The protozoan Trypanosoma brucei sp. cause diseases in humans and animals. Studies of T. brucei cell biology have revealed unique features, such as major endocytic events being limited to a single ...region, and mitochondrial genome segregation mediated via basal bodies. Further understanding of trypanosome cell biology can be facilitated with super-resolution fluorescence microscopy. Lack of a plasma membrane probe for fixed trypanosomes remains a persistent problem in need of a working solution. Herein, we report protocols developed using mCLING in super-resolution structured illumination fluorescence microscopy (SR-SIM). mCLING comprehensively labels flagellar membranes, including nascent intracellular stages. To extend its usefulness for trypanosome biology we optimized mCLING in combination with organelle-specific antibodies for immunofluorescence of basal bodies or mitochondria. Then in work with live trypanosomes, we demonstrated internalization of mCLING into endocytic stations that overlap with LysoTracker in acidic organelles. Greater detail of the intracellular location of mCLING was obtained with SR-SIM after pulsing trypanosomes with the probe, and allowing continuous uptake of fluorescent concanavalin A (ConA) destined for lysosomes. In most cases, ConA and mCLING vesicles were juxtaposed but not coincident. A video of the complete image stack at the 15 min time point shows zones of mCLING staining surrounding patches of ConA, consistent with persistence of mCLING in membranes of compartments that contain luminal ConA. In summary, these studies establish mCLING as a versatile trypanosome membrane probe compatible with super-resolution microscopy that can be used for detailed analysis of flagellar membrane biogenesis. In addition, mCLING can be used for immunofluorescence in fixed, permeabilized trypanosomes. Its robust staining of the plasma membrane eliminates a need to overlay transmitted light images on fluorescence pictures obtained from widefield, confocal, or super-resolution microscopy.
The single mitochondrial nucleoid (kinetoplast) of Trypanosoma brucei is found proximal to a basal body (mature (mBB)/probasal body (pBB) pair). Kinetoplast inheritance requires synthesis of, and ...scission of kinetoplast DNA (kDNA) generating two kinetoplasts that segregate with basal bodies into daughter cells. Molecular details of kinetoplast scission and the extent to which basal body separation influences the process are unavailable. To address this topic, we followed basal body movements in bloodstream trypanosomes following depletion of protein kinase TbCK1.2 which promotes kinetoplast division. In control cells we found that pBBs are positioned 0.4 um from mBBs in G1, and they mature after separating from mBBs by at least 0.8 um: mBB separation reaches ~2.2 um. These data indicate that current models of basal body biogenesis in which pBBs mature in close proximity to mBBs may need to be revisited. Knockdown of TbCK1.2 produced trypanosomes containing one kinetoplast and two nuclei (1K2N), increased the percentage of cells with uncleaved kDNA 400%, decreased mBB spacing by 15%, and inhibited cytokinesis 300%. We conclude that (a) separation of mBBs beyond a threshold of 1.8 um correlates with division of kDNA, and (b) TbCK1.2 regulates kDNA scission. We propose a Kinetoplast Division Factor hypothesis that integrates these data into a pathway for biogenesis of two daughter mitochondrial nucleoids.
Kinetoplast Division Factors in a Trypanosome Mensa-Wilmot, Kojo; Hoffman, Benjamin; Wiedeman, Justin ...
Trends in parasitology,
February 2019, 2019-02-00, 20190201, Letnik:
35, Številka:
2
Journal Article
Recenzirano
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Inheritance of the single mitochondrial nucleoid (kinetoplast) in the trypanosome requires numerous proteins, many of whose precise roles are unclear. By considering kinetoplast DNA (kDNA) as a ...template for cleavage into two equal-size networks, we predicted sets of mutant kinetoplasts associated with defects in each of the five steps in the kinetoplast cycle. Comparison of these kinetoplasts with those obtained after gene knockdowns enabled assignment of proteins to five classes – kDNA synthesis, site of scission selection, scission, separation, and partitioning. These studies highlight how analysis of mutant kinetoplast phenotypes may be used to predict functional categories of proteins involved in the biogenesis of kinetoplasts.
Kinetoplasts (mitochondrial genome nucleoids) are important in bloodstream trypanosomes for the establishment of mitochondrial membrane potential.
Many proteins involved in segregation of kinetoplasts have been identified.
A region between a kinetoplast and basal bodies is described as a tripartite attachment complex (TAC).
A set of TAC-associated proteins (TACAPs) has been proposed as the machinery for kinetoplast segregation.
Subcomplexes of TACAPs that form in vivo have been described.
Several proteins that do not associate with TAC are involved in the maintenance of the kinetoplast.
New kinetoplast-associated proteins have been identified.
We are approaching an exciting period in the field when a molecular understanding of how all aspects of kinetoplast biogenesis are executed seems achievable.
Human African trypanosomiasis (HAT) is a deadly neglected tropical disease caused by the protozoan parasite Trypanosoma brucei. During the course of screening a collection of diverse nitrogenous ...heterocycles, we discovered two novel compounds that contain the tetracyclic core of the Yohimbine and Corynanthe alkaloids, were potent inhibitors of T. brucei proliferation and T. brucei methionyl-tRNA synthetase (TbMetRS) activity. Inspired by these key findings, we prepared several novel series of hydroxyalkyl δ-lactam, δ-lactam, and piperidine analogs and tested their anti-trypanosomal activity. A number of inhibitors were more potent against T. brucei than these initial hits with one hydroxyalkyl δ-lactam derivative being 25-fold more effective in our assay. Surprisingly, most of these active compounds failed to inhibit TbMetRS. This work underscores the importance of verifying, irrespective of close structural similarities, that new compounds designed from a lead with a known biological target engage the putative binding site.
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Inheritance of the single mitochondrial nucleoid (kinetoplast) in the trypanosome requires numerous proteins many of whose precise roles are unclear. By considering kinetoplast DNA (kDNA) as a ...template for cleavage into two equal-size networks, we predicted sets of mutant kinetoplasts associated with defects in each of five steps in the kinetoplast cycle. Comparison of these kinetoplasts with those obtained after gene knockdowns enabled assignment of proteins to five classes - kDNA synthesis, site of scission selection, scission, separation, and partitioning. These studies highlight how analysis of mutant kinetoplast phenotypes may be used to predict functional categories of proteins involved in biogenesis of kinetoplasts.
Trypanosoma brucei is a protozoan responsible for the disease Human African Trypanosomiasis. Fluorescence microscopy has proven to be an invaluable asset for revealing cellular morphology of the ...trypanosome. Recent advances in super-resolution microscopy have allowed the observation of details previously only seen in electron microscopy techniques. But the dearth of fluorescent membrane probes compatible with super-resolution microscopy has limited its effectiveness in studies of membrane biology. In Chapter 2 we present optimized protocols for the use of mCLING and report its utility in both fixed and live trypanosomes as a super resolution-compatible fluorescent marker of the plasma membrane, flagellar membrane, and endocytic vesicles. In Chapter 3, we report an effort to characterize signaling pathways of trypanosome casein kinase TbCK1.2. TbCK1.2 regulates kinetoplast division and cytokinesis and is essential for cell viability. Delineation of protein kinase signaling pathways in the trypanosome is hampered by several factors. First, 60% of proteins in T. brucei lack homologs in model eukaryotes. Second, substrates for T. brucei kinases have not been defined experimentally. Combined, these facts preclude bioinformatic approaches for kinasepathway prediction. Here, we present a bottom-up approach to characterize the signaling pathways of TbCK1.2 and identify its endogenous kinase substrates. The strategies presented in this study may find general use for identifying proteins involved in pathways regulated by protein kinases in other obscure eukaryotes. In Chapter 4, we identify evidence of proteome adaptation to loss of the TbCK1.2 pathway protein BBP59. Most genes in T. brucei are not required for proliferation of BSF trypanosomes, but few studies have directly asked “why?”. Here we begin to address this question by studying trypanosomes lacking a BBP59 gene and identifying alterations in the proteome that may promote survival. Our results suggest that the concepts of “essential” and “non-essential” genes may need to be re-evaluated. Modern proteomic techniques have enabled and simplified highly sensitive, quantitative, global proteomic studies that can easily identify “silent” changes in the proteome associated with gene knockout. We propose a molecular definition of “essential gene” as one whose loss necessitates proteome remodeling for survival.
Discovery of new chemotherapeutic lead agents can be accelerated by optimizing chemotypes proven to be effective in other diseases to act against parasites. One such medicinal chemistry campaign has ...focused on optimizing the anilinoquinazoline drug lapatinib (1) and the alkynyl thieno3,2-dpyrimidine hit GW837016X (NEU-391, 3) into leads for antitrypanosome drugs. We now report the structure–activity relationship studies of 3 and its analogs against Trypanosoma brucei, which causes human African trypanosomiasis (HAT). The series was also tested against Trypanosoma cruzi, Leishmania major, and Plasmodium falciparum. In each case, potent antiparasitic hits with acceptable toxicity margins over mammalian HepG2 and NIH3T3 cell lines were identified. In a mouse model of HAT, 3 extended life of treated mice by 50%, compared to untreated controls. At the cellular level, 3 inhibited mitosis and cytokinesis in T. brucei. Thus, the alkynylthieno3,2-dpyrimidine chemotype is an advanced hit worthy of further optimization as a potential chemotherapeutic agent for HAT.
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