Flagellum Attachment Research Articles

Novel Bilobe Components in Trypanosoma brucei Identified Using Proximity-Dependent Biotinylation

The trypanosomes are a family of parasitic protists of which the African trypanosome, Trypanosoma brucei, is the best characterized. The complex and highly ordered cytoskeleton of T. brucei has been shown to play vital roles in its biology but remains difficult to study, in large part owing to the intractability of its constituent proteins. Existing methods of protein identification, such as bioinformatic analysis, generation of monoclonal antibody panels, proteomics, affinity purification, and yeast two-hybrid screens, all have drawbacks. Such deficiencies-troublesome proteins and technical limitations-are common not only to T. brucei but also to many other protists, many of which are even less well studied. Proximity-dependent biotin identification (BioID) is a recently developed technique that allows forward screens for interaction partners and near neighbors in a native environment with no requirement for solubility in nonionic detergent. As such, it is extremely well suited to the exploration of the cytoskeleton. In this project, BioID was adapted for use in T. brucei. The trypanosome bilobe, a discrete cytoskeletal structure with few known protein components, represented an excellent test subject. Use of the bilobe protein TbMORN1 as a probe resulted in the identification of seven new bilobe constituents and two new flagellum attachment zone proteins. This constitutes the first usage of BioID on a largely uncharacterized structure, and demonstrates its utility in identifying new components of such a structure. This remarkable success validates BioID as a new tool for the study of unicellular eukaryotes in particular and the eukaryotic cytoskeleton in general.

Morphology of the trypanosome bilobe, a novel cytoskeletal structure.

The trypanosome bilobe is a cytoskeletal structure of unclear function. To date, four proteins have been shown to localize stably to it: TbMORN1, TbLRRP1, TbCentrin2, and TbCentrin4. In this study, a combination of immunofluorescence microscopy and electron microscopy was used to explore the morphology of the bilobe and its relationship to other nearby cytoskeletal structures in the African trypanosome procyclic trypomastigote. The use of detergent/salt-extracted flagellum preparations was found to be an effective way of discerning features of the cytoskeletal ultrastructure that are normally obscured. TbMORN1 and TbCentrin4 together define a hairpin structure comprising an arm of TbCentrin4 and a fishhook of TbMORN1. The two arms flank a specialized microtubule quartet and the flagellum attachment zone filament, with TbMORN1 running alongside the former and TbCentrin4 alongside the latter. The hooked part of TbMORN1 sits atop the flagellar pocket collar marked by TbBILBO1. The TbMORN1 bilobe occasionally exhibits tendrillar extensions that seem to be connected to the basal and probasal bodies. The TbMORN1 molecules present on these tendrils undergo higher rates of turnover than those for molecules on the main bilobe structure. These observations have been integrated with previous detailed descriptions of the cytoskeletal elements in trypanosome cells.

An intracellular membrane junction consisting of flagellum adhesion glycoproteins links flagellum biogenesis to cell morphogenesis in Trypanosoma brucei

African trypanosomes have a single, membrane-bounded flagellum that is attached to the cell cortex by membrane adhesion proteins and an intracellular flagellum attachment zone (FAZ) complex. The coordinated assembly of flagellum and FAZ, during the cell cycle and the life cycle development, plays a pivotal role in organelle positioning, cell division and cell morphogenesis. To understand how the flagellum and FAZ assembly are coordinated, we examined the domain organization of the flagellum adhesion protein 1 (FLA1), a glycosylated, transmembrane protein essential for flagellum attachment and cell division. By immunoprecipitation of a FLA1-truncation mutant that mislocalized to the flagellum, a novel FLA1-binding protein (FLA1BP) was identified in procyclic Trypanosoma brucei. The interaction between FLA1 on the cell membrane and FLA1BP on the flagellum membrane acts like a molecular zipper, joining flagellum membrane to cell membrane and linking flagellum biogenesis to FAZ elongation. By coordinating flagellum and FAZ assembly during the cell cycle, morphology information is transmitted from the flagellum to the cell body.

An orphan kinesin in trypanosomes cooperates with a kinetoplastid-specific kinesin to maintain cell morphology through regulating subpellicular microtubules

Microtubules are a vital part of the cytoskeleton of eukaryotic cells and are involved in various cellular processes. The cytoskeleton of Trypanosoma brucei is characterized by an array of subpellicular microtubules and is essential for maintenance of cell shape and polarity, but little is known about the regulation of the assembly and organization of the subpellicular microtubule corset. Here, we report that the orphan kinesin TbKIN-D regulates the organization of subpellicular microtubules and is required for maintaining cell morphology. TbKIN-D possesses in vitro ATPase activity, associates with cytoskeletal microtubules and is distributed throughout the cytoskeleton at all cell cycle stages. RNAi of TbKIN-D disrupts the organization of the subpellicular microtubule corset and distorts cell morphology, resulting in round cells with an elongated posterior filled with newly assembled microtubules. Depletion of TbKIN-D also abolishes the segregation of organelles and cytoskeletal structures, suggesting that cellular morphogenesis is essential for proper organelle segregation. Moreover, TbKIN-D deficiency impairs the attachment of the new flagellum without compromising the formation of the flagellum attachment zone. Finally, we identified TbKIN-C, a kinetoplastid-specific kinesin known to regulate subpellicular microtubules and cell morphogenesis in T. brucei, as a partner of TbKIN-D. Further, we demonstrate that interaction between TbKIN-C and TbKIN-D requires the coiled-coil motifs in the C-termini of both proteins. Altogether, our results suggest that TbKIN-D cooperates with TbKIN-C to maintain cell morphology by regulating the organization of the subpellicular microtubule corset.

A Trypanosoma brucei Protein Required for Maintenance of the Flagellum Attachment Zone and Flagellar Pocket ER Domains

Trypanosomes and Leishmanias are important human parasites whose cellular architecture is centred on the single flagellum. In trypanosomes, this flagellum is attached to the cell along a complex flagellum attachment zone (FAZ), comprising flagellar and cytoplasmic components, the integrity of which is required for correct cell morphogenesis and division. The cytoplasmic FAZ cytoskeleton is conspicuously associated with a sheet of endoplasmic reticulum termed the ‘FAZ ER’. In the present work, 3D electron tomography of bloodstream form trypanosomes was used to clarify the nature of the FAZ ER. We also identified TbVAP, a T. brucei protein whose knockdown by RNAi in procyclic form cells leads to a dramatic reduction in the FAZ ER, and in the ER associated with the flagellar pocket. TbVAP is an orthologue of VAMP-associated proteins (VAPs), integral ER membrane proteins whose mutation in humans has been linked to familial motor neuron disease. The localisation of tagged TbVAP and the phenotype of TbVAP RNAi in procyclic form trypanosomes are consistent with a function for TbVAP in the maintenance of sub-populations of the ER associated with the FAZ and the flagellar pocket. Nevertheless, depletion of TbVAP did not affect cell viability or cell cycle progression.

A coiled-coil- and C2-domain-containing protein is required for FAZ assembly and cell morphology in Trypanosoma brucei

Trypanosoma brucei, a flagellated protozoan parasite causing human sleeping sickness, relies on a subpellicular microtubule array for maintenance of cell morphology. The flagellum is attached to the cell body through a poorly understood flagellum attachment zone (FAZ), and regulates cell morphogenesis using an unknown mechanism. Here we identified a new FAZ component, CC2D, which contains coiled-coil motifs followed by a C-terminal C2 domain. T. brucei CC2D is present on the FAZ filament, FAZ-juxtaposed ER membrane and the basal bodies. Depletion of CC2D inhibits the assembly of a new FAZ filament, forming a FAZ stub with a relatively fixed size at the base of a detached, but otherwise normal, flagellum. Inhibition of new FAZ formation perturbs subpellicular microtubule organization and generates short daughter cells. The cell length shows a strong linear correlation with FAZ length, in both control cells and in cells with inhibited FAZ assembly. Together, our data support a direct function of FAZ assembly in determining new daughter cell length by regulating subpellicular microtubule synthesis.

The Repetitive Cytoskeletal Protein H49 of Trypanosoma cruzi Is a Calpain-Like Protein Located at the Flagellum Attachment Zone

Background Trypanosoma cruzi has a single flagellum attached to the cell body by a network of specialized cytoskeletal and membranous connections called the flagellum attachment zone. Previously, we isolated a DNA fragment (clone H49) which encodes tandemly arranged repeats of 68 amino acids associated with a high molecular weight cytoskeletal protein. In the current study, the genomic complexity of H49 and its relationships to the T. cruzi calpain-like cysteine peptidase family, comprising active calpains and calpain-like proteins, is addressed. Immunofluorescence analysis and biochemical fractionation were used to demonstrate the cellular location of H49 proteins.Methods and FindingsAll of H49 repeats are associated with calpain-like sequences. Sequence analysis demonstrated that this protein, now termed H49/calpain, consists of an amino-terminal catalytic cysteine protease domain II, followed by a large region of 68-amino acid repeats tandemly arranged and a carboxy-terminal segment carrying the protease domains II and III. The H49/calpains can be classified as calpain-like proteins as the cysteine protease catalytic triad has been partially conserved in these proteins. The H49/calpains repeats share less than 60% identity with other calpain-like proteins in Leishmania and T. brucei, and there is no immunological cross reaction among them. It is suggested that the expansion of H49/calpain repeats only occurred in T. cruzi after separation of a T. cruzi ancestor from other trypanosomatid lineages. Immunofluorescence and immunoblotting experiments demonstrated that H49/calpain is located along the flagellum attachment zone adjacent to the cell body.ConclusionsH49/calpain contains large central region composed of 68-amino acid repeats tandemly arranged. They can be classified as calpain-like proteins as the cysteine protease catalytic triad is partially conserved in these proteins. H49/calpains could have a structural role, namely that of ensuring that the cell body remains attached to the flagellum by connecting the subpellicular microtubule array to it.

The structural basis of localizing polo-like kinase to the flagellum attachment zone in Trypanosoma brucei.

The polo-like kinase in the deep branching eukaryote Trypanosoma brucei (TbPlk) has many unique features. Unlike all the other polo-like kinases known to associate with the nucleus and controlling both mitosis and cytokinesis, TbPlk localizes to the flagellum attachment zone (FAZ) and regulates only cytokinesis in T. brucei. TbPlk was, however, previously found capable of complementing all the multiple Plk (Cdc5) functions in Saccharomyces cerevisiae, indicating that it has acquired all the functions of Cdc5. In the present study, Cdc5 tagged with an enhanced yellow fluorescence protein (EYFP) localized exclusively in the FAZ of T. brucei, suggesting that the unusual localization and limited function of TbPlk are probably attributed to the particular environment in T. brucei cells. Structural basis for the FAZ localization of TbPlk was further investigated with TbPlk and TbPlk mutants tagged with EYFP and expressed in T. brucei. The results indicated that a kinase-inactive mutant N169A and a TbPlk mutant with the entire kinase domain (KD) deleted both localized to the FAZ. Substantial association with FAZ was also maintained when one of the two polo-boxes (PB1 or 2) or the linker region between them was deleted from TbPlk. But a deletion of both polo-boxes led to a complete exclusion of the protein from FAZ. All the deletion mutants retained the kinase activity, further indicating that the TbPlk kinase function does not play a role for FAZ localization. The two polo boxes in TbPlk are most likely instrumental in localizing the protein to FAZ through potential interactions with certain FAZ structural component(s). A putative cryptic bipartite nuclear targeting signal was identified in TbPlk, which was capable of directing TbPlk into the nucleus when either the kinase activity was lost or the PB1 was deleted from the protein.

Replication of the ERES:Golgi junction in bloodstream‐form African trypanosomes

The biogenesis of the ER Exit Site/Golgi Junction (EGJ) in bloodstream-form African trypanosomes is investigated using tagged markers for ER Exit Sites, the Golgi and the bilobe structure. The typical pattern is two EGJ in G1 phase (1 kinetoplast/1 nucleus, 1K1N) through S-phase (2K1N), duplication to four EGJ in post-mitotic cells (2K2N) and segregation of two EGJ to each daughter. Lesser cell percentages have elevated EGJ copy numbers in all stages, and blocking cell cycle progression results in even higher copy numbers. EGJs are closely aligned with the flagellar attachment zone (FAZ) indicating nucleation on the FAZ-associated ER (FAZ:ER). Only the most posterior EGJ in each cell is in proximity to the bilobe, which is located at the base of the FAZ filament near the mouth of the flagellar pocket. These results indicate that EGJ replication in bloodstream trypanosomes is not tightly coupled to the cell cycle. Furthermore, segregation of EGJ is not obligately mediated by the bilobe, rather assembly of the EGJ on the FAZ:ER, which is coupled to the flagellar cytoskeleton, apparently ensures segregation with fidelity during cytokinesis. These findings differ markedly from procyclic-form trypanosomes, and models highlighting these stage-specific differences in EGJ biogenesis are proposed.

Independent Analysis of the Flagellum Surface and Matrix Proteomes Provides Insight into Flagellum Signaling in Mammalian-infectious Trypanosoma brucei

The flagellum of African trypanosomes is an essential and multifunctional organelle that functions in motility, cell morphogenesis, and host-parasite interaction. Previous studies of the trypanosome flagellum have been limited by the inability to purify flagella without first removing the flagellar membrane. This limitation is particularly relevant in the context of studying flagellum signaling, as signaling requires surface-exposed proteins in the flagellar membrane and soluble signaling proteins in the flagellar matrix. Here we employ a combination of genetic and mechanical approaches to purify intact flagella from the African trypanosome, Trypanosoma brucei, in its mammalian-infectious stage. We combined flagellum purification with affinity-purification of surface-exposed proteins to conduct independent proteomic analyses of the flagellum surface and matrix fractions. The proteins identified encompass a broad range of molecular functionalities, including many predicted to function in signaling. Immunofluorescence and RNA interference studies demonstrate flagellum localization and function for proteins identified and provide insight into mechanisms of flagellum attachment and motility. The flagellum surface proteome includes many T. brucei-specific proteins and is enriched for proteins up-regulated in the mammalian-infectious stage of the parasite life-cycle. The combined results indicate that the flagellum surface presents a diverse and dynamic host-parasite interface that is well-suited for host-parasite signaling.

Molecular bases of cytoskeleton plasticity during the Trypanosoma brucei parasite cycle

African trypanosomes are flagellated protozoan parasites responsible for sleeping sickness and transmitted by tsetse flies. The accomplishment of their parasite cycle requires adaptation to highly diverse environments. These transitions take place in a strictly defined order and are accompanied by spectacular morphological modifications in cell size, shape and positioning of organelles. To understand the molecular bases of these processes, parasites isolated from different tissues of the tsetse fly were analysed by immunofluorescence with markers for specific cytoskeleton components and by a new immunofluorescence-based assay for evaluation of the cell volume. The data revealed striking differences between proliferative stages found in the midgut or in the salivary glands and the differentiating stage occurring in the proventriculus. Cell proliferation was characterized by a significant increase in cell volume, by a pronounced cell elongation marked by microtubule extension at the posterior end, and by the production of a new flagellum similar to the existing one. In contrast, the differentiating stage found in the proventriculus does not display any increase in cell volume neither in cell length, but is marked by a profound remodelling of the posterior part of the cytoskeleton and by changes in molecular composition and/or organization of the flagellum attachment zone.

Attachment of flagellum to the cell body is important to the kinetics of transferrin uptake by Trypanosoma cruzi

The flagellar pocket and the cytostome are surface domains of Trypanosoma cruzi epimastigote involved in acquisition of nutrients. The cytostome is physically connected to the flagellar complex. To investigate if this association plays a role in endocytosis in T. cruzi, the endocytic activity in wild type and gp72 null mutant (flagellum-cell body attachment region is absent) epimastigotes was compared. Both wild type and mutant cells were incubated with transferrin conjugated with Alexa 543 or gold particles over different time periods and thereafter qualitatively and quantitatively analyzed by flow cytometry and transmission electron microscopy. Flow cytometry analysis showed a reduction in transferrin uptake by null mutant after 30 min of incubation. In addition, at this time period, signals detected by fluorescence microscopy were slightly lower in null mutant cells. At lower incubation times, no differences between wild type and mutant epimastigotes could be observed. Quantitative data obtained by morphometric and flow cytometry analysis suggested that the speed of the endocytic process in the null mutant was similar to wild type cells, although null mutants were not able to bind cargo and therefore internalize as much as wild type epimastigotes. Our observations suggest that the physical association between cytostome and the flagellar complex plays a role in endocytosis efficiency by epimastigotes of T. cruzi.

A Comparative Proteomic Analysis Reveals a New Bi-Lobe Protein Required for Bi-Lobe Duplication and Cell Division in Trypanosoma brucei

A Golgi-associated bi-lobed structure was previously found to be important for Golgi duplication and cell division in Trypanosoma brucei. To further understand its functions, comparative proteomics was performed on extracted flagellar complexes (including the flagellum and flagellum-associated structures such as the basal bodies and the bi-lobe) and purified flagella to identify new bi-lobe proteins. A leucine-rich repeats containing protein, TbLRRP1, was characterized as a new bi-lobe component. The anterior part of the TbLRRP1-labeled bi-lobe is adjacent to the single Golgi apparatus, and the posterior side is tightly associated with the flagellar pocket collar marked by TbBILBO1. Inducible depletion of TbLRRP1 by RNA interference inhibited duplication of the bi-lobe as well as the adjacent Golgi apparatus and flagellar pocket collar. Formation of a new flagellum attachment zone and subsequent cell division were also inhibited, suggesting a central role of bi-lobe in Golgi, flagellar pocket collar and flagellum attachment zone biogenesis.

Polo-Like Kinase Guides Cytokinesis in Trypanosoma brucei through an Indirect Means

Polo-like kinase in Trypanosoma brucei (TbPLK) is confined to the flagellum attachment zone (FAZ) and regulates only cytokinetic initiation. However, it apparently diffuses into the cytoplasm before the trans-localization of chromosomal passenger complex (CPC) from the midzone of central spindle to FAZ, which is known to be required for initiating cytokinesis. Synchronized T. brucei procyclic cells treated with a TbPLK inhibitor, GW843682X (GW), in late S phase were found to go through a full cell cycle at a normal pace before being arrested at cytokinetic initiation in the second cycle. However, synchronized cells treated with GW in G(1) phase were arrested at cytokinetic initiation within the first cell cycle, suggesting that inhibition of TbPLK at its emergence blocks cytokinesis within the same cell cycle. To rule out potential off-target effects from GW, TbPLK RNA interference (RNAi) was induced to deplete TbPLK, and the progression of synchronized cells from late S phase was also found to be arrested at cytokinetic initiation within the first cell cycle. Apparently, TbPLK has accomplished its role in guiding cytokinesis before the late S phase, presumably by phosphorylating a certain substrate(s) during S phase, which may play a critical role in initiating the subsequent cytokinesis.

The Aurora Kinase in Trypanosoma brucei Plays Distinctive Roles in Metaphase-Anaphase Transition and Cytokinetic Initiation

Aurora B kinase is an essential regulator of chromosome segregation with the action well characterized in eukaryotes. It is also implicated in cytokinesis, but the detailed mechanism remains less clear, partly due to the difficulty in separating the latter from the former function in a growing cell. A chemical genetic approach with an inhibitor of the enzyme added to a synchronized cell population at different stages of the cell cycle would probably solve this problem. In the deeply branched parasitic protozoan Trypanosoma brucei, an Aurora B homolog, TbAUK1, was found to control both chromosome segregation and cytokinetic initiation by evidence from RNAi and dominant negative mutation. To clearly separate these two functions, VX-680, an inhibitor of TbAUK1, was added to a synchronized T. brucei procyclic cell population at different cell cycle stages. The unique trans-localization pattern of the chromosomal passenger complex (CPC), consisting of TbAUK1 and two novel proteins TbCPC1 and TbCPC2, was monitored during mitosis and cytokinesis by following the migration of the proteins tagged with enhanced yellow fluorescence protein in live cells with time-lapse video microscopy. Inhibition of TbAUK1 function in S-phase, prophase or metaphase invariably arrests the cells in the metaphase, suggesting an action of TbAUK1 in promoting metaphase-anaphase transition. TbAUK1 inhibition in anaphase does not affect mitotic exit, but prevents trans-localization of the CPC from the spindle midzone to the anterior tip of the new flagellum attachment zone for cytokinetic initiation. The CPC in the midzone is dispersed back to the two segregated nuclei, while cytokinesis is inhibited. In and beyond telophase, TbAUK1 inhibition has no effect on the progression of cytokinesis or the subsequent G1, S and G2 phases until a new metaphase is attained. There are thus two clearly distinct points of TbAUK1 action in T. brucei: the metaphase-anaphase transition and cytokinetic initiation. This is the first time to our knowledge that the dual functions of an Aurora B homolog is dissected and separated into two clearly distinct time frames in a cell cycle.

The Glycosylphosphatidylinositol-PLC in Trypanosoma brucei Forms a Linear Array on the Exterior of the Flagellar Membrane Before and After Activation

Bloodstream forms of Trypanosoma brucei contain a glycosylphosphatidylinositol-specific phospholipase C (GPI-PLC) that cleaves the GPI-anchor of the variable surface glycoprotein (VSG). Its location in trypanosomes has been controversial. Here, using confocal microscopy and surface labelling techniques, we show that the GPI-PLC is located exclusively in a linear array on the outside of the flagellar membrane, close to the flagellar attachment zone, but does not co-localize with the flagellar attachment zone protein, FAZ1. Consequently, the GPI-PLC and the VSG occupy the same plasma membrane leaflet, which resolves the topological problem associated with the cleavage reaction if the VSG and the GPI-PLC were on opposite sides of the membrane. The exterior location requires the enzyme to be tightly regulated to prevent VSG release under basal conditions. During stimulated VSG release in intact cells, the GPI-PLC did not change location, suggesting that the release mechanism involves lateral diffusion of the VSG in the plane of the membrane to the fixed position of the GPI-PLC.

The bilobe structure of Trypanosoma brucei contains a MORN-repeat protein

The Golgi of the kinetoplastid parasite Trypanosoma brucei is closely apposed to a bilobe structure containing TbCentrin2 and TbCentrin4 in procyclic cells. However, both are additionally localized to the basal bodies. Here we report the characterization of a membrane occupation and recognition nexus (MORN)-repeat protein, TbMORN1, present at the bilobe but not at the basal body. The anterior part of the TbMORN1 structure partially overlapped with the flagellar attachment zone while the posterior part overlapped with the flagellar pocket. Depletion studies using RNAi showed that there was a modest growth inhibition in procyclic cells but lethality in bloodstream cells, showing that it is an essential protein in the bloodstream form of the organism. TbMORN1 appears to be a useful marker for the bilobe in T. brucei.

Three-dimensional cellular architecture of the flagellar pocket and associated cytoskeleton in trypanosomes revealed by electron microscope tomography

This study uses electron tomography linked to a variety of other EM methods to provide an integrated view of the flagellar pocket and basal body area of the African trypanosome procyclic trypomastigote. We reveal the pocket as an asymmetric membranous 'balloon' with two boundary structures. One of these - the collar - defines the flagellum exit point. The other defines the entry point of the flagellum into the pocket and consists of both an internal transitional fibre array and an external membrane collarette. A novel set of nine radial fibres is described in the basal body proximal zone. The pocket asymmetry is invariably correlated with the position of the probasal body and Golgi. The neck region, just distal to the flagellum exit site, is a specialised area of membrane associated with the start of the flagellum attachment zone and signifies the point where a special set of four microtubules, nucleated close to the basal bodies, joins the subpellicular array. The neck region is also associated with the single Golgi apparatus of the cell. The flagellar exit point interrupts the subpellicular microtubule array with discrete endings of microtubules at the posterior side. Overall, our studies reveal a highly organised, yet dynamic, area of cytoplasm and will be informative in understanding its function.

KMP-11, a Basal Body and Flagellar Protein, Is Required for Cell Division in Trypanosoma brucei

Kinetoplastid membrane protein 11 (KMP-11) has been identified as a flagellar protein and is conserved among kinetoplastid parasites, but its potential function remains unknown. In a recent study, we identified KMP-11 as a microtubule-bound protein localizing to the flagellum as well as the basal body in both procyclic and bloodstream forms of Trypanosoma brucei (Z. Li, J. H. Lee, F. Chu, A. L. Burlingame, A. Gunzl, and C. C. Wang, PLoS One 3:e2354, 2008). Silencing of KMP-11 by RNA interference inhibited basal body segregation and cytokinesis in both forms and resulted in multiple nuclei of various sizes, indicating a continuous, albeit somewhat defective, nuclear division while cell division was blocked. KMP-11 knockdown in the procyclic form led to severely compromised formation of the new flagellum attachment zone (FAZ) and detachment of the newly synthesized flagellum. However, a similar phenotype was not observed in the bloodstream form depleted of KMP-11. Thus, KMP-11 is a flagellar protein playing critical roles in regulating cytokinesis in both forms of the trypanosomes. Its distinct roles in regulating FAZ formation in the two forms may provide a clue to the different mechanisms of cytokinetic initiation in procyclic and bloodstream trypanosomes.

Polo-Like Kinase Is Expressed in S/G 2 /M Phase and Associated with the Flagellum Attachment Zone in both Procyclic and Bloodstream Forms of Trypanosoma brucei

Trypanosoma brucei, the etiologic agent of African sleeping sickness, divides into insect (procyclic) and bloodstream forms. These two forms are subject to distinct cell cycle regulations, with cytokinesis controlled primarily by basal body/kinetoplast segregation in the procyclic form but by mitosis in the bloodstream form. Polo-like kinases (PLKs), known to play essential roles in regulating both mitosis and cytokinesis among eukaryotes, have a homologue in T. brucei, TbPLK, which regulates only cytokinesis. In our previous study, overexpressed triply hemagglutinin-tagged TbPLK (TbPLK-3HA) in the procyclic form localized to a mid-dorsal point and the anterior tip of the cell along the flagellum attachment zone (FAZ). In our current study, TbPLK-3HA expressed at the endogenous level was identified at the same dorsal location of both procyclic and bloodstream forms, albeit it was no longer detectable at the anterior tip of the cell. Endogenously expressed TbPLK fused with an enhanced yellow fluorescent protein (EYFP) localized to the same dorsal location along the FAZs in living procyclic and bloodstream cells. Fluorescence-activated cell sorter analysis of hydroxyurea-synchronized procyclic cells revealed that TbPLK-EYFP emerges during S phase, persists through G(2)/M phase, and vanishes in G(1) phase. An indicated TbPLK-EYFP association with the FAZs of G(2)/M cells may thus represent a timely localization to a potential initiation site of cytokinesis, which agrees with the recognized role of TbPLK in cytokinetic initiation.