Revealing spatio-temporal dynamics with long-term trypanosomatid live-cell imaging.

Trypanosoma brucei, the causative agent of human African trypanosomiasis, is highly motile and must be able to move in all three dimensions for reliable cell division. These characteristics make long-term microscopic imaging of live T. brucei cells challenging, which has limited our understanding of important cellular events. To address this issue, we devised an imaging approach that confines cells in small volumes within cast agarose microwells that can be imaged continuously for up to 24 h. Individual T. brucei cells were imaged through multiple rounds of cell division with high spatial and temporal resolution. We developed a strategy that employs in-well "sentinel" cells to monitor potential imaging toxicity during loss-of-function experiments such as small-molecule inhibition and RNAi. Using our approach, we show that the asymmetric daughter cells produced during T. brucei division subsequently divide at different rates, with the old-flagellum daughter cell dividing first. The flagellar detachment phenotype that appears during inhibition of the Polo-like kinase homolog TbPLK occurs in a stepwise fashion, with the new flagellum initially linked by its tip to the old, attached flagellum. We probe the feasibility of a previously proposed "back-up" cytokinetic mechanism and show that cells that initiate this process do not appear to complete cell division. This live-cell imaging method will provide a novel avenue for studying a wide variety of cellular events in trypanosomatids that have previously been inaccessible.

The Trypanosoma brucei subpellicular microtubule array is organized into functionally discrete subdomains defined by microtubule associated proteins.

Microtubules are inherently dynamic cytoskeletal polymers whose length and organization can be altered to perform essential functions in eukaryotic cells, such as providing tracks for intracellular trafficking and forming the mitotic spindle. Microtubules can be bundled to create more stable structures that collectively propagate force, such as in the flagellar axoneme, which provides motility. The subpellicular microtubule array of the protist parasite Trypanosoma brucei, the causative agent of African sleeping sickness, is a remarkable example of a highly specialized microtubule bundle. It is comprised of a single layer of microtubules that are crosslinked to each other and to the overlying plasma membrane. The array microtubules appear to be highly stable and remain intact throughout the cell cycle, but very little is known about the pathways that tune microtubule properties in trypanosomatids. Here, we show that the subpellicular microtubule array is organized into subdomains that consist of differentially localized array-associated proteins at the array posterior, middle, and anterior. The array-associated protein PAVE1 stabilizes array microtubules at the cell posterior and is essential for maintaining its tapered shape. PAVE1 and the newly identified protein PAVE2 form a complex that binds directly to the microtubule lattice, demonstrating that they are a true kinetoplastid-specific MAP. TbAIR9, which localizes to the entirety of the subpellicular array, is necessary for maintaining the localization of array-associated proteins within their respective subdomains of the array. The arrangement of proteins within the array likely tunes the local properties of array microtubules and creates the asymmetric shape of the cell, which is essential for parasite viability.

TbSmee1 regulates hook complex morphology and the rate of flagellar pocket uptake in Trypanosoma brucei.

Trypanosoma brucei uses multiple mechanisms to evade detection by its insect and mammalian hosts. The flagellar pocket (FP) is the exclusive site of uptake from the environment in trypanosomes and shields receptors from exposure to the host. The FP neck is tightly associated with the flagellum via a series of cytoskeletal structures that include the hook complex (HC) and the centrin arm. These structures are implicated in facilitating macromolecule entry into the FP and nucleating the flagellum attachment zone (FAZ), which adheres the flagellum to the cell surface. TbSmee1 (Tb927.10.8820) is a component of the HC and a putative substrate of polo-like kinase (TbPLK), which is essential for centrin arm and FAZ duplication. We show that depletion of TbSmee1 in the insect-resident (procyclic) form of the parasite causes a 40% growth decrease and the appearance of multinucleated cells that result from defective cytokinesis. Cells lacking TbSmee1 contain HCs with aberrant morphology and show delayed uptake of both fluid-phase and membrane markers. TbPLK localization to the tip of the new FAZ is also blocked. These results argue that TbSmee1 is necessary for maintaining HC morphology, which is important for the parasite's ability to take up molecules from its environment.

Identification of TOEFAZ1-interacting proteins reveals key regulators of Trypanosoma brucei cytokinesis.

The protist parasite Trypanosoma brucei is an obligate extracellular pathogen that retains its highly polarized morphology during cell division and has evolved a novel cytokinetic process independent of non-muscle myosin II. The polo-like kinase homolog TbPLK is essential for transmission of cell polarity during division and for cytokinesis. We previously identified a putative TbPLK substrate named Tip of the Extending FAZ 1 (TOEFAZ1) as an essential kinetoplastid-specific component of the T. brucei cytokinetic machinery. We performed a proximity-dependent biotinylation identification (BioID) screen using TOEFAZ1 as a means to identify additional proteins that are involved in cytokinesis. Using quantitative proteomic methods, we identified nearly 500 TOEFAZ1-proximal proteins and characterized 59 in further detail. Among the candidates, we identified an essential putative phosphatase that regulates the expression level and localization of both TOEFAZ1 and TbPLK, a previously uncharacterized protein that is necessary for the assembly of a new cell posterior, and a microtubule plus-end directed orphan kinesin that is required for completing cleavage furrow ingression. The identification of these proteins provides new insight into T. brucei cytokinesis and establishes TOEFAZ1 as a key component of this essential and uniquely configured process in kinetoplastids.

A functional analysis of TOEFAZ1 uncovers protein domains essential for cytokinesis in Trypanosoma brucei.

The parasite Trypanosoma brucei is highly polarized, including a flagellum that is attached along the cell surface by the flagellum attachment zone (FAZ). During cell division, the new FAZ positions the cleavage furrow, which ingresses from the anterior tip of the cell towards the posterior. We recently identified TOEFAZ1 (for 'Tip of the Extending FAZ protein 1') as an essential protein in trypanosome cytokinesis. Here, we analyzed the localization and function of TOEFAZ1 domains by performing overexpression and RNAi complementation experiments. TOEFAZ1 comprises three domains with separable functions: an N-terminal α-helical domain that may be involved in FAZ recruitment, a central intrinsically disordered domain that keeps the morphogenic kinase TbPLK at the new FAZ tip, and a C-terminal zinc finger domain necessary for TOEFAZ1 oligomerization. Both the N-terminal and C-terminal domains are essential for TOEFAZ1 function, but TbPLK retention at the FAZ is not necessary for cytokinesis. The feasibility of alternative cytokinetic pathways that do not employ TOEFAZ1 are also assessed. Our results show that TOEFAZ1 is a multimeric scaffold for recruiting proteins that control the timing and location of cleavage furrow ingression.

Polo-like kinase is necessary for flagellum inheritance in Trypanosoma brucei.

Polo-like kinases play an important role in a variety of mitotic events in mammalian cells, ranging from centriole separation and chromosome congression to abscission. To fulfill these roles, Polo-like kinase homologs move to different cellular locations as the cell cycle progresses, starting at the centrosome, progressing to the spindle poles and then the midbody. In the protist parasite Trypanosoma brucei, the single polo-like kinase homolog T. brucei PLK (TbPLK) is essential for cytokinesis and is necessary for the correct duplication of a centrin-containing cytoskeletal structure known as the bilobe. We show that TbPLK has a dynamic localization pattern during the cell cycle. The kinase localizes to the basal body, which nucleates the flagellum, and then successively localizes to a series of cytoskeletal structures that regulate the position and attachment of the flagellum to the cell body. The kinase localizes to each of these structures as they are duplicating. TbPLK associates with a specialized set of microtubules, known as the microtubule quartet, which might transport the kinase during its migration. Depletion of TbPLK causes defects in basal body segregation and blocks the duplication of the regulators that position the flagellum, suggesting that its presence on these structures might be necessary for their proper biogenesis. TbPLK migrates throughout the cell in T. brucei, but the specific locations to which it targets and its functions are geared towards the inheritance of a properly positioned and attached flagellum.

Morphogenesis in Trypanosoma cruzi epimastigotes proceeds via a highly asymmetric cell division.

Trypanosoma cruzi is a protist parasite that is the causative agent of Chagas disease, a neglected tropical disease endemic to the Americas. T. cruzi cells are highly polarized and undergo morphological changes as they cycle within their insect and mammalian hosts. Work on related trypanosomatids has described cell division mechanisms in several life-cycle stages and identified a set of essential morphogenic proteins that serve as markers for key events during trypanosomatid division. Here, we use Cas9-based tagging of morphogenic genes, live-cell imaging, and expansion microscopy to study the cell division mechanism of the insect-resident epimastigote form of T. cruzi, which represents an understudied trypanosomatid morphotype. We find that T. cruzi epimastigote cell division is highly asymmetric, producing one daughter cell that is significantly smaller than the other. Daughter cell division rates differ by 4.9 h, which may be a consequence of this size disparity. Many of the morphogenic proteins identified in T. brucei have altered localization patterns in T. cruzi epimastigotes, which may reflect fundamental differences in the cell division mechanism of this life cycle stage, which widens and shortens the cell body to accommodate the duplicated organelles and cleavage furrow rather than elongating the cell body along the long axis of the cell, as is the case in life-cycle stages that have been studied in T. brucei. This work provides a foundation for further investigations of T. cruzi cell division and shows that subtle differences in trypanosomatid cell morphology can alter how these parasites divide.

Morphogenesis in Trypanosoma cruzi epimastigotes proceeds via a highly asymmetric cell division.

Trypanosoma cruzi is a protist parasite that is the causative agent of Chagas' disease, a neglected tropical disease endemic to the Americas. T. cruzi cells are highly polarized and undergo morphological changes as they cycle within their insect and mammalian hosts. Work on related trypanosomatids has described cell division mechanisms in several life-cycle stages and identified a set of essential morphogenic proteins that serve as markers for key events during trypanosomatid division. Here, we use Cas9-based tagging of morphogenic genes, live-cell imaging, and expansion microscopy to study the cell division mechanism of the insect-resident epimastigote form of T. cruzi, which represents an understudied trypanosomatid morphotype. We find that T. cruzi epimastigote cell division is highly asymmetric, producing one daughter cell that is significantly smaller than the other. Daughter cell division rates differ by 4.9 h, which may be a consequence of this size disparity. Many of the morphogenic proteins identified in T. brucei have altered localization patterns in T. cruzi epimastigoes, which may reflect fundamental differences in the cell division mechanism of this life cycle stage, which widens and shortens the cell body to accommodate the duplicated organelles and cleavage furrow rather than elongating the cell body along the long axis of the cell, as is the case in life-cycle stages that have been studied in T. brucei. This work provides a foundation for further investigations of T. cruzi cell division and shows that subtle differences in trypansomatid cell morphology can alter how these parasites divide.

Cytokinesis in Trypanosoma brucei relies on an orphan kinesin that dynamically crosslinks microtubules.

Many single-celled eukaryotes have complex cell morphologies defined by microtubules arranged into higher-order structures. The auger-like shape of the parasitic protist Trypanosoma brucei (T. brucei) is mediated by a parallel array of microtubules that underlies the plasma membrane. The subpellicular array must be partitioned and segregated using a microtubule-based mechanism during cell division. We previously identified an orphan kinesin, KLIF, that localizes to the ingressing cleavage furrow and is essential for the completion of cytokinesis. We have characterized the biophysical properties of a truncated KLIF construct in vitro to gain mechanistic insight into the function of this novel kinesin. We find that KLIF is a non-processive dimeric kinesin that dynamically crosslinks microtubules. Microtubules crosslinked by KLIF in an antiparallel orientation are translocated relative to one another, while microtubules crosslinked parallel to one another remain static, resulting in the formation of organized parallel bundles. In addition, we find that KLIF stabilizes the alignment of microtubule plus ends. These features provide a mechanistic understanding for how KLIF functions to form a new pole of aligned microtubule plus ends that defines the shape of the new cell posterior, which is an essential requirement for the completion of cytokinesis in T. brucei.

Cell division: Naegleria bundles up for mitosis.

How well do we understand the range of mechanisms used by eukaryotes for mitosis? A new study in a highly divergent eukaryote shows that unusual tubulin isoforms can create a mitotic spindle exclusively out of microtubule bundles.

The roles of enzyme localisation and complex formation in glycan assembly within the Golgi apparatus.

Cell surface glycans govern numerous cell-cell interactions are therefore key determinants of multicellular biology. They originate from biosynthetic pathways comprising an assembly line of glycosyltransferases within the Golgi compartment. Although the mechanisms of Golgi enzyme localisation are still under debate, the distribution of these enzymes among the Golgi cisternae can dictate the overall structures produced by the cell. Fine-tuning of glycan biosynthetic pathways is further accomplished by specific associations among glycosyltransferases. Together, localisation and association govern the assembly of complex glycans and thereby regulate interactions at the cell surface.

Alternate histories of cytokinesis: lessons from the trypanosomatids.

Popular culture has recently produced several "alternate histories" that describe worlds where key historical events had different outcomes. Beyond entertainment, asking "could this have happened a different way?" and "what would the consequences be?" are valuable approaches for exploring molecular mechanisms in many areas of research, including cell biology. Analogous to alternate histories, studying how the evolutionary trajectories of related organisms have been selected to provide a range of outcomes can tell us about the plasticity and potential contained within the genome of the ancestral cell. Among eukaryotes, a group of model organisms has been employed with great success to identify a core, conserved framework of proteins that segregate the duplicated cellular organelles into two daughter cells during cell division, a process known as cytokinesis. However, these organisms provide relatively sparse sampling across the broad evolutionary distances that exist, which has limited our understanding of the true potential of the ancestral eukaryotic toolkit. Recent work on the trypanosomatids, a group of eukaryotic parasites, exemplifies alternate historical routes for cytokinesis that illustrate the range of eukaryotic diversity, especially among unicellular organisms.

More than Microtubules: The Structure and Function of the Subpellicular Array in Trypanosomatids.

The subpellicular microtubule array defines the wide range of cellular morphologies found in parasitic kinetoplastids (trypanosomatids). Morphological studies have characterized array organization, but little progress has been made towards identifying the molecular mechanisms that are responsible for array differentiation during the trypanosomatid life cycle, or the apparent stability and longevity of array microtubules. In this review, we outline what is known about the structure and biogenesis of the array, with emphasis on Trypanosoma brucei, Trypanosoma cruzi, and Leishmania, which cause life-threatening diseases in humans and livestock. We highlight unanswered questions about this remarkable cellular structure that merit new consideration in light of our recently improved understanding of how the 'tubulin code' influences microtubule dynamics to generate complex cellular structures.

Regulating cell surface glycosylation with a small-molecule switch.

Correct localization of Golgi-resident enzymes is essential for the formation of specific glycan epitopes. In this chapter, we describe a method to control the localization, and thus the activity, of an individual glycosyltransferase by administration of a small molecule. Our method takes advantage of the modularity of most Golgi-resident enzymes, which are composed of localization and catalytic domains. These domains can be physically separated and fused to the small molecule binding proteins FRB and FKBP, which dimerize in the presence of rapamycin. In this way, rapamycin serves as a "switch" for enzyme activity.

A unified approach towards Trypanosoma brucei functional genomics using Gibson assembly.

Trypanosoma brucei is the causative agent of human African trypanosomiasis and nagana in cattle. Recent advances in high throughput phenotypic and interaction screens have identified a wealth of novel candidate proteins for diverse functions such as drug resistance, life cycle progression, and cytoskeletal biogenesis. Characterization of these proteins will allow a more mechanistic understanding of the biology of this important pathogen and could identify novel drug targets. However, methods for rapidly validating and prioritizing these potential targets are still being developed. While gene tagging via homologous recombination and RNA interference are available in T. brucei, a general strategy for creating the most effective constructs for these approaches is lacking. Here, we adapt Gibson assembly, a one-step isothermal process that rapidly assembles multiple DNA segments in a single reaction, to create endogenous tagging, overexpression, and long hairpin RNAi constructs that are compatible with well-established T. brucei vectors. The generality of the Gibson approach has several advantages over current methodologies and substantially increases the speed and ease with which these constructs can be assembled.

Proteomic identification of novel cytoskeletal proteins associated with TbPLK, an essential regulator of cell morphogenesis in Trypanosoma brucei.

Trypanosoma brucei is the causative agent of African sleeping sickness, a devastating disease endemic to sub-Saharan Africa with few effective treatment options. The parasite is highly polarized, including a single flagellum that is nucleated at the posterior of the cell and adhered along the cell surface. These features are essential and must be transmitted to the daughter cells during division. Recently we identified the T. brucei homologue of polo-like kinase (TbPLK) as an essential morphogenic regulator. In the present work, we conduct proteomic screens to identify potential TbPLK binding partners and substrates to better understand the molecular mechanisms of kinase function. These screens identify a cohort of proteins, most of which are completely uncharacterized, which localize to key cytoskeletal organelles involved in establishing cell morphology, including the flagella connector, flagellum attachment zone, and bilobe structure. Depletion of these proteins causes substantial changes in cell division, including mispositioning of the kinetoplast, loss of flagellar connection, and prevention of cytokinesis. The proteins identified in these screens provide the foundation for establishing the molecular networks through which TbPLK directs cell morphogenesis in T. brucei.

Polo-like kinase phosphorylation of bilobe-resident TbCentrin2 facilitates flagellar inheritance in Trypanosoma brucei.

In the protist parasite Trypanosoma brucei, the single Polo-like kinase (TbPLK) controls the inheritance of a suite of organelles that help position the parasite's single flagellum. These include the basal bodies, the bilobe, and the flagellar attachment zone (FAZ). TbCentrin2 was previously shown to be a target for TbPLK in vitro, and this is extended in this study to in vivo studies, highlighting a crucial role for serine 54 in the N-terminal domain. Duplication of the bilobe correlates with the presence of TbPLK and phospho-TbCentrin2, identified using phosphospecific antiserum. Mutation of S54 leads to slow growth (S54A) or no growth (S54D), the latter suggesting that dephosphorylation is needed to complete bilobe duplication and subsequent downstream events necessary for flagellum inheritance.

An analogue-sensitive approach identifies basal body rotation and flagellum attachment zone elongation as key functions of PLK in Trypanosoma brucei.

Polo-like kinases are important regulators of cell division, playing diverse roles in mitosis and cytoskeletal inheritance. In the parasite Trypanosoma brucei, the single PLK homologue TbPLK is necessary for the assembly of a series of essential organelles that position and adhere the flagellum to the cell surface. Previous work relied on RNA interference or inhibitors of undefined specificity to inhibit TbPLK, both of which have significant experimental limitations. Here we use an analogue-sensitive approach to selectively and acutely inhibit TbPLK. T. brucei cells expressing only analogue-sensitive TbPLK (TbPLK(as)) grow normally, but upon treatment with inhibitor develop defects in flagellar attachment and cytokinesis. TbPLK cannot migrate effectively when inhibited and remains trapped in the posterior of the cell throughout the cell cycle. Using synchronized cells, we show that active TbPLK is a direct requirement for the assembly and extension of the flagellum attachment zone, which adheres the flagellum to the cell surface, and for the rotation of the duplicated basal bodies, which positions the new flagellum so that it can extend without impinging on the old flagellum. This approach should be applicable to the many kinases found in the T. brucei genome that lack an ascribed function.

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

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.

TbG63, a golgin involved in Golgi architecture in Trypanosoma brucei.

Golgins are coiled-coil proteins that have been implicated in the structure and function of the Golgi complex. Here, we identify and characterize a trypanosomal golgin, TbG63, showing that it has a C-terminal membrane anchor and an N-terminus that projects into the cytoplasm. TbG63 in procyclic parasites is localized to the Golgi and interacts with the active, GTP-form of TbRab1A. Overexpression of TbG63 has dramatic effects on Golgi architecture -- effects that require the N-terminus -- whereas depletion has little, if any, effect on the growth rate. By contrast, in the bloodstream form of the parasite, depletion of TbG63 slows growth, although it has no obvious effect on the transport of a variant surface glycoprotein (VSG) or on Golgi structure. TbG63 might be a useful tool to study the structure and functioning of the Golgi complex.