Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localisation and dynamics
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos, instrument model
ORGANISM(S): Trypanosoma Brucei
SUBMITTER: 
Philippe Bastin
LAB HEAD: Philippe Bastin
PROVIDER: PXD000051 | Pride | 2018-12-17
REPOSITORIES: Pride
Publications
Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics.
Subota Ines I Julkowska Daria D Vincensini Laetitia L Reeg Nele N Buisson Johanna J Blisnick Thierry T Huet Diego D Perrot Sylvie S Santi-Rocca Julien J Duchateau Magalie M Hourdel Véronique V Rousselle Jean-Claude JC Cayet Nadège N Namane Abdelkader A Chamot-Rooke Julia J Bastin Philippe P
Molecular & cellular proteomics : MCP 20140416 7
Cilia and flagella are complex organelles made of hundreds of proteins of highly variable structures and functions. Here we report the purification of intact flagella from the procyclic stage of Trypanosoma brucei using mechanical shearing. Structural preservation was confirmed by transmission electron microscopy that showed that flagella still contained typical elements such as the membrane, the axoneme, the paraflagellar rod, and the intraflagellar transport particles. It also revealed that fl ...[more]
Similar Datasets
Project description:Cilia and flagella serve as cellular antennae that translate sensory information into cellular responses. In sperm, a single chemoattractant molecule triggers signaling events that evoke a Ca2+ response and chemotactic steering. Key components of the chemotactic signaling pathway from sperm of the sea urchin Arbacia punctulata were investigated. Shotgun proteomics employing SDS-PAGE LC-MS/MS and MudPIT provided the proteome inventory of sperm flagella.
2019-12-11 | PXD015332 | Pride
Project description:The salt acclimation process of the euryhaline model cyanobacterium Synechocystis sp. PCC 6803 was analyzed by combining transcriptomic, proteomic and metabolomic methods. The comparison of salt-induced proteome and transcriptome changes revealed that most stably up-regulated proteins also showed elevated mRNA levels. The Pearson correlation coefficient for salt-induced abundance changes of 1749 transcript/protein pairs was r = 0.58. In addition to the rapid and stable upregulation of compatible solute biochemistry, a dynamic reorganization of the transcriptome occurred during the first hours after salt shock, which probably involves the action of small regulatory RNAs. Based on these data, an extended salt stimulon can be defined comprising many proteins directly or indirectly related to compatible solute metabolism, ion and water movements as well as a defined set of small regulatory RNAs. Our comprehensive data set provides the basis for future attempts to engineer cyanobacterial salt tolerance and to search for processes regulating this important environmental acclimation process.
2022-02-17 | PXD026118 | Pride
Project description:The estrogen receptor a (ERa) is a ligand-regulated transcription factor. However, a wide variety of other extracellular signals can activate ERa in the absence of estrogen. The impact of these alternate modes of activation on gene expression profiles has not been characterized. We show that estrogen, growth factors and cAMP elicit surprisingly distinct ERa-dependent transcriptional responses in human MCF7 breast cancer cells. In response to growth factors and cAMP, ERa primarily activates and represses genes, respectively. The combined treatments with the antiestrogen tamoxifen and cAMP or growth factors regulate yet other sets of genes. In many cases, tamoxifen is perverted to an agonist, potentially mimicking what is happening in certain tamoxifen-resistant breast tumors and emphasizing the importance of the cellular signaling environment. Using a computational analysis, we predicted that a Hox protein might be involved in mediating such combinatorial effects, and then confirmed it experimentally. Although both tamoxifen and cAMP block the proliferation of MCF7 cells, their combined application stimulates it, and this can be blocked with a dominant-negative Hox mutant. Thus, the activating signal dictates both target gene selection and regulation by ERa, and this has consequences on global gene expression patterns that may be relevant to understanding the progression of ERa-dependent carcinomas. Keywords: treatment response 10 treatment conditions (3 biological replicates for each, totalling 30 individual samples), of which 3 were untreated controls. Each replicate was hybridized to a separate chip, totalling 27 cDNA slides representing 9 unique conditions.
2008-05-22 | E-GEOD-10466 | biostudies-arrayexpress
Project description:To study the regulatory outcome of Hfq-mediated sRNA-target interactions, we measured the change in gene expression following overexpression of each of five well-established sRNAs: GcvB, MicA, ArcZ, RyhB and CyaR. For each of the studied sRNAs we applied RNA-seq to two E. coli K-12 MG1655 strains: (1) a WT strain or a strain deleted of the sRNA gene; (2) a strain overexpressing the sRNA, either artificially from a plasmid or from the endogenous sRNA gene by changing the growth condition. For GcvB induction, the E. coli K-12 MG1655 Z1 gcvB::Cm, pZA12-gcvB and the E. coli K-12 MG1655 Z1 gcvB::Cm, pTP-011 strains were used. For MicA overexpression the E. coli K-12 MG1655lacIq pBRplac, pEF21-Hfq and the E. coli K-12 MG1655lacIq pMicA, pEF21-Hfq strains were used. For ArcZ induction the E. coli K-12 MG1655 Z1 arcZ::Cm, pZE12-ArcZ and the E. coli K-12 MG1655 Z1 arcZ::Cm, pJV300 strains were used. For RyhB overexpression the E. coli K-12 MG1655 ryhB::Cm and the E. coli K-12 MG1655 were used. For CyaR induction the E. coli K-12 MG1655 Z1 cyaR::Cm, pZE12-CyaR and the E. coli K-12 MG1655 Z1 cyaR::Cm, pJV300 strains were used. All E. coli strains used in this study were grown overnight in Luria Bertani (LB) medium at 37 °C with shaking (200 r.p.m.), diluted 100-fold in fresh LB medium, and re-grown with shaking at 37 °C to exponential phase, for GcvB (OD600 = 0.3) and for RyhB (OD600 = 0.5), or to stationary phase, for ArcZ WT, ArcZ M1, ArcZ M2 and CyaR (OD600 = 1.0) and for MicA (grown for 6 hr). For induction of ArcZ and GcvB, IPTG was added (1mM, 20 min). MicA was constitutively expressed and further induced at the end of growth with IPTG (1mM, 20 min). For induction of RyhB, the iron chelator 2,2'-Dipyridyl was added (200 μM, 30 min).
2020-03-03 | E-MTAB-8229 | biostudies-arrayexpress
Project description:Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC.
2023-06-23 | PXD041551 | Pride
Project description:Single-cell RNA sequencing (10X) analyses of human somitoids, novel organoids that periodically form somite-like structures from human iPS cells. Somitoids made with and without Matrigel were compared. Somitoids treated with different concentrations of CHIR (WNT signaling activator) during the initial 2 day-culture were also compared.
2022-04-07 | E-MTAB-11334 | biostudies-arrayexpress
Project description:The ring-shaped cohesin complex links sister chromatids until their timely segregation during mitosis. Cohesin is enriched at centromeres, where it provides the cohesive counter-force to bi-polar tension produced by the mitotic spindle. As a consequence of spindle tension, centromeric sequences transiently split in pre-anaphase cells, in some organisms up to several micrometeres. This M-bM-^@M-^Xcentromere breathingM-bM-^@M-^Y presents a paradox, how sister sequences separate where cohesin is most enriched. We now show that in the budding yeast S. cerevisiae, cohesin binding diminishes over centromeric sequences that split during breathing. We see no evidence for cohesin translocation to surrounding sequences, suggesting that cohesin is removed from centromeres during breathing. Two pools of cohesin can be distinguished. Cohesin loaded before DNA replication, that has established sister chromatid cohesion, disappears during breathing. In contrast, cohesin loaded after DNA replication is partly retained. As sister centromeres re-associate after transient separation, cohesin is re-loaded in a manner independent of the canonical cohesin loader Scc2/Scc4. Efficient centromere re-association requires the cohesion establishment factor Eco1, suggesting that re-establishment of sister chromatid cohesion contributes to the dynamic behaviour of centromeres in mitosis. These findings provide new insights into cohesin behaviour at centromeres. Keywords: ChIP-chip SAMPLES Origin of each biological sample: Saccharomyces cerevisiae (W303background) Growth conditions: Cells containing the MET3-CDC20 allele were grown at 25M-BM-0C in SC medium lacking methionine with 2% raffinose or 2% glucose as the carbon source (Uhlmann et al. 2000). Cultures were synchronized in G1 with M-NM-1-factor (5 M-NM-<g/ml) and released into medium containing 100 mM hydroxyurea (HU) for arrest in early S-phase. For arrest in metaphase, cells were released from M-NM-1-factor or HU arrest by filtration and re-suspension in YP medium supplemented with 5 mM methionine to deplete Cdc20. To inactivate cohesin loading, cells harbouring the temperature sensitive scc2-4 allele (Ciosk et al. 2000) were shifted to the restrictive temperature of 35M-BM-0C for 1 hour before release. To prevent metaphase spindle assembly, or to disassemble already formed spindles, 7.5 M-NM-<g/ml nocodazole was added to the medium for 1 hour. To wash out nocodazole, cells were filtered again, washed, and re-suspended in fresh medium lacking nocodazole for 1 hour. Expression of Scc1 to induce differentially epitope-tagged cohesin under control of the GAL1 promoter, or of Cdc14, was achieved by addition of 2% galactose for 1 hour to cultures grown in raffinose-containing medium. EXPERIMENTAL DESIGN Experiment type: ChIP-chip = Chromatin immunoprecipitation (ChIP) followed by hybridization to a high-density oligonucleotide microarray (chip). Number of hybridizations performed: 13 ChIP-chip samples; 2 SUPernatant samples Analysis: ChIP samples were normalized against SUPernatant fractions Quality control: Confirmation of results by duplication of experiments, by usage of different epitope tags for the same protein, and by usage of different subunits of the same protein complex. Also, whole cell extract, SUPernatant, and ChIP fractions were checked by Western blotting. Protocols: ChIP-chip and hybridization protocols were performed as previously described (Katou et al. 2003; Lengronne et al. 2004; Lengronne et al. 2006). A summary is provided below: ChIP. Cells were grown under the conditions described above and cross-linked with 1% formaldehyde. Cells were disrupted using a Multi-Beads Shocker, and whole cell extracts were sonicated to obtain 400-600 bp genomic DNA fragments. Chromatin immunoprecipitation (ChIP) was performed with anti-HA antibody or anti-PK monoclonal antibody coupled to protein A Dynabeads. Immunprecipates were eluted and incubated overnight at 65M-BM-:C to reverse the cross-link. The immunoprecipitated genomic DNA was then incubated with proteinase K, extracted twice with phenol/chloroform/isoamylalcohol, precipitated, resuspended in TE and incubated with RNaseA. The DNA was purified, concentrated by ethanol precipitation and amplified by PCR after random priming. 10ug of the amplified DNA was digested with DNase I and end-labelled using TdT Terminal Transferase and Biotin-N6-ddATP. Hybridization and scanning. Samples were hybridized in buffer containing SSPE, TritonX-100, denatured salmon sperm DNA and biotin-labelled control oligonucleotide, oligo B2. Samples were denatured at 95M-BM-:C for 10 minutes before being hybridized to the arrays for 16 hours at 42M-BM-:C in a GeneChip Hybridization Oven 640. The washing and scanning protocol (FlexMidi_euk2v3_450), provided by Affymetrix, was performed automatically on a GeneChip Fluidics Station 450. Arrays were scanned using the GeneChip Scanner 3000 7G, and primary analysis of tiling chip data was performed using the Affymetrix GeneChip Operating Software (GCOS). ARRAY DESIGN General array design: in situ synthesized arrays from Affymetrix Location and ID of each spot on arrays: available upon request from Affymetrix Probe type: oligonucleotide Availability of arrays: commercially available from Affymetrix S. cerevisiae (Chromosome VI) rikDACF, P/N# 510636 S. cerevisiae T iling Array (Forward), P/N# 520286 REFERENCES Ciosk R, Shirayama M, Shevchenko A, Tanaka T, Toth A, Shevchenko A, Nasmyth K (2000) Cohesin's binding to chromosomes depends on a separate complex consisting of Scc2 and Scc4 proteins. Mol Cell 5: 1-20 Katou Y, Kanoh Y, Bandoh M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K, Shirahige K (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424: 1078-1083 Lengronne A, Katou Y, Mori S, Yokobayashi S, Kelly GP, Itoh T, Watanabe Y, Shirahige K, Uhlmann F (2004) Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature 430: 573-578 Lengronne A, McIntyre J, Katou Y, Kanoh Y, Hopfner K-P, Shirahige K, Uhlmann F (2006) Establishment of sister chromatid cohesion at the S. cerevisiae replication fork. Mol Cell 23: 787-799 Uhlmann F, Wernic D, Poupart M-A, Koonin EV, Nasmyth K (2000) Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103: 375-386
2007-09-29 | E-GEOD-8661 | biostudies-arrayexpress
Project description:The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 and an isogenic ramA gene knockout mutant which contained ramA upon an IPTG-inducible over-expression plasmid.
2009-12-31 | E-MEXP-2195 | biostudies-arrayexpress
Project description:AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana (Hybridisations done at NASC). The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Series 1: Growth conditions: Sterilized seeds will be stratified at 4degreeC for 3 days, exposed to white light for 2 h to induce germination, and grown on MS agar plates (0.9 % agar) without sugar in total darkness for 4 days at 22degreeC. Seedlings will then be transferred to the light conditions described for each slide below for 1 h (to identify early induced genes) and 4 hrs (maximum expression of the first initial light response of most target genes), respectively. All light treatments will be done in parallel to minimize the number of dark controls. All samples will be done in triplicate and only with shoots. Experimenter name = Thomas Kretsch; Experimenter institute = AtGenExpress Experiment Overall Design: 48 samples were used in this experiment
2008-06-13 | E-GEOD-5617 | biostudies-arrayexpress
Project description:This study newly identified Tripelennamine (TA) as an inhibitor of yeast meiosis and sporulation. To examine if and how exposure of sporulating yeast cells to TA influences the viability of the cells a homozygous deletion pool experiment was conducted.
2010-03-31 | E-MEXP-2535 | biostudies-arrayexpress