Characterization of two novel proteins involved in mitochondrial DNA anchoring

AbstractTrypanosoma brucei is a single celled eukaryotic parasite in the group of the Excavates. T. brucei cells harbor a single mitochondrion with a singular mitochondrial genome, that consists of a unique network of thousands of interwoven circular DNA molecule copies and is termed the kinetoplast DNA (kDNA). To ensure proper inheritance of the kDNA to the daughter cells the genome is linked to the basal body, the master organizer of the cell cycle in trypanosomes. The structure connecting the basal body and kDNA is termed the tripartite attachment complex (TAC). Using a combination of proteomics and RNAi (depletomics) we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and Tb927.11.16120 as novel candidates of a structure that connects the TAC to the kDNA. Both proteins localize in the region of the unilateral filaments between TAC102 and the kDNA and depletion of each leads to a strong kDNA loss phenotype. TbmtHMG44 and Tb927.11.16120 stably associate with extracted flagella, even after DNase treatment however they do require the kDNA for initial assembly. Furthermore we demonstrate that recombinant Tb927.11.16120 is a DNA binding protein and thus a promising candidate to link the TAC to the kDNA.

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Timing of nuclear and kinetoplast DNA replication and early morphological events in the cell cycle of Trypanosoma brucei

Journal of Cell Science ◽

10.1242/jcs.95.1.49 ◽

1990 ◽

Vol 95 (1) ◽

pp. 49-57 ◽

Cited By ~ 1

Author(s):

R. Woodward ◽

K. Gull

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Trypanosoma Brucei ◽

Basal Body ◽

Unit Cell ◽

Kinetoplast Dna ◽

Interphase Cell ◽

Body Formation ◽

Timing Of Initiation ◽

Single Mitochondrion

We have used immunofluorescent detection of 5-bromo-2-deoxyuridine-substituted DNA in order to determine the timing of initiation and the duration of nuclear and kinetoplast S-phases within the procyclic stage of the Trypanosoma brucei cell cycle. Both nuclear and kinetoplast S-phases were shown to be periodic, occupying 0.18 and 0.12 of the unit cell cycle, respectively. In addition, initiation of both of these S-phases were in approximate synchrony, differing by only 0.03 of the unit cell cycle. We have also used a monoclonal antibody that recognises the basal bodies of T. brucei in order to visualise cells possessing a new pro-basal body and hence determine the time of pro-basal body formation within the cell cycle. Pro-basal body formation occurred within a few minutes of the initiation of nuclear S-phase, at 0.41 of the unit cell cycle. This provides detection of the earliest known cell cycle event in T. brucei at the level of the light microscope. Cell cycle events including initiation of nuclear and kinetoplast DNA replication and pro-basal body formation may be strictly coordinated in T. brucei in order to maintain the precise single-mitochondrion (kinetoplast), singleflagellum status of the interphase cell.

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Proximity Interactions among Basal Body Components in Trypanosoma brucei Identify Novel Regulators of Basal Body Biogenesis and Inheritance

mBio ◽

10.1128/mbio.02120-16 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 19

Author(s):

Hung Quang Dang ◽

Qing Zhou ◽

Veronica W. Rowlett ◽

Huiqing Hu ◽

Kyu Joon Lee ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Basal Body ◽

Functional Characterization ◽

Body Protein ◽

Superresolution Microscopy ◽

Novel Proteins ◽

Organelle Segregation ◽

Body Components ◽

Flagellar Axoneme

ABSTRACT The basal body shares similar architecture with centrioles in animals and is involved in nucleating flagellar axonemal microtubules in flagellated eukaryotes. The early-branching Trypanosoma brucei possesses a motile flagellum nucleated from the basal body that consists of a mature basal body and an adjacent pro-basal body. Little is known about the basal body proteome and its roles in basal body biogenesis and flagellar axoneme assembly in T. brucei . Here, we report the identification of 14 conserved centriole/basal body protein homologs and 25 trypanosome-specific basal body proteins. These proteins localize to distinct subdomains of the basal body, and several of them form a ring-like structure surrounding the basal body barrel. Functional characterization of representative basal body proteins revealed distinct roles in basal body duplication/separation and flagellar axoneme assembly. Overall, this work identified novel proteins required for basal body duplication and separation and uncovered new functions of conserved basal body proteins in basal body duplication and separation, highlighting an unusual mechanism of basal body biogenesis and inheritance in this early divergent eukaryote. IMPORTANCE The basal body in the early-branching protozoan Trypanosoma brucei nucleates flagellum assembly and also regulates organelle segregation, cell morphogenesis, and cell division. However, the molecular composition and the assembly process of the basal body remain poorly understood. Here, we identify 14 conserved basal body proteins and 25 trypanosome-specific basal body proteins via bioinformatics, localization-based screening, and proximity-dependent biotin identification. We further localized these proteins to distinct subdomains of the basal body by using fluorescence microscopy and superresolution microscopy, discovered novel regulators of basal body duplication and separation, and uncovered new functions of conserved basal body proteins in basal body duplication and separation. This work lays the foundation for dissecting the mechanisms underlying basal body biogenesis and inheritance in T. brucei .

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

PLoS ONE ◽

10.1371/journal.pone.0249908 ◽

2021 ◽

Vol 16 (4) ◽

pp. e0249908

Author(s):

Catherine Sullenberger ◽

Benjamin Hoffman ◽

Justin Wiedeman ◽

Gaurav Kumar ◽

Kojo Mensa-Wilmot

Keyword(s):

Trypanosoma Brucei ◽

Basal Body ◽

Basal Bodies ◽

Kinetoplast Dna ◽

Body Movements ◽

Mitochondrial Nucleoid ◽

Daughter Cells ◽

Close Proximity ◽

Division Factor ◽

Mitochondrial Nucleoids

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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Characterization of Two NMN Deamidase Mutants as Possible Probes for an NMN Biosensor

International Journal of Molecular Sciences ◽

10.3390/ijms22126334 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6334

Author(s):

Alessandra Camarca ◽

Gabriele Minazzato ◽

Angela Pennacchio ◽

Alessandro Capo ◽

Adolfo Amici ◽

...

Keyword(s):

Enzymatic Reaction ◽

Promising Candidate ◽

Beneficial Effects ◽

Steady State Fluorescence ◽

Nicotinamide Mononucleotide ◽

Recognition Elements ◽

Steady State Fluorescence Spectroscopy ◽

Similar Affinity ◽

Micromolar Range

Nicotinamide mononucleotide (NMN) is a key intermediate in the nicotinamide adenine dinucleotide (NAD+) biosynthesis. Its supplementation has demonstrated beneficial effects on several diseases. The aim of this study was to characterize NMN deamidase (PncC) inactive mutants to use as possible molecular recognition elements (MREs) for an NMN-specific biosensor. Thermal stability assays and steady-state fluorescence spectroscopy measurements were used to study the binding of NMN and related metabolites (NaMN, Na, Nam, NR, NAD, NADP, and NaAD) to the PncC mutated variants. In particular, the S29A PncC and K61Q PncC variant forms were selected since they still preserve the ability to bind NMN in the micromolar range, but they are not able to catalyze the enzymatic reaction. While S29A PncC shows a similar affinity also for NaMN (the product of the PncC catalyzed reaction), K61Q PncC does not interact significantly with it. Thus, PncC K61Q mutant seems to be a promising candidate to use as specific probe for an NMN biosensor.

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The Double-Edged Sword in Pathogenic Trypanosomatids: The Pivotal Role of Mitochondria in Oxidative Stress and Bioenergetics

BioMed Research International ◽

10.1155/2014/614014 ◽

2014 ◽

Vol 2014 ◽

pp. 1-14 ◽

Cited By ~ 47

Author(s):

Rubem Figueiredo Sadok Menna-Barreto ◽

Solange Lisboa de Castro

Keyword(s):

Trypanosoma Brucei ◽

Cell Signalling ◽

Oxygen Species ◽

Kinetoplast Dna ◽

Excellent Candidate ◽

Causative Agents ◽

Parasite Biology ◽

Oxidative Regulation ◽

Single Mitochondrion

The pathogenic trypanosomatidsTrypanosoma brucei,Trypanosoma cruzi, andLeishmaniaspp. are the causative agents of African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. These diseases are considered to be neglected tropical illnesses that persist under conditions of poverty and are concentrated in impoverished populations in the developing world. Novel efficient and nontoxic drugs are urgently needed as substitutes for the currently limited chemotherapy. Trypanosomatids display a single mitochondrion with several peculiar features, such as the presence of different energetic and antioxidant enzymes and a specific arrangement of mitochondrial DNA (kinetoplast DNA). Due to mitochondrial differences between mammals and trypanosomatids, this organelle is an excellent candidate for drug intervention. Additionally, during trypanosomatids’ life cycle, the shape and functional plasticity of their single mitochondrion undergo profound alterations, reflecting adaptation to different environments. In an uncoupling situation, the organelle produces high amounts of reactive oxygen species. However, these species role in parasite biology is still controversial, involving parasite death, cell signalling, or even proliferation. Novel perspectives on trypanosomatid-targeting chemotherapy could be developed based on better comprehension of mitochondrial oxidative regulation processes.

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Facile Synthesis of Cu2O Nanocubes in Triblock Copolymer Solutions

Solid State Phenomena ◽

10.4028/www.scientific.net/ssp.121-123.275 ◽

2007 ◽

Vol 121-123 ◽

pp. 275-278

Author(s):

Jin Hua Jiang ◽

Qiu Ming Gao

Keyword(s):

Triblock Copolymer ◽

Solar Spectrum ◽

Blue Shift ◽

Edge Length ◽

Promising Candidate ◽

Organic Species ◽

Transmission Electron ◽

Ft Ir ◽

Average Edge Length

Cuprous oxide and related materials in nanosizes are of much interest and investigated extensively recently. It is reported here that cubic Cu2O nanocubes were synthesized successfully in aqueous solutions at room temperature in air condition. Copper (II) salts in water were reduced with ascorbate acid in air, using the nonionic pluronic amphiphilic triblock copolymer EO20PO70EO20 (P123) as the template-directing and protecting agent. The average edge length of the cubes varied from 50 to 100 nm. Transmission electron microscopy (TEM) has been used for the shape and structural characterization of the obtained Cu2O nanocubes. The UV-Vis spectra showed an obvious blue-shift (0.53 eV), compared to the band gap of the bulk Cu2O crystal, which makes it a promising candidate in solar energy conversion since this sample can make use of higher energy visible rays of solar spectrum. In the FT-IR spectra the peak of Cu-O bond for the Cu2O is clearly distinguished and several weak peaks of the C-H, C-C and C=O bonds for the organic species can also be detectable, implying a little P123 residua in the products. The effect of the triblock copolymer P123 on the growth of the Cu2O nanocubes is discussed.

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Fabrication and Characterization of Al/NiO Energetic Nanomultilayers

Journal of Nanomaterials ◽

10.1155/2015/964135 ◽

2015 ◽

Vol 2015 ◽

pp. 1-6 ◽

Cited By ~ 3

Author(s):

YiChao Yan ◽

Wei Shi ◽

HongChuan Jiang ◽

Jie Xiong ◽

WanLi Zhang ◽

...

Keyword(s):

Photoelectron Spectroscopy ◽

Total Thickness ◽

X Ray Diffraction ◽

Dc Magnetron Sputtering ◽

Metallic Oxide ◽

Promising Candidate ◽

X Ray ◽

Thermal Boundary Conductance ◽

Fabrication And Characterization

The redox reaction between Al and metallic oxide has its advantage compared with intermetallic reaction and Al/NiO nanomutlilayers are a promising candidate for enhancing the performance of energetic igniter. Al/NiO nanomutlilayers with different modulation periods are prepared on alumina substrate by direct current (DC) magnetron sputtering. The thicknesses of each period are 250 nm, 500 nm, 750 nm, 1000 nm, and 1500 nm, respectively, and the total thickness is 3 μm. The X-ray diffraction (XRD) and scanning electron microscope (SEM) results of the as-deposited Al/NiO nanomutlilayers show that the NiO films are amorphous and the layered structures are clearly distinguished. The X-ray photoelectron spectroscopy (XPS) demonstrates that the thickness of Al2O3increases on the side of Al monolayer after annealing at 450°C. The thermal diffusion time becomes greater significantly as the amount of thermal boundary conductance across the interfaces increases with relatively smaller modulation period. Differential scanning calorimeter (DSC) curve suggests that the energy release per unit mass is below the theoretical heat of the reaction due to the nonstoichiometric ratio between Al and NiO and the presence of impurities.

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