Basal body movements as a mechanism for mitochondrial genome segregation in the trypanosome cell cycle

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The Trypanosoma brucei AIR9-like protein is cytoskeleton-associated and is required for nucleus positioning and accurate cleavage furrow placement

AIR9 is a cytoskeleton-associated protein in Arabidopsis thaliana with roles in cytokinesis and cross wall maturation, and reported homologues in land plants and excavate protists, including trypanosomatids. We show that the Trypanosoma brucei AIR9-like protein, TbAIR9, is also cytoskeleton-associated and colocalises with the subpellicular microtubules. We find it to be expressed in all life cycle stages and show that it is essential for normal proliferation of trypanosomes in vitro. Depletion of TbAIR9 from procyclic trypanosomes resulted in increased cell length due to increased microtubule extension at the cell posterior. Additionally, the nucleus was re-positioned to a location posterior to the kinetoplast, leading to defects in cytokinesis and the generation of aberrant progeny. In contrast, in bloodstream trypanosomes, depletion of TbAIR9 had little effect on nucleus positioning, but resulted in aberrant cleavage furrow placement and the generation of non-equivalent daughter cells following cytokinesis. Our data provide insight into the control of nucleus positioning in this important pathogen and emphasise differences in the cytoskeleton and cell cycle control between two life cycle stages of the T. brucei parasite

Intramitochondrial Localization of Universal Minicircle Sequence-Binding Protein, a Trypanosomatid Protein That Binds Kinetoplast Minicircle Replication Origins

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase β, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication

Reliability approach for safe designing on a locking system

The aim of this work is to predict the failure probability of a locking system. This failure probability is assessed using complementary methods: the First-Order Reliability Method (FORM) and Second-Order Reliability Method (SORM) as approximated methods, and Monte Carlo simulations as the reference method. Both types are implemented in a specific software [Phimeca software. Software for reliability analysis developed by Phimeca Engineering S.A.] used in this study. For the Monte Carlo simulations, a response surface, based on experimental design and finite element calculations [Abaqus/Standard User’s Manuel vol. I.], is elaborated so that the relation between the random input variables and structural responses could be established. Investigations of previous reliable methods on two configurations of the locking system show the large sturdiness of the first one and enable design improvements for the second one

The Leishmania ARL-1 and Golgi Traffic

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study

A monoclonal antibody marker for the exclusion-zone filaments of Trypanosoma brucei

<p>Abstract</p> <p>Background</p> <p><it>Trypanosoma brucei </it>is a haemoflagellate pathogen of man, wild animals and domesticated livestock in central and southern Africa. In all life cycle stages this parasite has a single mitochondrion that contains a uniquely organised genome that is condensed into a flat disk-like structure called the kinetoplast. The kinetoplast is essential for insect form procyclic cells and therefore is a potential drug target. The kinetoplast is unique in nature because it consists of novel structural proteins and thousands of circular, interlocking, DNA molecules (kDNA). Secondly, kDNA replication is critically timed to coincide with nuclear S phase and new flagellum biogenesis. Thirdly, the kinetoplast is physically attached to the flagellum basal bodies <it>via </it>a structure called the tripartite attachment complex (TAC). The TAC consists of unilateral filaments (within the mitochondrion matrix), differentiated mitochondrial membranes and exclusion-zone filaments that extend from the distal end of the basal bodies. To date only one protein, p166, has been identified to be a component of the TAC.</p> <p>Results</p> <p>In the work presented here we provide data based on a novel EM technique developed to label and characterise cytoskeleton structures in permeabilised cells without extraction of mitochondrion membranes. We use this protocol to provide data on a new monoclonal antibody reagent (Mab 22) and illustrate the precise localisation of basal body-mitochondrial linker proteins. Mab 22 binds to these linker proteins (exclusion-zone filaments) and provides a new tool for the characterisation of cytoskeleton mediated kinetoplast segregation.</p> <p>Conclusion</p> <p>The antigen(s) recognised by Mab 22 are cytoskeletal, insensitive to extraction by high concentrations of non-ionic detergent, extend from the proximal region of basal bodies and bind to the outer mitochondrial membrane. This protein(s) is the first component of the TAC exclusion-zone fibres to be identified. Mab 22 will therefore be important in characterising TAC biogenesis.</p

Structural and functional studies of the first tripartite protein complex at the Trypanosoma brucei flagellar pocket collar

The flagellar pocket (FP) is the only endo-and exocytic organelle in most trypanosomes and, as such, is essential throughout the life cycle of the parasite. The neck of the FP is maintained enclosed around the flagellum via the flagellar pocket collar (FPC). The FPC is a macromolecular cytoskeletal structure and is essential for the formation of the FP and cytokinesis. FPC biogenesis and structure are poorly understood, mainly due to the lack of information on FPC composition. To date, only two FPC proteins, BILBO1 and FPC4, have been characterized. BILBO1 forms a molecular skeleton upon which other FPC proteins can, theoretically, dock onto. We previously identified FPC4 as the first BILBO1 interacting partner and demonstrated that its C-terminal domain interacts with the BILBO1 N-terminal domain (NTD). Here, we report by yeast two-hybrid, bioinformatics, functional and structural studies the characterization of a new FPC component and BILBO1 partner protein, BILBO2 (Tb927.6.3240). Further, we demonstrate that BILBO1 and BILBO2 share a homologous NTD and that both domains interact with FPC4. We have determined a 1.9 resolution crystal structure of the BILBO2 NTD in complex with the FPC4 BILBO1-binding domain. Together with mutational analyses, our studies reveal key residues for the function of the BILBO2 NTD and its interaction with FPC4 and evidenced a tripartite interaction between BILBO1, BILBO2, and FPC4. Our work sheds light on the first atomic structure of an FPC protein complex and represents a significant step in deciphering the FPC function in Trypanosoma brucei and other pathogenic kinetoplastids.Pourquoi et comment les trypanosomes construisent un Collier de la Poche FlagellaireAlliance française contre les maladies parasitaire

Structural studies of the shortest extended synaptotagmin with only two C2 domains from Trypanosoma brucei

Alliance française contre les maladies parasitaire

LHC as πp\pi p and ππ\pi\pi Collider

We propose an experiment at the LHC with leading neutron production.The latter can be used to extract from it the total $\pi^+ p$ cross-sections. With two leading neutrons we can get access to the total $\pi^+\pi^+$ cross-sections. In this note we give some estimates and discuss related problems and prospects.Comment: 22 pages, 18 figures, 8 tables, to be publishe

Photoproduction of D∗±D^{*\pm} mesons associated with a leading neutron

The photoproduction of $D^{*\pm} (2010)$ mesons associated with a leading neutron has been observed with the ZEUS detector in $ep$ collisions at HERA using an integrated luminosity of 80 pb$^{-1}$. The neutron carries a large fraction, {$x_L>0.2$}, of the incoming proton beam energy and is detected at very small production angles, {$\theta_n<0.8$ mrad}, an indication of peripheral scattering. The $D^*$ meson is centrally produced with pseudorapidity {$|\eta| 1.9$ GeV}, which is large compared to the average transverse momentum of the neutron of 0.22 GeV. The ratio of neutron-tagged to inclusive $D^*$ production is $8.85\pm 0.93({\rm stat.})^{+0.48}_{-0.61}({\rm syst.})\%$ in the photon-proton center-of-mass energy range {$130 <W<280$ GeV}. The data suggest that the presence of a hard scale enhances the fraction of events with a leading neutron in the final state.Comment: 28 pages, 4 figures, 2 table