microscopic Imaging of Trypanosoma brucei

Protozoan parasites are fearsome pathogens responsible for a substantial proportion of human mortality, morbidity, and economic hardship. The principal disease agents are members of the orders Apicomplexa (Plasmodium, Toxoplasma, Eimeria) and Kinetoplastida (Trypanosomes, Leishmania). The majority of humans are at risk from infection from one or more of these organisms, with profound effects on the economy, social structure and quality of life in endemic areas; Plasmodium itself accounts for over one million deaths per annum, and an estimated 4 x 10(7) disability-adjusted life years (DALYs), whereas the Kinetoplastida are responsible for over 100,000 deaths per annum and 4 x 10(6) DALYs. Current control strategies are failing due to drug resistance and inadequate implementation of existing public health strategies. Trypanosoma brucei, the African Trypanosome, has emerged as a favored model system for the study of basic cell biology in Kinetoplastida, because of several recent technical advances (transfection, inducible expression systems, and RNA interference), and these advantages, together with genome sequencing efforts are widely anticipated to provide new strategies of therapeutic intervention. Here we describe a suite of methods that have been developed for the microscopic analysis of T. brucei at the light and ultrastructural levels, an essential component of analysis of gene function and hence identification of therapeutic targets.

Vaccinia Protein F12 Has Structural Similarity to Kinesin Light Chain and Contains a Motor Binding Motif Required for Virion Export

Vaccinia virus (VACV) uses microtubules for export of virions to the cell surface and this process requires the viral protein F12. Here we show that F12 has structural similarity to kinesin light chain (KLC), a subunit of the kinesin-1 motor that binds cargo. F12 and KLC share similar size, pI, hydropathy and cargo-binding tetratricopeptide repeats (TPRs). Moreover, molecular modeling of F12 TPRs upon the crystal structure of KLC2 TPRs showed a striking conservation of structure. We also identified multiple TPRs in VACV proteins E2 and A36. Data presented demonstrate that F12 is critical for recruitment of kinesin-1 to virions and that a conserved tryptophan and aspartic acid (WD) motif, which is conserved in the kinesin-1-binding sequence (KBS) of the neuronal protein calsyntenin/alcadein and several other cellular kinesin-1 binding proteins, is essential for kinesin-1 recruitment and virion transport. In contrast, mutation of WD motifs in protein A36 revealed they were not required for kinesin-1 recruitment or IEV transport. This report of a viral KLC-like protein containing a KBS that is conserved in several cellular proteins advances our understanding of how VACV recruits the kinesin motor to virions, and exemplifies how viruses use molecular mimicry of cellular components to their advantage.

An Evolutionarily Conserved Coiled-Coil Protein Implicated in Polycystic Kidney Disease Is Involved in Basal Body Duplication and Flagellar Biogenesis in Trypanosoma brucei†

ABSTRACT Trypanosoma brucei is a flagellated protozoan with a highly polarized cellular structure. TbLRTP is a trypanosomal protein containing multiple SDS22-class leucine-rich repeats and a coiled-coil domain with high similarity to a mammalian testis-specific protein of unknown function. Homologues are present in a wide range of higher eukaryotes including zebra fish, where the gene product has been implicated in polycystic kidney disease. Western blot analysis and immunofluorescence with antibodies against recombinant TbLRTP indicate that the protein is expressed throughout the trypanosome life cycle and localizes to distal zones of the basal bodies. Overexpression and RNA interference demonstrate that TbLRTP is important for faithful basal body duplication and flagellum biogenesis. Expression of excess TbLRTP suppresses new flagellum assembly, while reduction of TbLRTP protein levels often results in the biogenesis of additional flagellar axonemes and paraflagellar rods that, most remarkably, are intracellular and fully contained within the cytoplasm. The mutant flagella are devoid of membrane and are often associated with four microtubules in an arrangement similar to that observed in the normal flagellar attachment zone. Aberrant basal body and flagellar biogenesis in TbLRTP mutants also influences cell size and cytokinesis. These findings demonstrate that TbLRTP suppresses basal body replication and subsequent flagellar biogenesis and indicate a critical role for the LRTP family of proteins in the control of the cell cycle. These data further underscore the role of aberrant flagellar biogenesis as a disease mechanism.

TbRAB18, a developmentally regulated Golgi GTPase from Trypanosoma brucei.

The trypanosomal secretory system is broadly similar to that of higher eukaryotes as proteins enter the system via the endoplasmic reticulum and are transported to the Golgi complex for elaboration of glycan chains. Importantly N-glycan processing is stage specific with only the bloodstream form (BSF) processing beyond the oligomannose form. Increased complexity of the BSF Golgi apparatus, as evidenced by morphological studies, may underpin this higher activity, but few trypanosome-specific Golgi proteins have been described that may play a role in this developmental alteration. Here we describe a novel member of the T. brucei Rab family, TbRAB18, which is stage-regulated and highly expressed in the BSF whilst barely detectable in the insect stage. This stage-specific expression suggests the presence of a TbRAB18-dependent transport pathway required for survival in the mammalian host. Furthermore, data indicate that TbRAB18 localises to membranes in close juxtaposition to structures stained with BODIPY-ceramide, a Golgi marker. Wild type TbRAB18, ectopically expressed in insect stage cells colocalises with TbRAB31, and hence is targeted to the Golgi complex, consistent with the location of the endogenous protein in the bloodstream form, whilst GTP and GDP-locked mutant isoforms demonstrate distinct localisations, suggesting that Golgi-targetting of TbRAB18 is nucleotide-state dependent. We also find that ectopic expression of TbRAB18 wild type and mutant isoforms has no detectable effect on the synthetic anteriograde trafficking probe, TbBiPN. Finally, the location, and hence function, of TbRAB18 are distinct from the closest metazoan homologue, murine Rab18; the latter protein is involved in endocytic transport pathways whilst clearly TbRAB18 is not. Our data indicate further complexity in the evolution of small GTPases, and highlight the need for robust functional data prior to assignment of members of complex gene families.

Vaccinia virus protein complex F12/E2 interacts with kinesin light chain isoform 2 to engage the kinesin-1 motor complex.

During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface. Deletion of the F12L or E2L genes causes a severe inhibition of IEV transport and a tiny plaque size. Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress. The A36 protein is present in the outer membrane of IEVs, and over-expressed fragments of this protein interact with kinesin light chain (KLC). However, no interaction of F12 or E2 with the kinesin complex has been reported hitherto. Here the F12/E2 complex is shown to associate with kinesin-1 through an interaction of E2 with the C-terminal tail of KLC isoform 2, which varies considerably between different KLC isoforms. siRNA-mediated knockdown of KLC isoform 1 increased IEV transport to the cell surface and virus plaque size, suggesting interaction with KLC isoform 1 is somehow inhibitory of IEV transport. In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways. Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously. These observations suggest redundancy in the mechanisms used for IEV egress, with involvement of KLC isoforms 1 and 2, and provide evidence of interaction of F12/E2 complex with the kinesin-1 complex.

High quality DEM generation from PCIAS

This paper presents an efficient Phase Correlation based Image Analysis System (PCIAS) for high quality DEM generation. A multi-resolution phase correlation based disparity estimation and refinement algorithm has been implemented in PCIAS. It can easily cope with the precise disparity estimation from sub-pixel to very large disparity range with varying baseline/distance ratio in vertical or slightly oblique view stereo imaging. The PCIAS is now a fully operational, professional C++ software package equipped with a robust phase correlation engine, which is among the most advanced technology for sub-pixel image feature shift analysis, and is able to achieve <;1/50th pixel accuracy in dense disparity map estimation. Our experiment indicates PCIAS can generate high quality DEM from very narrow baseline satellite image pairs with view angle difference as small as just 1 degree.

PCIAS Subpixel Technology

PCIAS represents Phase Correlation based Image Analysis System. It is a professional C++ software package to deliver advanced subpixel technology that has been developed during the last several years supported by the SEAS DTC (System Engineering for Autonomous Systems Defence Technology Centre). This paper provides and overview of the PCIAS. After a brief introduction to the principles and algorithms, the paper illustrates, with examples, the major applications based on high precision sub-pixel disparity estimation.

Achievements and Experiences from a Grid-Based Earthquake Analysis and Modelling Study

We have developed and used a grid-based geoinformatics infrastructure and analytical methods for investigating the relationship between macro and microscale earthquake deformational processes by linking geographically distributed and computationally intensive earthquake monitoring and modelling tools. Using this infrastructure, measurement of lateral co-seismic deformation is carried out with imageodesy algorithms running on servers at the London eScience Centre. The resultant deformation field is used to initialise geomechanical simulations of the earthquake deformation running on supercomputers based at the University of Oklahoma. This paper describes the details of our work, summarizes our scientific results and details our experiences from implementing and testing the distributed infrastructure and analysis workflow.