AEE788 Inhibits Basal Body Assembly and Blocks DNA Replication in the African Trypanosome

Sullenberger, Catherine; Piqué, Daniel G.; Ogata, Yuko; Mensa-Wilmot, Kojo

doi:10.1124/mol.116.106906

Cited by 10 publications

(33 citation statements)

References 105 publications

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“…Formation of a nascent flagellum is detected by mCLING staining. Prior to division of a kinetoplast [ 3 , 14 ], a new flagellum protrudes from a newly-formed flagellar pocket alongside the old flagellum [ 15 ] ( Fig 1C ) and continues to elongate through the period when the kinetoplast divides ( Fig 1D–1F ). The T .…”

Section: Resultsmentioning

confidence: 99%

“…Bloodstream form T . brucei Lister 427 was cultured in HMI-9 media [ 10 ] in log-phase growth (≤10 6 cells/mL) [ 3 ].…”

Section: Methodsmentioning

confidence: 99%

“…antigenic variation, RNA editing, trans-splicing, and catenated mitochondrial DNA). Fluorescence microscopy is frequently used to visualize cells, localize trypanosome proteins [ 3 ], and detect small molecule drugs intracellularly [ 4 ]. Resolution of the technique is constrained by the diffraction limit of visible light, as it cannot distinguish fluorescent objects within ≈200 nm lateral to each other (reviewed in [ 5 ]).…”

Section: Introductionmentioning

confidence: 99%

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A fixable probe for visualizing flagella and plasma membranes of the African trypanosome

Wiedeman

Mensa-Wilmot

2018

PLoS ONE

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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.

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Section: Resultsmentioning

confidence: 99%

“…Bloodstream form T . brucei Lister 427 was cultured in HMI-9 media [ 10 ] in log-phase growth (≤10 6 cells/mL) [ 3 ].…”

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A fixable probe for visualizing flagella and plasma membranes of the African trypanosome

Wiedeman

Mensa-Wilmot

2018

PLoS ONE

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“…Additional evidence has been reported in support of AEE788 T. brucei BSF proliferation inhibition. AEE788, evaluated in T. brucei (strain RUMP528 or Lister 427), has demonstrated an EC 50 value of 5 μ m between 4 and 9 h of treatment, and beyond 9 h the drug (5 μ m ) inhibited Tb Tf endocytosis …”

Section: Human Kinase Inhibitor Repurposing In Trypanosomatidsmentioning

confidence: 99%

Repurposing of Human Kinase Inhibitors in Neglected Protozoan Diseases

et al. 2017

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Human African trypanosomiasis (HAT), Chagas disease, and leishmaniasis belong to a group of infectious diseases known as neglected tropical diseases and are induced by infection with protozoan parasites named trypanosomatids. Drugs in current use have several limitations, and therefore new candidate drugs are required. The majority of current therapeutic trypanosomatid targets are enzymes or cell‐surface receptors. Among these, eukaryotic protein kinases are a major group of protein targets whose modulation may be beneficial for the treatment of neglected tropical protozoan diseases. This review summarizes the finding of new hit compounds for neglected tropical protozoan diseases, by repurposing known human kinase inhibitors on trypanosomatids. Kinase inhibitors are grouped by human kinase family and discussed according to the screening (target‐based or phenotypic) reported for these compounds on trypanosomatids. This collection aims to provide insight into repurposed human kinase inhibitors and their importance in the development of new chemical entities with potential beneficial effects on the diseases caused by trypanosomatids.

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“…Kinetoplast division is detected in G2. Mitosis in 2K1N trypanosomes produces 2K2N cells that after cytokinesis yield two 1K1N cells [22,23].…”

Section: Introductionmentioning

confidence: 99%

Casein kinase TbCK1.2 regulates division of kinetoplast DNA, and movement of basal bodies in the African trypanosome

et al. 2021

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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.

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AEE788 Inhibits Basal Body Assembly and Blocks DNA Replication in the African Trypanosome

Cited by 10 publications

References 105 publications

A fixable probe for visualizing flagella and plasma membranes of the African trypanosome

A fixable probe for visualizing flagella and plasma membranes of the African trypanosome

Repurposing of Human Kinase Inhibitors in Neglected Protozoan Diseases

Casein kinase TbCK1.2 regulates division of kinetoplast DNA, and movement of basal bodies in the African trypanosome

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