Whole cell reconstructions of Leishmania mexicana through the cell cycle.

The unicellular parasite Leishmania has a precisely defined cell architecture that is inherited by each subsequent generation, requiring a highly coordinated pattern of duplication and segregation of organelles and cytoskeletal structures. A framework of nuclear division and morphological changes is known from light microscopy, yet this has limited resolution and the intrinsic organisation of organelles within the cell body and their manner of duplication and inheritance is unknown. Using volume electron microscopy approaches, we have produced three-dimensional reconstructions of different promastigote cell cycle stages to give a spatial and quantitative overview of organelle positioning, division and inheritance. The first morphological indications seen in our dataset that a new cell cycle had begun were the assembly of a new flagellum, the duplication of the contractile vacuole and the increase in volume of the nucleus and kinetoplast. We showed that the progression of the cytokinesis furrow created a specific pattern of membrane indentations, while our analysis of sub-pellicular microtubule organisation indicated that there is likely a preferred site of new microtubule insertion. The daughter cells retained these indentations in their cell body for a period post-abscission. By comparing cultured and sand fly derived promastigotes, we found an increase in the number and overall volume of lipid droplets in the promastigotes from the sand fly, reflecting a change in their metabolism to ensure transmissibility to the mammalian host. Our insights into the cell cycle mechanics of Leishmania will support future molecular cell biology analyses of these parasites.

Nucleolar targeting in an early-branching eukaryote suggests a general mechanism for ribosome protein sorting.

The compartmentalised eukaryotic cell demands accurate targeting of proteins to the organelles in which they function, whether membrane-bound (like the nucleus) or non-membrane-bound (like the nucleolus). Nucleolar targeting relies on positively charged localisation signals and has received rejuvenated interest since the widespread recognition of liquid-liquid phase separation (LLPS) as a mechanism contributing to nucleolus formation. Here, we exploit a new genome-wide analysis of protein localisation in the early-branching eukaryote Trypanosoma brucei to analyse general nucleolar protein properties. T. brucei nucleolar proteins have similar properties to those in common model eukaryotes, specifically basic amino acids. Using protein truncations and addition of candidate targeting sequences to proteins, we show both homopolymer runs and distributed basic amino acids give nucleolar partition, further aided by a nuclear localisation signal (NLS). These findings are consistent with phase separation models of nucleolar formation and physical protein properties being a major contributing mechanism for eukaryotic nucleolar targeting, conserved from the last eukaryotic common ancestor. Importantly, cytoplasmic ribosome proteins, unlike mitochondrial ribosome proteins, have more basic residues - pointing to adaptation of physicochemical properties to assist segregation.

The single flagellum of Leishmania has a fixed polarisation of its asymmetric beat.

Eukaryotic flagella undertake different beat types as necessary for different functions; for example, the Leishmania parasite flagellum undergoes a symmetric tip-to-base beat for forward swimming and an asymmetric base-to-tip beat to rotate the cell. In multi-ciliated tissues or organisms, the asymmetric beats are coordinated, leading to movement of the cell, organism or surrounding fluid. This coordination involves a polarisation of power stroke direction. Here, we asked whether the asymmetric beat of the single Leishmania flagellum also has a fixed polarisation. We developed high frame rate dual-colour fluorescence microscopy to visualise flagellar-associated structures in live swimming cells. This showed that the asymmetric Leishmania beat is polarised, with power strokes only occurring in one direction relative to the asymmetric flagellar machinery. Polarisation of bending was retained in deletion mutants whose flagella cannot beat but have a static bend. Furthermore, deletion mutants for proteins required for asymmetric extra-axonemal and rootlet-like flagellum-associated structures also retained normal polarisation. Leishmania beat polarisation therefore likely arises from either the nine-fold rotational symmetry of the axoneme structure or is due to differences between the outer doublet decorations.

Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections.

The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasite's life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single paralysed mutant lacking the central pair protein PF16 or an uncoordinated swimmer lacking the axonemal protein MBO2 showed that these promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.

Direction of flagellum beat propagation is controlled by proximal/distal outer dynein arm asymmetry.

The 9 + 2 axoneme structure of the motile flagellum/cilium is an iconic, apparently symmetrical cellular structure. Recently, asymmetries along the length of motile flagella have been identified in a number of organisms, typically in the inner and outer dynein arms. Flagellum-beat waveforms are adapted for different functions. They may start either near the flagellar tip or near its base and may be symmetrical or asymmetrical. We hypothesized that proximal/distal asymmetry in the molecular composition of the axoneme may control the site of waveform initiation and the direction of waveform propagation. The unicellular eukaryotic pathogens Trypanosoma brucei and Leishmania mexicana often switch between tip-to-base and base-to-tip waveforms, making them ideal for analysis of this phenomenon. We show here that the proximal and distal portions of the flagellum contain distinct outer dynein arm docking-complex heterodimers. This proximal/distal asymmetry is produced and maintained through growth by a concentration gradient of the proximal docking complex, generated by intraflagellar transport. Furthermore, this asymmetry is involved in regulating whether a tip-to-base or base-to-tip beat occurs, which is linked to a calcium-dependent switch. Our data show that the mechanism for generating proximal/distal flagellar asymmetry can control waveform initiation and propagation direction.

Use of chiral cell shape to ensure highly directional swimming in trypanosomes.

Swimming cells typically move along a helical path or undergo longitudinal rotation as they swim, arising from chiral asymmetry in hydrodynamic drag or propulsion bending the swimming path into a helix. Helical paths are beneficial for some forms of chemotaxis, but why asymmetric shape is so prevalent when a symmetric shape would also allow highly directional swimming is unclear. Here, I analyse the swimming of the insect life cycle stages of two human parasites; Trypanosoma brucei and Leishmania mexicana. This showed quantitatively how chirality in T. brucei cell shape confers highly directional swimming. High speed videomicrographs showed that T. brucei, L. mexicana and a T. brucei RNAi morphology mutant have a range of shape asymmetries, from wild-type T. brucei (highly chiral) to L. mexicana (near-axial symmetry). The chiral cells underwent longitudinal rotation while swimming, with more rapid longitudinal rotation correlating with swimming path directionality. Simulation indicated hydrodynamic drag on the chiral cell shape caused rotation, and the predicted geometry of the resulting swimming path matched the directionality of the observed swimming paths. This simulation of swimming path geometry showed that highly chiral cell shape is a robust mechanism through which microscale swimmers can achieve highly directional swimming at low Reynolds number. It is insensitive to random variation in shape or propulsion (biological noise). Highly symmetric cell shape can give highly directional swimming but is at risk of giving futile circular swimming paths in the presence of biological noise. This suggests the chiral T. brucei cell shape (associated with the lateral attachment of the flagellum) may be an adaptation associated with the bloodstream-inhabiting lifestyle of this parasite for robust highly directional swimming. It also provides a plausible general explanation for why swimming cells tend to have strong asymmetries in cell shape or propulsion.

Intraflagellar transport speed is sensitive to genetic and mechanical perturbations to flagellar beating.

Two sets of motor proteins underpin motile cilia/flagella function. The axoneme-associated inner and outer dynein arms drive sliding of adjacent axoneme microtubule doublets to periodically bend the flagellum for beating, while intraflagellar transport (IFT) kinesins and dyneins carry IFT trains bidirectionally along the axoneme. Despite assembling motile cilia and flagella, IFT train speeds have only previously been quantified in immobilized flagella-mechanical immobilization or genetic paralysis. This has limited investigation of the interaction between IFT and flagellar beating. Here, in uniflagellate Leishmania parasites, we use high-frequency, dual-color fluorescence microscopy to visualize IFT train movement in beating flagella. We discovered that adhesion of flagella to a microscope slide is detrimental, reducing IFT train speed and increasing train stalling. In flagella free to move, IFT train speed is not strongly dependent on flagella beat type; however, permanent disruption of flagella beating by deletion of genes necessary for formation or regulation of beating showed an inverse correlation of beat frequency and IFT train speed.

TrypTag.org: from images to discoveries using genome-wide protein localisation in Trypanosoma brucei.

TrypTag was a 4-year project to tag the N- and C-termini of almost all Trypanosoma brucei proteins with a fluorescent protein and record the subcellular localisation through images and manual annotation. We highlight the new routes to cell biological discovery this transformative resource is enabling for parasitologists and cell biologists.

Comprehensive sub-mitochondrial protein map of the parasitic protist Trypanosoma brucei defines critical features of organellar biology.

We have generated a high-confidence mitochondrial proteome (MitoTag) of the Trypanosoma brucei procyclic stage containing 1,239 proteins. For 337 of these, a mitochondrial localization had not been described before. We use the TrypTag dataset as a foundation and take advantage of the properties of the fluorescent protein tag that causes aberrant but fortuitous accumulation of tagged matrix and inner membrane proteins near the kinetoplast (mitochondrial DNA). Combined with transmembrane domain predictions, this characteristic allowed categorization of 1,053 proteins into mitochondrial sub-compartments, the detection of unique matrix-localized fucose and methionine synthesis, and the identification of new kinetoplast proteins, which showed kinetoplast-linked pyrimidine synthesis. Moreover, disruption of targeting signals by tagging allowed mapping of the mode of protein targeting to these sub-compartments, identifying a set of C-tail anchored outer mitochondrial membrane proteins and mitochondrial carriers likely employing multiple target peptides. This dataset represents a comprehensive, updated mapping of the mitochondrion.

Genome-wide subcellular protein map for the flagellate parasite Trypanosoma brucei.

Trypanosoma brucei is a model trypanosomatid, an important group of human, animal and plant unicellular parasites. Understanding their complex cell architecture and life cycle is challenging because, as with most eukaryotic microbes, ~50% of genome-encoded proteins have completely unknown functions. Here, using fluorescence microscopy and cell lines expressing endogenously tagged proteins, we mapped the subcellular localization of 89% of the T. brucei proteome, a resource we call TrypTag. We provide clues to function and define lineage-specific organelle adaptations for parasitism, mapping the ultraconserved cellular architecture of eukaryotes, including the first comprehensive 'cartographic' analysis of the eukaryotic flagellum, which is vital for morphogenesis and pathology. To demonstrate the power of this resource, we identify novel organelle subdomains and changes in molecular composition through the cell cycle. TrypTag is a transformative resource, important for hypothesis generation for both eukaryotic evolutionary molecular cell biology and fundamental parasite cell biology.

Stage-specific transcription activator ESB1 regulates monoallelic antigen expression in Trypanosoma brucei.

Variant surface glycoprotein (VSG) coats bloodstream form Trypanosoma brucei parasites, and monoallelic VSG expression underpins the antigenic variation necessary for pathogenicity. One of thousands of VSG genes is transcribed by RNA polymerase I in a singular nuclear structure called the expression site body (ESB), but how monoallelic VSG transcription is achieved remains unclear. Using a localization screen of 153 proteins we found one, ESB-specific protein 1 (ESB1), that localized only to the ESB and is expressed only in VSG-expressing life cycle stages. ESB1 associates with DNA near the active VSG promoter and is necessary for VSG expression, with overexpression activating inactive VSG promoters. Mechanistically, ESB1 is necessary for recruitment of a subset of ESB components, including RNA polymerase I, revealing that the ESB has separately assembled subdomains. Because many trypanosomatid parasites have divergent ESB1 orthologues yet do not undergo antigenic variation, ESB1 probably represents an important class of transcription regulators.

A resource for improved predictions of Trypanosoma and Leishmania protein three-dimensional structure.

AlphaFold2 and RoseTTAfold represent a transformative advance for predicting protein structure. They are able to make very high-quality predictions given a high-quality alignment of the protein sequence with related proteins. These predictions are now readily available via the AlphaFold database of predicted structures and AlphaFold or RoseTTAfold Colaboratory notebooks for custom predictions. However, predictions for some species tend to be lower confidence than model organisms. Problematic species include Trypanosoma cruzi and Leishmania infantum: important unicellular eukaryotic human parasites in an early-branching eukaryotic lineage. The cause appears to be due to poor sampling of this branch of life (Discoba) in the protein sequences databases used for the AlphaFold database and ColabFold. Here, by comprehensively gathering openly available protein sequence data for Discoba species, significant improvements to AlphaFold2 protein structure prediction over the AlphaFold database and ColabFold are demonstrated. This is made available as an easy-to-use tool for the parasitology community in the form of Colaboratory notebooks for generating multiple sequence alignments and AlphaFold2 predictions of protein structure for Trypanosoma, Leishmania and related species.

Therapeutics-how to treat phase separation-associated diseases.

Liquid-liquid phase separation has drawn attention as many neurodegeneration or cancer-associated proteins are able to form liquid membraneless compartments (condensates) by liquid-liquid phase separation. Furthermore, there is rapidly growing evidence that disease-associated mutation or post-translational modification of these proteins causes aberrant location, composition or physical properties of the condensates. It is ambiguous whether aberrant condensates are always causative in disease mechanisms, however they are likely promising potential targets for therapeutics. The conceptual framework of liquid-liquid phase separation provides opportunities for novel therapeutic approaches. This review summarises how the extensive recent advances in understanding control of nucleation, growth and composition of condensates by protein post-translational modification has revealed many possibilities for intervention by conventional small molecule enzyme inhibitors. This includes the first proof-of-concept examples. However, understanding membraneless organelle formation as a physical chemistry process also highlights possible physicochemical mechanisms of intervention. There is huge demand for innovation in drug development, especially for challenging diseases of old age including neurodegeneration and cancer. The conceptual framework of liquid-liquid phase separation provides a new paradigm for thinking about modulating protein function and is very different from enzyme lock-and-key or structured binding site concepts and presents new opportunities for innovation.

Cellular landmarks of Trypanosoma brucei and Leishmania mexicana.

The kinetoplastids Trypanosoma brucei and Leishmania mexicana are eukaryotes with a highly structured cellular organisation that is reproduced with great fidelity in each generation. The pattern of signal from a fluorescently tagged protein can define the specific structure/organelle that this protein localises to, and can be extremely informative in phenotype analysis in experimental perturbations, life cycle tracking, post-genomic assays and functional analysis of organelles. Using the vast coverage of protein subcellular localisations provided by the TrypTag project, an ongoing project to determine the localisation of every protein encoded in the T. brucei genome, we have generated an inventory of reliable reference organelle markers for both parasites that combines epifluorescence images with a detailed description of the key features of each localisation. We believe this will be a useful comparative resource that will enable researchers to quickly and accurately pinpoint the localisation of their proteins of interest and will provide cellular markers for many types of cell biology studies. We see this as another important step in the post-genomic era analyses of these parasites, in which ever expanding datasets generate numerous candidates to analyse. Adoption of these reference proteins by the community is likely to enhance research studies and enable better comparison of data.

Analyzing the dynamics of cell cycle processes from fixed samples through ergodic principles.

Tools to analyze cyclical cellular processes, particularly the cell cycle, are of broad value for cell biology. Cell cycle synchronization and live-cell time-lapse observation are widely used to analyze these processes but are not available for many systems. Simple mathematical methods built on the ergodic principle are a well-established, widely applicable, and powerful alternative analysis approach, although they are less widely used. These methods extract data about the dynamics of a cyclical process from a single time-point "snapshot" of a population of cells progressing through the cycle asynchronously. Here, I demonstrate application of these simple mathematical methods to analysis of basic cyclical processes--cycles including a division event, cell populations undergoing unicellular aging, and cell cycles with multiple fission (schizogony)--as well as recent advances that allow detailed mapping of the cell cycle from continuously changing properties of the cell such as size and DNA content. This includes examples using existing data from mammalian, yeast, and unicellular eukaryotic parasite cell biology. Through the ongoing advances in high-throughput cell analysis by light microscopy, electron microscopy, and flow cytometry, these mathematical methods are becoming ever more important and are a powerful complementary method to traditional synchronization and time-lapse cell cycle analysis methods.

Scanning and three-dimensional electron microscopy methods for the study of Trypanosoma brucei and Leishmania mexicana flagella.

Three-dimensional electron microscopy tools have revolutionized our understanding of cell structure and molecular complexes in biology. Here, we describe methods for studying flagellar ultrastructure and biogenesis in two unicellular parasites-Trypanosoma brucei and Leishmania mexicana. We describe methods for the preparation of these parasites for scanning electron microscopy cellular electron tomography, and serial block face scanning electron microscopy (SBFSEM). These parasites have a highly ordered cell shape and form, with a defined positioning of internal cytoskeletal structures and organelles. We show how knowledge of these can be used to dissect cell cycles in both parasites and identify the old flagellum from the new in T. brucei. Finally, we demonstrate the use of SBFSEM three-dimensional models for analysis of individual whole cells, demonstrating the excellent potential this technique has for future studies of mutant cell lines.

The limits on trypanosomatid morphological diversity.

Cell shape is one, often overlooked, way in which protozoan parasites have adapted to a variety of host and vector environments and directional transmissions between these environments. Consequently, different parasite life cycle stages have characteristic morphologies. Trypanosomatid parasites are an excellent example of this in which large morphological variations between species and life cycle stage occur, despite sharing well-conserved cytoskeletal and membranous structures. Here, using previously published reports in the literature of the morphology of 248 isolates of trypanosomatid species from different hosts, we perform a meta-analysis of the occurrence and limits on morphological diversity of different classes of trypanosomatid morphology (trypomastigote, promastigote, etc.) in the vertebrate bloodstream and invertebrate gut environments. We identified several limits on cell body length, cell body width and flagellum length diversity which can be interpreted as biomechanical limits on the capacity of the cell to attain particular dimensions. These limits differed for morphologies with and without a laterally attached flagellum which we suggest represent two morphological superclasses, the 'juxtaform' and 'liberform' superclasses. Further limits were identified consistent with a selective pressure from the mechanical properties of the vertebrate bloodstream environment; trypanosomatid size showed limits relative to host erythrocyte dimensions. This is the first comprehensive analysis of the limits of morphological diversity in any protozoan parasite, revealing the morphogenetic constraints and extrinsic selection pressures associated with the full diversity of trypanosomatid morphology.