scholarly journals Structure of the trypanosome paraflagellar rod and insights into non-planar motility of eukaryotic cells

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Jiayan Zhang ◽  
Hui Wang ◽  
Simon Imhof ◽  
Xueting Zhou ◽  
Shiqing Liao ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Disease Transmission ◽  
Electron Tomography ◽  
Signal Transmission ◽  
Protozoan Parasite ◽  
Empty Space ◽  
Eukaryotic Cells ◽  
Critical Gap ◽  
Proximal Zone ◽  
Paracrystalline Array

AbstractEukaryotic flagella (synonymous with cilia) rely on a microtubule-based axoneme, together with accessory filaments to carryout motility and signaling functions. While axoneme structures are well characterized, 3D ultrastructure of accessory filaments and their axoneme interface are mostly unknown, presenting a critical gap in understanding structural foundations of eukaryotic flagella. In the flagellum of the protozoan parasite Trypanosoma brucei (T. brucei), the axoneme is accompanied by a paraflagellar rod (PFR) that supports non-planar motility and signaling necessary for disease transmission and pathogenesis. Here, we employed cryogenic electron tomography (cryoET) with sub-tomographic averaging, to obtain structures of the PFR, PFR-axoneme connectors (PACs), and the axonemal central pair complex (CPC). The structures resolve how the 8 nm repeat of the axonemal tubulin dimer interfaces with the 54 nm repeat of the PFR, which consist of proximal, intermediate, and distal zones. In the distal zone, stacked “density scissors” connect with one another to form a “scissors stack network (SSN)” plane oriented 45° to the axoneme axis; and ~370 parallel SSN planes are connected by helix-rich wires into a paracrystalline array with ~90% empty space. Connections from these wires to the intermediate zone, then to overlapping layers of the proximal zone and to the PACs, and ultimately to the CPC, point to a contiguous pathway for signal transmission. Together, our findings provide insights into flagellum-driven, non-planar helical motility of T. brucei and have broad implications ranging from cell motility and tensegrity in biology, to engineering principles in bionics.

scholarly journals Nuclear envelope budding is an evolutionary conserved phenomenon

2018 ◽  
Author(s):  
Dimitra Panagaki ◽  
Richard Neutze ◽  
Johanna L. Höög
Keyword(s):  
Nuclear Envelope ◽  
Trypanosoma Brucei ◽  
Electron Tomography ◽  
Human Cell Line ◽  
Eukaryotic Cells ◽  
Nuclear Pores ◽  
Dna Transport ◽  
Parasitic Protist ◽  
3D Architecture ◽  
Cellular Dna

AbstractEukaryotic cells are defined by the compartmentalization of the cytoplasm into organelles, the largest of which is the nucleus, which contains the cellular DNA. Transport into and out of the nucleus is highly regulated and is traditionally thought to occur solely through nuclear pores. However, a small number of papers has repeatedly shown vesicular budding from the nuclear envelopes in different organisms. We used electron microscopy to identify such nuclear envelope budding events in a human cell line,Caenorhabditis elegansworms, the two yeastsSaccharomyces cerevisiaeandSchizosaccharomyces pombeand the parasitic protistTrypanosoma brucei. Progressing to electron tomography, the finer details of the 3D architecture of such budding events was revealed. We summarize all the organisms in which this mode of translocation over the nuclear envelope has been observed and conclude that this may be a fundamental, evolutionary conserved mechanism of transport inside eukaryotic cells.

scholarly journals APEX2 proximity proteomics resolves flagellum subdomains and identifies flagellum tip-specific proteins in Trypanosoma brucei

2020 ◽  
Author(s):  
Daniel E. Vélez-Ramírez ◽  
Michelle M. Shimogawa ◽  
Sunayan Ray ◽  
Andrew Lopez ◽  
Shima Rayatpisheh ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Trypanosoma Brucei ◽  
Model Organism ◽  
Protein Composition ◽  
Principal Component ◽  
Protozoan Parasite ◽  
Sleeping Sickness ◽  
Critical Gap ◽  
Flagellar Membrane ◽  
And Function

ABSTRACTTrypanosoma brucei is the protozoan parasite responsible for sleeping sickness, a lethal vector-borne disease. T. brucei has a single flagellum that plays critical roles in parasite biology, transmission and pathogenesis. An emerging concept in flagellum biology is that the organelle is organized into subdomains, each having specialized composition and function. Overall flagellum proteome has been well-studied, but a critical gap in knowledge is the protein composition of individual flagellum subdomains. We have therefore used APEX-based proximity proteomics to examine protein composition of T. brucei flagellum subdomains. To assess effectiveness of APEX-based proximity labeling, we fused APEX2 to the DRC1 subunit of the nexin-dynein regulatory complex, an axonemal complex distributed along the flagellum. We found that DRC1-APEX2 directs flagellum-specific biotinylation and purification of biotinylated proteins yields a DRC1 “proximity proteome” showing good overlap with proteomes obtained from purified axonemes. We next employed APEX2 fused to a flagellar membrane protein that is restricted to the flagellum tip, adenylate cyclase 1 (AC1), or a flagellar membrane protein that is excluded from the flagellum tip, FS179. Principal component analysis demonstrated the pools of biotinylated proteins in AC1-APEX2 and FS179-APEX2 samples are distinguished from each other. Comparing proteins in these two pools allowed us to identify an AC1 proximity proteome that is enriched for flagellum tip proteins and includes several proteins involved in signal transduction. Our combined results demonstrate that APEX2-based proximity proteomics is effective in T. brucei and can be used to resolve proteome composition of flagellum subdomains that cannot themselves be readily purified.IMPORTANCESleeping sickness is a neglected tropical disease, caused by the protozoan parasite Trypanosoma brucei. The disease disrupts the sleep-wake cycle, leading to coma and death if left untreated. T. brucei motility, transmission, and virulence depend on its flagellum (aka cilium), which consists of several different specialized subdomains. Given the essential and multifunctional role of the T. brucei flagellum, there is need of approaches that enable proteomic analysis of individual subdomains. Our work establishes that APEX2 proximity labeling can, indeed, be implemented in the biochemical environment of T. brucei, and has allowed identification of proximity proteomes for different subdomains. This capacity opens the possibility to study the composition and function of other compartments. We further expect that this approach may be extended to other eukaryotic pathogens, and will enhance the utility of T. brucei as a model organism to study ciliopathies, heritable human diseases in which cilia function is impaired.

scholarly journals Cryo electron tomography with volta phase plate reveals novel structural foundations of the 96-nm axonemal repeat in the pathogen Trypanosoma brucei

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Simon Imhof ◽  
Jiayan Zhang ◽  
Hui Wang ◽  
Khanh Huy Bui ◽  
Hoangkim Nguyen ◽  
...  
Keyword(s):  
Life Cycle ◽  
High Resolution ◽  
Trypanosoma Brucei ◽  
Electron Tomography ◽  
Protozoan Parasite ◽  
Economic Importance ◽  
Phase Plate ◽  
Life Cycle Stages ◽  
Cryo Electron Tomography ◽  
Regulatory Complex

The 96-nm axonemal repeat includes dynein motors and accessory structures as the foundation for motility of eukaryotic flagella and cilia. However, high-resolution 3D axoneme structures are unavailable for organisms among the Excavates, which include pathogens of medical and economic importance. Here we report cryo electron tomography structures of the 96-nm repeat from Trypanosoma brucei, a protozoan parasite in the Excavate lineage that causes African trypanosomiasis. We examined bloodstream and procyclic life cycle stages, and a knockdown lacking DRC11/CMF22 of the nexin dynein regulatory complex (NDRC). Sub-tomogram averaging yields a resolution of 21.8 Å for the 96-nm repeat. We discovered several lineage-specific structures, including novel inter-doublet linkages and microtubule inner proteins (MIPs). We establish that DRC11/CMF22 is required for the NDRC proximal lobe that binds the adjacent doublet microtubule. We propose that lineage-specific elaboration of axoneme structure in T. brucei reflects adaptations to support unique motility needs in diverse host environments.

scholarly journals Metabolic reprogramming during the Trypanosoma brucei life cycle

F1000Research ◽  
2017 ◽  
Vol 6 ◽  
pp. 683 ◽  
Author(s):  
Terry K. Smith ◽  
Frédéric Bringaud ◽  
Derek P. Nolan ◽  
Luisa M. Figueiredo
Keyword(s):  
Life Cycle ◽  
Trypanosoma Brucei ◽  
Model Organism ◽  
Protozoan Parasite ◽  
Tsetse Fly ◽  
Metabolic Reprogramming ◽  
Mammalian Host ◽  
Insect Vector ◽  
Environmental Signals ◽  
Cellular Metabolic Activity

Cellular metabolic activity is a highly complex, dynamic, regulated process that is influenced by numerous factors, including extracellular environmental signals, nutrient availability and the physiological and developmental status of the cell. The causative agent of sleeping sickness, Trypanosoma brucei, is an exclusively extracellular protozoan parasite that encounters very different extracellular environments during its life cycle within the mammalian host and tsetse fly insect vector. In order to meet these challenges, there are significant alterations in the major energetic and metabolic pathways of these highly adaptable parasites. This review highlights some of these metabolic changes in this early divergent eukaryotic model organism.

scholarly journals Polo-like kinase is required for Golgi and bilobe biogenesis in Trypanosoma brucei

2008 ◽  
Vol 181 (3) ◽  
pp. 431-438 ◽  
Author(s):  
Christopher L. de Graffenried ◽  
Helen H. Ho ◽  
Graham Warren
Keyword(s):  
Cell Cycle ◽  
Endoplasmic Reticulum ◽  
Trypanosoma Brucei ◽  
Protozoan Parasite ◽  
Controlled Assembly

A bilobed structure marked by TbCentrin2 regulates Golgi duplication in the protozoan parasite Trypanosoma brucei. This structure must itself duplicate during the cell cycle for Golgi inheritance to proceed normally. We show here that duplication of the bilobed structure is dependent on the single polo-like kinase (PLK) homologue in T. brucei (TbPLK). Depletion of TbPLK leads to malformed bilobed structures, which is consistent with an inhibition of duplication and an increase in the number of dispersed Golgi structures with associated endoplasmic reticulum exit sites. These data suggest that the bilobe may act as a scaffold for the controlled assembly of the duplicating Golgi.

scholarly journals Structure-based drug design against Trypanosoma brucei methionyl-tRNA synthetase

2014 ◽  
Vol 70 (a1) ◽  
pp. C708-C708
Author(s):  
Cho Yeow Koh ◽  
Jasmine Nguyen ◽  
Sayaka Shibata ◽  
Zhongsheng Zhang ◽  
Ranae Ranade ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Trypanosoma Brucei ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protozoan Parasite ◽  
Trna Synthetase ◽  
Structure Based Drug Design ◽  
Chemical Structures ◽  
Ic50 Values ◽  
Methionyl Trna Synthetase

Infection by the protozoan parasite Trypanosoma brucei causes human African trypanosomiasis, commonly known as sleeping sickness. The disease is fatal without treatment; yet, current therapeutic options for the disease are inadequate due to toxicity, difficulty in administration and emerging resistance. Therefore, methionyl-tRNA synthetase of T. brucei (TbMetRS) is targeted for the development of new antitrypanosomal drugs. We have recently completed a high-throughput screening campaign against TbMetRS using a 364,131 compounds library in The Scripps Research Institute Molecular Screening Center. Here we outline our strategy to integrate the power of crystal structures with high-throughput screening in a drug discovery project. We applied the rapid crystal soaking procedure to obtain structures of TbMetRS in complex with inhibitors reported earlier[1] to approximately 70 high-throughput screening hits. This resulted in more than 20 crystal structures of TbMetRS·hit complexes. These hits cover a large diversity of chemical structures with IC50 values between 200 nM and 10 µM. Based on the solved structures and existing knowledge drawn from other in-house inhibitors, the IC50 value of the most promising hit has been improved. Further development of the compounds into potent TbMetRS inhibitors with desirable pharmacokinetic properties is on-going and will continue to benefit from information derived from crystal structures.

The structural mechanics of cell division in Trypanosoma brucei

2008 ◽  
Vol 36 (3) ◽  
pp. 421-424 ◽  
Author(s):  
Sue Vaughan ◽  
Keith Gull
Keyword(s):  
Cell Division ◽  
Trypanosoma Brucei ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Single Copy ◽  
Structural Mechanics ◽  
Sub Saharan Africa ◽  
Mammalian Host

Undoubtedly, there are fundamental processes driving the structural mechanics of cell division in eukaryotic organisms that have been conserved throughout evolution and are being revealed by studies on organisms such as yeast and mammalian cells. Precision of structural mechanics of cytokinesis is however probably no better illustrated than in the protozoa. A dramatic example of this is the protozoan parasite Trypanosoma brucei, a unicellular flagellated parasite that causes a devastating disease (African sleeping sickness) across Sub-Saharan Africa in both man and animals. As trypanosomes migrate between and within a mammalian host and the tsetse vector, there are periods of cell proliferation and cell differentiation involving at least five morphologically distinct cell types. Much of the existing cytoskeleton remains intact during these processes, necessitating a very precise temporal and spatial duplication and segregation of the many single-copy organelles. This structural precision is aiding progress in understanding these processes as we apply the excellent reverse genetics and post-genomic technologies available in this system. Here we outline our current understanding of some of the structural aspects of cell division in this fascinating organism.

Human African trypanosomiasis

2010 ◽  
pp. 1119-1127
Author(s):  
August Stich
Keyword(s):  
West Africa ◽  
East Africa ◽  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Protozoan Parasite ◽  
Sleeping Sickness ◽  
Tsetse Flies ◽  
African Trypanosomiasis ◽  
Tropical Africa

Human African trypanosomiasis (HAT, sleeping sickness) is caused by two subspecies of the protozoan parasite Trypanosoma brucei: T. b. rhodesiense is prevalent in East Africa among many wild and domestic mammals; T. b. gambiense causes an anthroponosis in Central and West Africa. The disease is restricted to tropical Africa where it is transmitted by the bite of infected tsetse flies (...

Human African trypanosomiasis

2020 ◽  
pp. 1451-1459
Author(s):  
Reto Brun ◽  
Johannes Blum
Keyword(s):  
Trypanosoma Brucei ◽  
Human African Trypanosomiasis ◽  
Specific Treatment ◽  
Protozoan Parasite ◽  
Control Measures ◽  
Tsetse Flies ◽  
African Trypanosomiasis ◽  
Control Programmes ◽  
The 1960S ◽  
Stage 1

Human African trypanosomiasis (sleeping sickness) is caused by subspecies of the protozoan parasite Trypanosoma brucei. The disease is restricted to tropical Africa where it is transmitted by the bite of infected tsetse flies (Glossina spp.). Control programmes in the 1960s were very effective, but subsequent relaxation of control measures led to recurrence of epidemic proportions in the 1980s and 1990s. Control is now being regained. Untreated human African trypanosomiasis is almost invariably fatal. Specific treatment depends on the trypanosome subspecies and the stage of the disease. Drugs used for stage 1 include pentamidine and suramin, and for stage 2 include melarsoprol, eflornithine, and nifurtimox, but regimens are not standardized, and treatment is difficult and dangerous; all of the drugs used have many side effects, some potentially lethal.

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