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Keith Gull - One of the best experts on this subject based on the ideXlab platform.
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Protein diversity in discrete structures at the distal tip of the trypanosome Flagellum.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Vladimir Varga, Flavia Moreira-leite, Neil Portman, Keith GullAbstract:The distal end of the Eukaryotic Flagellum/cilium is important for axonemal growth and signaling and has distinct biomechanical properties. Specific Flagellum tip structures exist, yet their composition, dynamics, and functions are largely unknown. We used biochemical approaches to identify seven constituents of the flagella connector at the tip of an assembling trypanosome Flagellum and three constituents of the axonemal capping structure at the tips of both assembling and mature flagella. Both tip structures contain evolutionarily conserved as well as kinetoplastid-specific proteins, and component assembly into the structures occurs very early during Flagellum extension. Localization and functional studies reveal that the flagella connector membrane junction is attached to the tips of extending microtubules of the assembling Flagellum by a kinesin-15 family member. On the opposite side, a kinetoplastid-specific kinesin facilitates attachment of the junction to the microtubules in the mature Flagellum. Functional studies also suggest roles of several other components and the definition of subdomains in the tip structures.
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the paraflagellar rod of kinetoplastid parasites from structure to components and function
International Journal for Parasitology, 2010Co-Authors: Neil Portman, Keith GullAbstract: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. One of the best studied of these structures is the paraflagellar rod of kinetoplastid protozoa in which the morphological characteristics have been well defined and some of the major protein constituents have been identified. 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.
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A protein-protein interaction map of the Trypanosoma brucei paraflagellar rod.
PLoS ONE, 2009Co-Authors: Sylvain Lacomble, Neil Portman, Keith GullAbstract:We have conducted a protein interaction study of components within a specific sub-compartment of a Eukaryotic Flagellum. The trypanosome Flagellum contains a para-crystalline extra-axonemal structure termed the paraflagellar rod (PFR) with around forty identified components. We have used a Gateway cloning approach coupled with yeast two-hybrid, RNAi and 2D DiGE to define a protein-protein interaction network taking place in this structure. We define two clusters of interactions; the first being characterised by two proteins with a shared domain which is not sufficient for maintaining the interaction. The other cohort is populated by eight proteins, a number of which possess a PFR domain and sub-populations of this network exhibit dependency relationships. Finally, we provide clues as to the structural organisation of the PFR at the molecular level. This multi-strand approach shows that protein interactome data can be generated for insoluble protein complexes.
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combining rna interference mutants and comparative proteomics to identify protein components and dependences in a Eukaryotic Flagellum
Journal of Biological Chemistry, 2009Co-Authors: Neil Portman, Paul G. Mckean, Sylvain Lacomble, Benjamin Thomas, Keith GullAbstract:Eukaryotic flagella from organisms such as Trypanosoma brucei can be isolated and their protein components identified by mass spectrometry. Here we used a comparative approach utilizing two-dimensional difference gel electrophoresis and isobaric tags for relative and absolute quantitation to reveal protein components of flagellar structures via ablation by inducible RNA interference mutation. By this approach we identified 20 novel components of the paraflagellar rod (PFR). Using epitope tagging we validated a subset of these as being present within the PFR by immunofluorescence. Bioinformatic analysis of the PFR cohort reveals a likely calcium/calmodulin regulatory/signaling linkage between some components. We extended the RNA interference mutant/comparative proteomic analysis to individual novel components of our PFR proteome, showing that the approach has the power to reveal dependences between subgroups within the cohort.
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Trypanosome IFT mutants provide insight into the motor location for mobility of the flagella connector and flagellar membrane formation
Journal of Cell Science, 2006Co-Authors: Jacqueline A. Davidge, Michael L. Ginger, Paul G. Mckean, Emma Chambers, Harriet A. Dickinson, Katie Towers, Keith GullAbstract:The flagella connector (FC) of procyclic trypanosomes is a mobile, transmembrane junction important in providing cytotactic morphogenetic information to the daughter cell. Quantitative analyses of FC positioning along the old Flagellum, involving direct observations and use of the MPM2 anti-phosphoprotein monoclonal reveals a `stop point' is reached on the old Flagellum which correlates well with the initiation of basal body migration and kinetoplast segregation. This demonstrates further complexities of the FC and its movement in morphogenetic events in trypanosomes than have hitherto been described. We used intraflagellar transport RNAi mutants to ablate the formation of a new Flagellum. Intriguingly the FC could still move, indicating that a motor function beyond the new Flagellum is sufficient to move it. When such a FC moves, it drags a sleeve of new flagellar membrane out of the flagellar pocket. This axoneme-less flagellar membrane maintains appropriate developmental relationships to the cell body including following the correct helical path and being connected to the internal cytoskeleton by macula adherens junctions. Movement of the FC in the apparent absence of intraflagellar transport raises the possibility of a new form of motility within a Eukaryotic Flagellum.
Neil Portman - One of the best experts on this subject based on the ideXlab platform.
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Protein diversity in discrete structures at the distal tip of the trypanosome Flagellum.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Vladimir Varga, Flavia Moreira-leite, Neil Portman, Keith GullAbstract:The distal end of the Eukaryotic Flagellum/cilium is important for axonemal growth and signaling and has distinct biomechanical properties. Specific Flagellum tip structures exist, yet their composition, dynamics, and functions are largely unknown. We used biochemical approaches to identify seven constituents of the flagella connector at the tip of an assembling trypanosome Flagellum and three constituents of the axonemal capping structure at the tips of both assembling and mature flagella. Both tip structures contain evolutionarily conserved as well as kinetoplastid-specific proteins, and component assembly into the structures occurs very early during Flagellum extension. Localization and functional studies reveal that the flagella connector membrane junction is attached to the tips of extending microtubules of the assembling Flagellum by a kinesin-15 family member. On the opposite side, a kinetoplastid-specific kinesin facilitates attachment of the junction to the microtubules in the mature Flagellum. Functional studies also suggest roles of several other components and the definition of subdomains in the tip structures.
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the paraflagellar rod of kinetoplastid parasites from structure to components and function
International Journal for Parasitology, 2010Co-Authors: Neil Portman, Keith GullAbstract: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. One of the best studied of these structures is the paraflagellar rod of kinetoplastid protozoa in which the morphological characteristics have been well defined and some of the major protein constituents have been identified. 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.
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A protein-protein interaction map of the Trypanosoma brucei paraflagellar rod.
PLoS ONE, 2009Co-Authors: Sylvain Lacomble, Neil Portman, Keith GullAbstract:We have conducted a protein interaction study of components within a specific sub-compartment of a Eukaryotic Flagellum. The trypanosome Flagellum contains a para-crystalline extra-axonemal structure termed the paraflagellar rod (PFR) with around forty identified components. We have used a Gateway cloning approach coupled with yeast two-hybrid, RNAi and 2D DiGE to define a protein-protein interaction network taking place in this structure. We define two clusters of interactions; the first being characterised by two proteins with a shared domain which is not sufficient for maintaining the interaction. The other cohort is populated by eight proteins, a number of which possess a PFR domain and sub-populations of this network exhibit dependency relationships. Finally, we provide clues as to the structural organisation of the PFR at the molecular level. This multi-strand approach shows that protein interactome data can be generated for insoluble protein complexes.
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combining rna interference mutants and comparative proteomics to identify protein components and dependences in a Eukaryotic Flagellum
Journal of Biological Chemistry, 2009Co-Authors: Neil Portman, Paul G. Mckean, Sylvain Lacomble, Benjamin Thomas, Keith GullAbstract:Eukaryotic flagella from organisms such as Trypanosoma brucei can be isolated and their protein components identified by mass spectrometry. Here we used a comparative approach utilizing two-dimensional difference gel electrophoresis and isobaric tags for relative and absolute quantitation to reveal protein components of flagellar structures via ablation by inducible RNA interference mutation. By this approach we identified 20 novel components of the paraflagellar rod (PFR). Using epitope tagging we validated a subset of these as being present within the PFR by immunofluorescence. Bioinformatic analysis of the PFR cohort reveals a likely calcium/calmodulin regulatory/signaling linkage between some components. We extended the RNA interference mutant/comparative proteomic analysis to individual novel components of our PFR proteome, showing that the approach has the power to reveal dependences between subgroups within the cohort.
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Swimming with protists: perception, motility and Flagellum assembly
Nature Reviews Microbiology, 2008Co-Authors: Michael L. Ginger, Neil Portman, Paul G. MckeanAbstract:Flagella and cilia are used in unicellular and multicellular eukaryotes for fast cell motility, rapid movement of material over cell surfaces, cell feeding and cell division. The structure of the Eukaryotic Flagellum is not related to the structure of the prokaryotic Flagellum. The principal feature of most motile Eukaryotic flagella is the '9+2' microtubule axoneme. Some deviations from this canonical structure are known. Recent biochemical and proteomic studies have identified protist flagella as organelles with their own associated metabolism. The challenge is to understand the physiological functions of these unexpected metabolic pathways. Intraflagellar transport is a widely conserved mechanism by which flagella in many organisms are built. For some protists, studies in the areas of cell biology and comparative genomics are challenging our paradigms of Flagellum assembly. Protist flagella also function in response to, and in the initiation of, signal-transduction cascades. Flagellar or ciliary motility is important for cytokinesis in some protists. In the example of the African trypanosome, Trypanosoma brucei , small-molecule-dependent intervention could eventually afford new possibilities for drug design against sleeping sickness, a tropical disease of Sub-Saharan Africa. In unicellular and multicellular eukaryotes, fast cell motility and rapid movement of material over cell surfaces are often mediated by ciliary or flagellar beating. The conserved defining structure in most motile cilia and flagella is the '9+2' microtubule axoneme. Our general understanding of Flagellum assembly and the regulation of flagellar motility has been led by results from seminal studies of flagellate protozoa and algae. Here we review recent work relating to various aspects of protist physiology and cell biology. In particular, we discuss energy metabolism in Eukaryotic flagella, modifications to the canonical assembly pathway and Flagellum function in parasite virulence. The motility that is afforded by flagella and cilia is widespread among unicellular eukaryotes. In this Review, Ginger and colleagues describe the structure of the Flagellum, its metabolic activity, different synthesis pathways and flagellar functions that extend beyond motility.
Sarah E Reece - One of the best experts on this subject based on the ideXlab platform.
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The Role of Central Microtubules in the Beating of Eukaryotic Flagella, Revealed by High-Speed Holographic Microscopy
Biophysical Journal, 2014Co-Authors: Laurence G Wilson, Lucy M Carter, Sarah E ReeceAbstract:Understanding the mechanics of the Eukaryotic Flagellum is a key challenge in biophysics. As well as being of scientific interest, there are clear therapeutic applications, not least in reproductive medicine. The axoneme lies at the heart of the Flagellum, and its structure is known: nine microtubule doublets surround a central pair of microtubule singlets. The role of the central pair in this canonical ‘9+2’ arrangement has been the subject of speculation for some time, though they are known to assist in regulating the flagellar beat. Our group has developed high-speed holographic microscopy that allows us to numerically refocus a digital image off-line. By generating a stack of refocused images from each frame in a video, we obtain scans of the sample volume at the frame rate of our video camera. Effectively, this allows us to image 500-1000 volumes per second. By analyzing this volumetric data, we can measure the waveform of a Eukaryotic Flagellum to within 200 nm in three dimensions, and with millisecond time resolution. We have previously used this method to measure the waveform of a naturally occurring, isolated Flagellum: the microgamete of the rodent malaria parasite Plasmodium berghei. In order to assess the role of the central pair, we take advantage of a newly-available mutant strain that is lacking one or both central microtubules. By comparing the three-dimensional shape and movement observed in mutant flagella to that in the wild-type, we can get some insight into how the central pair helps to regulate the beating action.
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three dimensional dynamics of a Eukaryotic Flagellum revealed by high speed holographic microscopy
Biophysical Journal, 2013Co-Authors: Laurence G Wilson, Lucy M Carter, Wilson C K Poon, Sarah E ReeceAbstract:Understanding the mechanics of the Eukaryotic Flagellum is a key challenge in biophysics. As well as being of scientific interest, there are clear therapeutic applications, not least in reproductive medicine. The physics of swimming sperm has been studied for some time and there are several competing hypotheses for the underlying mechanics of the axoneme. A key aspect of this work remains unaddressed, however: almost all flagellar waveforms are three-dimensional. Comparing 2D theories with 2D images has met with some success, but for a complete understanding, the 3D nature of the beating must be considered. We have employed digital holographic microscopy to record the 3D beating of a gamete of Plasmodium berghei (a parasite associated with malaria in rodents). When coupled with high-speed imaging we are able to obtain a complete record of the flagellar waveform with excellent temporal and spatial resolution. This sperm is an interesting limiting case: it has no head, and almost no other accessory structures. Our analysis of the 3D beating pattern has implications for the proposed link between structural chirality in the axoneme and the chirality of flagellar waveforms.View Large Image | View Hi-Res Image | Download PowerPoint Slide
Kent L. Hill - One of the best experts on this subject based on the ideXlab platform.
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Motility and more: the Flagellum of Trypanosoma brucei
Nature Reviews Microbiology, 2014Co-Authors: Gerasimos Langousis, Kent L. HillAbstract:The protozoan parasite Trypanosoma brucei has a single Flagellum that is present in all of its different developmental stages. In this Review, Langousis and Hill discuss the structural and functional features of the Flagellum and highlight its central role in the virulence and transmission of this important human pathogen. The African trypanosome Trypanosoma brucei is a unicellular pathogen that causes lethal sleeping sickness in humans, which is a devastating and neglected tropical disease that is endemic to vast regions of Africa. T. brucei also infects wild and domestic livestock, which limits sustainable development, and it is thus considered to be both a cause and consequence of poverty. T. brucei has a single Flagellum that is present throughout the parasite and its life cycle. The Flagellum has conserved and unique features. It emerges from a membrane invagination at the posterior end of the cell and remains attached to the cell body for most of its length. The Flagellum contains cytoskeletal structures, which are ensheathed by a specialized flagellar membrane that interfaces with the external environment and that has a protein and lipid composition that is distinct from the rest of the cell surface. The T. brucei Flagellum has multiple functions and is essential for parasite motility, viability, transmission and pathogenesis. Flagellum-mediated motility is powered by the axoneme, which is a biological machine that converts dynein motor structural changes into Flagellum beating and parasite propulsion. T. brucei motility is crucial for movement through host tissues and provides a surprising immune-evasion mechanism. In addition to motility, the T. brucei Flagellum is an important morphogenetic hub that controls cell shape and size, directs organelle segregation and governs cell division. These functions are modulated during developmental transitions of the parasite and are achieved by the direct or indirect physical connections of the Flagellum to other cellular elements. The Flagellum is a crucial host–pathogen interface that has important roles in parasite transmission and virulence. Flagellar proteins mediate attachment to host tissues, carry out uptake of host growth factors and promote parasite survival by inhibiting host immunity. T. brucei is an excellent model system to study the biology of the highly conserved Eukaryotic Flagellum and offers valuable insights into how flagella assemble, move and sense the environment. Continued studies of the T.brucei Flagellum hold the promise of having a great impact on human health, as human flagella are paramount in human development and physiology. In addition, the flagella of many human pathogens are salient but unexplained structures that await further study. Trypanosoma brucei is a pathogenic unicellular eukaryote that infects humans and other mammals in sub-Saharan Africa. A central feature of trypanosome biology is the single Flagellum of the parasite, which is an essential and multifunctional organelle that facilitates cell propulsion, controls cell morphogenesis and directs cytokinesis. Moreover, the flagellar membrane is a specialized subdomain of the cell surface that mediates attachment to host tissues and harbours multiple virulence factors. In this Review, we discuss the structure, assembly and function of the trypanosome Flagellum, including canonical roles in cell motility as well as novel and emerging roles in cell morphogenesis and host–parasite interactions.
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CMF22 Is a Broadly Conserved Axonemal Protein and Is Required for Propulsive Motility in Trypanosoma brucei
Eukaryotic Cell, 2013Co-Authors: Hoangkim T. Nguyen, Gerasimos Langousis, Jaspreet Sandhu, Kent L. HillAbstract:ABSTRACT The Eukaryotic Flagellum (or cilium) is a broadly conserved organelle that provides motility for many pathogenic protozoa and is critical for normal development and physiology in humans. Therefore, defining core components of motile axonemes enhances understanding of Eukaryotic biology and provides insight into mechanisms of inherited and infectious diseases in humans. In this study, we show that component of motile flagella 22 (CMF22) is tightly associated with the flagellar axoneme and is likely to have been present in the last Eukaryotic common ancestor. The CMF22 amino acid sequence contains predicted IQ and ATPase associated with a variety of cellular activities (AAA) motifs that are conserved among CMF22 orthologues in diverse organisms, hinting at the importance of these domains in CMF22 function. Knockdown by RNA interference (RNAi) and rescue with an RNAi-immune mRNA demonstrated that CMF22 is required for propulsive cell motility in Trypanosoma brucei. Loss of propulsive motility in CMF22-knockdown cells was due to altered flagellar beating patterns, rather than flagellar paralysis, indicating that CMF22 is essential for motility regulation and likely functions as a fundamental regulatory component of motile axonemes. CMF22 association with the axoneme is weakened in mutants that disrupt the nexin-dynein regulatory complex, suggesting potential interaction with this complex. Our results provide insight into the core machinery required for motility of Eukaryotic flagella.
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CMF70 is a subunit of the dynein regulatory complex.
Journal of Cell Science, 2010Co-Authors: Zakayi P. Kabututu, Michelle Thayer, Jason H. Melehani, Kent L. HillAbstract:Flagellar motility drives propulsion of several important pathogens and is essential for human development and physiology. Motility of the Eukaryotic Flagellum requires coordinate regulation of thousands of dynein motors arrayed along the axoneme, but the proteins underlying dynein regulation are largely unknown. The dynein regulatory complex, DRC, is recognized as a focal point of axonemal dynein regulation, but only a single DRC subunit, trypanin/PF2, is currently known. The component of motile flagella 70 protein, CMF70, is broadly and uniquely conserved among organisms with motile flagella, suggesting a role in axonemal motility. Here we demonstrate that CMF70 is part of the DRC from Trypanosoma brucei. CMF70 is located along the Flagellum, co-sediments with trypanin in sucrose gradients and co-immunoprecipitates with trypanin. RNAi knockdown of CMF70 causes motility defects in a wild-type background and suppresses flagellar paralysis in cells with central pair defects, thus meeting the functional definition of a DRC subunit. Trypanin and CMF70 are mutually conserved in at least five of six extant Eukaryotic clades, indicating that the DRC was probably present in the last common Eukaryotic ancestor. We have identified only the second known subunit of this ubiquitous dynein regulatory system, highlighting the utility of combined genomic and functional analyses for identifying novel subunits of axonemal sub-complexes.
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Approaches for functional analysis of flagellar proteins in African trypanosomes.
Methods in Cell Biology, 2009Co-Authors: Michael Oberholzer, Miguel A. López, Katherine S. Ralston, Kent L. HillAbstract:The Eukaryotic Flagellum is a highly conserved organelle serving motility, sensory, and transport functions. Although genetic, genomic, and proteomic studies have led to the identification of hundreds of flagellar and putative flagellar proteins, precisely how these proteins function individually and collectively to drive Flagellum motility and other functions remains to be determined. In this chapter we provide an overview of tools and approaches available for studying Flagellum protein function in the protozoan parasite Trypanosoma brucei. We begin by outlining techniques for in vitro cultivation of both T. brucei life cycle stages, as well as transfection protocols for the delivery of DNA constructs. We then describe specific assays used to assess Flagellum function including Flagellum preparation and quantitative motility assays. We conclude the chapter with a description of molecular genetic approaches for manipulating gene function. In summary, the availability of potent molecular tools, as well as the health and economic relevance of T. brucei as a pathogen, combine to make the parasite an attractive and integral experimental system for the functional analysis of flagellar proteins.
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Functional genomics in Trypanosoma brucei identifies evolutionarily conserved components of motile flagella.
Journal of cell science, 2007Co-Authors: Desiree M Baron, Zakayi P. Kabututu, Katherine S. Ralston, Kent L. HillAbstract:Cilia and flagella are highly conserved, complex organelles involved in a variety of important functions. Flagella are required for motility of several human pathogens and ciliary defects lead to a variety of fatal and debilitating human diseases. Many of the major structural components of cilia and flagella are known, but little is known about regulation of flagellar beat. Trypanosoma brucei, the causative agent of African sleeping sickness, provides an excellent model for studying flagellar motility. We have used comparative genomics to identify a core group of 50 genes unique to organisms with motile flagella. These genes, referred to as T. brucei components of motile flagella (TbCMF) include 30 novel genes, and human homologues of many of the TbCMF genes map to loci associated with human ciliary diseases. To characterize TbCMF protein function we used RNA interference to target 41 TbCMF genes. Sedimentation assays and direct observation demonstrated clear motility defects in a majority of these knockdown mutants. Epitope tagging, fluorescence localization and biochemical fractionation demonstrated flagellar localization for several TbCMF proteins. Finally, ultrastructural analysis identified a family of novel TbCMF proteins that function to maintain connections between outer doublet microtubules, suggesting that they are the first identified components of nexin links. Overall, our results provide insights into the workings of the Eukaryotic Flagellum, identify several novel human disease gene candidates, reveal unique aspects of the trypanosome Flagellum and underscore the value of T. brucei as an experimental system for studying flagellar biology.
Alastair G. B. Simpson - One of the best experts on this subject based on the ideXlab platform.
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A natural toroidal microswimmer with a rotary Eukaryotic Flagellum
Nature Microbiology, 2019Co-Authors: Sebastian Hess, Laura Eme, Andrew J. Roger, Alastair G. B. SimpsonAbstract:We describe Idionectes vortex gen. nov., sp. nov., a unicellular microeukaryote that swims by continuous inversion of its surface, similar to a vortex ring. This previously unreported mode of motility approximates a hypothetical concept called the ‘toroidal swimmer’, in which a doughnut-shaped object rotates around its circular axis and travels in the opposite direction to its outer surface motion. During swimming, the Flagellum of Idionectes rotates relative to its cell body, which is normally a hallmark of prokaryotic rather than Eukaryotic flagella. Idionectes vortex is a flagellated unicellular microbial eukaryote with a unique mode of motility; it resembles a doughnut-shaped object that swims by continuous inversion of its surface, similar to a vortex ring.