An Alternative Model for the Role of RP2 Protein in Flagellum Assembly in the African Trypanosome

André, Jane; Kerry, Louise; Qi, Xin; Hawkins, Erica; Drižytė, Kristina; Ginger, Michael L.; McKean, Paul G.

doi:10.1074/jbc.m113.509521

Cited by 27 publications

(38 citation statements)

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“…Trypanosomes in G1 have a single mBB paired with an immature pBB, with each containing TbSAS6. TbRP2 (recognized by the antibody YL1/2) (Andre et al, 2014) is localized to transitional fibers, found only on mBBs (Andre et al, 2014). Maturation of the pBB is thought to precede assembly of new ones (Sherwin and Gull, 1989;Lacomble et al, 2010;Gluenz et al, 2011;Ikeda and de Graffenried, 2012).…”

Section: Resultsmentioning

confidence: 99%

“…Coverslips were briefly rinsed with PBS and rehydrated in blocking buffer [1% bovine serum albumin (BSA) (Sigma-Aldrich) in PBS] for 1 hour. Permeabilized trypanosomes were either costained with the primary antibodies YL1/2 (EMD Millipore, Billerica, MA) (Andre et al, 2014) and anti-TbSAS6 (Hu et al, 2015a) to detect basal bodies or stained with 20H5 (EMD Millipore) (He et al, 2005) for bilobes. The TbSAS6 antibody was a gift from Dr. Ziyin Li (University of Texas Health Science Center, San Antonio, TX).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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causes human African trypanosomiasis (HAT). The pyrrolopyrimidine AEE788 (a hit for anti-HAT drug discovery) associates with three trypanosome protein kinases. Herein we delineate the effects of AEE788 on using chemical biology strategies. AEE788 treatment inhibits DNA replication in the kinetoplast (mitochondrial nucleoid) and nucleus. In addition, AEE788 blocks duplication of the basal body and the bilobe without affecting mitosis. Thus, AEE788 prevents entry into the S-phase of the cell division cycle. To study the kinetics of early events in trypanosome division, we employed an "AEE788 block and release" protocol to stage entry into the S-phase. A time-course of DNA synthesis (nuclear and kinetoplast DNA), duplication of organelles (basal body, bilobe, kinetoplast, nucleus), and cytokinesis was obtained. Unexpected findings include the following: 1) basal body and bilobe duplication are concurrent; 2) maturation of probasal bodies, marked by TbRP2 recruitment, is coupled with nascent basal body assembly, monitored by localization of TbSAS6 at newly forming basal bodies; and 3) kinetoplast division is observed in G2 after completion of nuclear DNA synthesis. Prolonged exposure of trypanosomes to AEE788 inhibited transferrin endocytosis, altered cell morphology, and decreased cell viability. To discover putative effectors for the pleiotropic effects of AEE788, proteome-wide changes in protein phosphorylation induced by the drug were determined. Putative effectors include an SR protein kinase, bilobe proteins, TbSAS4, TbRP2, and BILBO-1. Loss of function of one or more of these effectors can, from published literature, explain the polypharmacology of AEE788 on trypanosome biology.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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“…Position within the cell cycle was determined based on the kDNA morphology (DAPI staining) and number of basal bodies (YL1/2 staining). YL1/2 recognizes RP2, a protein that localizes to transitional fibers and is therefore a marker only for mature basal bodies [41,42]…”

Section: Methodsmentioning

confidence: 99%

Identification of proteins involved inTrypanosoma bruceiDNA replication fork dynamics using nascent DNA proteomics

Rocha-Granados

Bermudez

Dodard

et al. 2018

Preprint

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29DNA replication, transcription and chromatin remodeling are coordinated to 30 ensure accurate duplication of genetic and epigenetic information. In regard to DNA 31 replication, trypanosomatid parasites such as Trypanosoma brucei display unusual 32 properties including significantly fewer origins of replication than model eukaryotes, a 33 highly divergent Origin Replication Complex (ORC), and an apparent lack of several 34 replication factor homologs. Although recent studies in T. brucei indicate functional links 35 among DNA replication, transcription, and antigenic variation, the underlying 36 mechanisms remain unknown. Here, we adapted an unbiased technology for the 37 identification of replication fork proteins called iPOND (isolation of proteins on nascent 38 DNA) to T. brucei, its first application to a parasite system. This led to the mass 39 spectrometric identification of core replication machinery and of proteins associated with 40 transcription, chromatin organization, and DNA repair that were enriched in the vicinity 41 of an unperturbed active replication fork. Of a total of 410 enriched proteins, among 42 which DNA polymerase  and replication factor C were scoring in the top, around 25%43 of the proteins identified were of unknown function and, therefore, have the potential to 44 be essential trypanosome-specific replication proteins. Initial characterization of a 45 protein annotated as a Replication Factor C subunit (Tb927.10.7990), and a protein of 46 unknown function (Tb927.3.5370) revealed that both proteins retain nuclear localization 47 throughout the cell cycle. While Tb927.3.5370 appeared to be a dispensable gene, 48 Tb927.10.7990 proved to be essential since its silencing caused a growth defect in 49 procyclic cells, accumulation of zoids and impaired DNA replication. Future studies on 50 the generated proteins list can contribute to the understanding of DNA replication 51 dynamics in T. brucei and how replication is coordinated with other cellular processes to 52 maintain genome integrity. 53 54 Introduction 55 Eukaryotic DNA replication is strictly coordinated and regulated by numerous 56 molecular machines to ensure genomic stability for future cell generations. DNA 57 replication initiation is coordinated with cell cycle progression through the multiprotein 58 Origin Recognition Complex (ORC) that plays an essential role by recruiting proteins 59 that lead to the assembly of the replicative machinery with the assistance of regulatory 60 components Cdc6 and Cdt1. The key factors Cdc45, the MCM replicative helicase 61 complex, and GINS proteins form the CMG complex that further recruits other 62 replication factors such as the clamp loader Replication factor C (RFC), the clamp 63 proliferating cell nuclear antigen (PCNA) and the three replicative DNA polymerases 64 (  ) leading to processive DNA replication [1,2]. 65 Instead of the archetypical Origin Recognition Complex (Orc1-6) found in model 66 eukaryotes, trypanosomes contain ORC1 and four other highly divergent ORC subunits ...

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“…YL1/2 an antibody targeting tyrosinated alpha tubulin and RP2, is located to the distal end of the basal body at the transitional fibers ( Fig. 1A; (34)(35)(36)). The individual TAC components were either tagged with different protein tags (myc, PTP or HA) or in the case of TAC102 we used a monoclonal α-TAC102 antibody (16).…”

Section: Relative Order Of the Tac Proteinsmentioning

confidence: 99%

A molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei

Hoffmann

Käser

Jakob

et al. 2017

Preprint

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In almost all eukaryotes mitochondria maintain their own genome. Despite the discovery more than 50 years ago still very little is known about how the genome is properly segregated during cell division.The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure proper segregation of the mitochondrial genome via the basal bodies movement, during cell cycle. Using super-resolution microscopy we precisely localize each of the currently known unique TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum towards the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on biochemical analysis the TAC consists of several non-overlapping subcomplexes suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC has an impact on mitochondrial organelle positioning however is not required for proper organelle biogenesis or segregation. Significance StatementMitochondrial genome replication and segregation are essential processes in most eukaryotic cells.While replication has been studied in some detail much less is known about the molecular machinery required distribute the replicated genomes. Using super-resolution microscopy in combination with molecular biology and biochemistry we show for the first time in which order the segregation machinery is assembled and that it is assembled de novo rather than in a semi conservative fashion in the single celled parasite Trypanosoma brucei. Furthermore, we demonstrate that the mitochondrial genome itself is not required for assembly to occur. It seems that the physical connection of the mitochondrial genome to cytoskeletal elements is a conserved feature in most eukaryotes, however the molecular components are highly diverse.

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An Alternative Model for the Role of RP2 Protein in Flagellum Assembly in the African Trypanosome

Cited by 27 publications

References 54 publications

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

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

Identification of proteins involved inTrypanosoma bruceiDNA replication fork dynamics using nascent DNA proteomics

A molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei

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