Characterization of a PKA-like kinase from Trypanosoma brucei

The protozoan parasite Trypanosoma brucei causes human sleeping sickness and Nagana in domestic animals and depends on the tsetse fly for dissemination. The complex T. brucei life cycle requires differentiation from the dividing long slender forms via the cell cycle arrested short stumpy forms (both in the mammalian bloodstream) to the procyclic forms of the insect vector. The signaling pathways that regulate differentiation are unknown but there is evidence for an involvement of cAMP. In search of the putative cAMP receptor, three catalytic and one regulatory PKA-like subunits have been previously cloned from T. brucei. The catalytic subunits possess all features of a classical PKA in terms of inhibitor and substrate specificity. It was shown that each of the catalytic PKAlike subunits binds to the regulatory subunit to form a dimeric PKA-like holoenzyme. Most surprisingly, we found that T. brucei PKA-like kinase, despite of its apparent similarities to a PKA, was not activated but instead inhibited by cAMP. Out of several other cyclic nucleotides that were tested on their effects on PKA-like kinase, only cGMP was able to activate the kinase, but in millimolar and thus most likely unphysiological concentrations. Assuming that the activation of PKA-like kinase might depend on its native, subcellular environment, an in vivo kinase assay was established in this work. It is based on the immunological detection of the phosphorylated form of the PKA reporter substrate VASP that was transgenically expressed in T. brucei. Interestingly, results from the in vivo assay did confirm the in vitro data, suggesting that T. brucei PKA-like kinase is in fact inhibited rather than activated by cAMP. Even though these findings challenge the original assumption that T. brucei PKA-like kinase transmits the differentation signal mimicked by cAMP antagonists, data from this work nevertheless provide evidence for an involvement of T. brucei PKA-like kinase in relaying extracellular cues. This is suggested from an increase in in vivo PKA activity in the presence of treatments that have either been shown to induce LS to SS differentiation (etazolate) or to participate in SS to PCF differentiation (cold shock, mild acid stress). In addition, in vivo PKA activity was stimulated with the PDE inhibitor dipyridamole and at hypoosmotic stress. In the context of a putative role for T. brucei PKA-like kinase in the regulation of differentiation, two of the catalytic isoforms (PKAC1 and PKAC2) were of particular interest. We found significant life cycle stage dependent differences in protein expression between the two almost identical isoforms. PKAC1 was nearly exclusively present in bloodstream forms and PKAC2 in procyclic cells. In addition, PKAC1, but not PKAC2 carries a phosphorylation that is restricted to the SS stage. This phosphorylation was mapped to the C-terminal threonine 324 by mass spectrometry. The functions of these life cycle stage dependent differences between PKAC1 and PKAC2 remain unknown. Reverse genetics did not reveal any functional differences between the isoforms, in fact, PKAC1 was even able to complement PKAC2 in procyclic PKAC2 knock-out cells. Results from several reverse genetic experiments indicate that T. brucei PKA-like kinase plays an important role in cell division. Depletion of either PKA-like subunit leads to a cytokinesis block. Depletion of the regulatory PKA-like subunit additionally results in altered basal body segregation. Given that 1) both cytokinesis and basal body movement had been previously suggested to be regulated by the trypanosomal flagellum (Kohl et al., 2003) and 2) the flagellum hosts T. brucei PKA-like kinase (C. Krumbholz, this lab) we propose that trypanosomal flagella act as signaling compartments for coordination of cell division

Plasticity of electric organ discharge waveform in the South African Bulldog fish, Marcusenius pongolensis: tradeoff between male attractiveness and predator avoidance?

Background: In adult male Marcusenius pongolensis the duration of their Electric Organ Discharge (EOD) pulses increases with body size over lifetime (267 to 818 microseconds, field-measured). Spawning males have been observed to exhibit an additional, temporary pulse duration increase which probably betters their mating success but increases predation risk by electroreceptive catfish. We here study the question of how the additional pulse duration increase is triggered and for how long it persists, in an attempt to understand the compromise between opposing selective forces. Results: Here, we demonstrate short-term plasticity in male EOD waveform in 10 captive M. pongolensis. An increase in EOD duration was experimentally evoked in two different ways: by exchanging the familiar neighbours of experimental subjects for stranger males that were separated by plastic mesh partitions, or by separating familiar fish by plastic mesh partitions introduced into their common tank. Both treatments evoked an increase of male EOD duration. Values exceeded those found in the non-reproductive season in nature. In one male the increase of EOD duration was 5.7fold, from 356 microseconds to 2029 microseconds. An increase in EOD duration was accompanied by a high level of aggression directed against the neighbours through the plastic mesh. With conditions remaining constant, EOD duration receded to 38 - 50 % of the maximum EOD duration after 10 weeks, or, more rapidly, when sensory contact between the fish was severely restricted by the introduction of a solid plastic wall. Conclusions: The short-term increase of EOD duration evoked by experimental manipulation of sensory contact with conspecifics through the plastic mesh, as reported here, resembled the changes in EOD waveform that accompanied reproduction in two captive males. Plasticity of the male EOD in pulse duration seems to be an adaptation for (1) securing a higher fitness by a sexually "attractive" long-duration EOD, while (2) limiting the risk of detection by electroreceptive predators, such as the sharptooth catfish, by receding to a shorter EOD as soon as reproduction is over

European Information System for Organic Markets (EISFOM QLK5-2002-02400): WP 2: “Data collection and processing systems (DCPS) for the conventional markets” and WP 3: “Data collection and processing systems for organic markets” = Deliverable D2

European markets for organic products are developing fast. In Europe, as other parts of the world, more and more farm land is being converted to organic production. In order to adjust production and consumption levels, detailed market information is needed, especially where decisions with a long-term impact need to be taken, for example on converting specific land or livestock enterprises requiring high levels of investment in glasshouses, housing, processing facilities etc. Since public subsidies (regional / national / European) are heavily involved in these investments, valid, accurate and up-to-date information is essential not only for farmers and growers, but also for policy-makers, consultants, processing industry etc. EU-research projects such as OFCAP (FAIR3-CT96-1794) and OMIaRD (QLK5-2000-01124) have shown that regional or national data gathering takes place in many countries, but often only very basic data are reported, such as certified organic holdings, land areas and livestock numbers. Important market data, e.g. the amount of production, consumption, international trade or producer and consumer prices, do not exist in most European countries. In some European countries there are only rough estimates of the levels of production and consumption. There is no standardization and data are seldom comparable. Furthermore, detailed information on specific commodities is missing. Hence, investment decisions are taken under conditions of great uncertainty. Likewise, if politicians want to support organic agriculture, they do not know whether it would be better to support production or consumption or to address problems in the marketing channel. The EU concerted action EISfOM (QLK5-2002-02400) (European Information System for Organic Markets) is attempting to take the first steps in solving these problems. The aim of this concerted action is to build up a framework for reporting valid and reliable data for relevant production and market sectors of the European organic sector in order to meet the needs of policy-makers, farmers, processors, wholesalers and other actors involved in organic markets. In order to reach this aim, this action was split into several workpackages. This report describes the approach and results of workpackages 2 and 3. In this first chapter the objective and general approach of these work packages are described. Chapters 2 and 3 provide an overview of international statistics and data collection systems within the food supply chain at the public and the private level. Chapter 4 describes national statistics and data collection systems within the food supply chain. In Chapter 5, an analysis and appraisal is made of the results with regard to organic data collection and processing systems (DCPSs) and their integration into existing common DCPSs. Chapter 6 draws several general conclusions. Two substantial annexes complete the report, one with the country reports on the situation of data collection and processing in all investigated countries and the other with the first and the second stage questionnaires covering the different data collection levels

Genome-wide in silico screen for CCCH-type zinc finger proteins of Trypanosoma brucei, Trypanosoma cruzi and Leishmania major.

BACKGROUND: CCCH type zinc finger proteins are RNA binding proteins with regulatory functions at all stages of mRNA metabolism. The best-characterized member, tritetraproline (TTP), binds to AU rich elements in 3' UTRs of unstable mRNAs, mediating their degradation. In kinetoplastids, CCCH type zinc finger proteins have been identified as being involved in the regulation of the life cycle and possibly the cell cycle. To date, no systematic listing of CCCH proteins in kinetoplastids is available. RESULTS: We have identified the complete set of CCCH type zinc finger proteins in the available genomes of the kinetoplastid protozoa Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. One fifths (20%) of all CCCH motifs fall into non-conventional classes and many had not been previously identified. One third of all CCCH proteins have more than one CCCH motif, suggesting multivalent RNA binding. One third have additional recognizable domains. The vast majority are unique to Kinetoplastida or to a subgroup within. Two exceptions are of interest: the putative orthologue of the mRNA nuclear export factor Mex67 and a 3'-5' exoribonuclease restricted to Leishmania species. CCCH motifs are absent from these proteins in other organisms and might be unique, novel features of the Kinetoplastida homologues. Of the others, several have a predicted, and in one case experimentally confirmed, connection to the ubiquitination pathways, for instance a HECT-type E3 ubiquitin ligase. The total number of kinetoplastid CCCH proteins is similar to the number in higher eukaryotes but lower than in yeast. A comparison of the genomic loci between the Trypanosomatidae homologues provides insight into both the evolution of the CCCH proteins as well as the CCCH motifs. CONCLUSION: This study provides the first systematic listing of the Kinetoplastida CCCH proteins. The number of CCCH proteins with more then one CCCH motif is larger than previously estimated, due to the identification of non-conventional CCCH motifs. Experimental approaches are now necessary to examine the functions of the many unique CCCH proteins as well as the function of the putative Mex67 and the Leishmania 3'-5' exoribonuclease.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Effects of resource availability and quality on soil microorganisms and their carbon assimilation

Soil microorganisms play a pivotal role in decomposition processes and therefore influence nutrient cycling and ecosystem function. Availability and quality of resources determines activity, growth and identity of substrate users. In agricultural systems, availability of resources is dependent on, for example, crop type, management, season, and depth. At depth substrate availability and microbial biomass decrease. However, there remain gaps in our understanding of C turnover in subsoil and how processes in the topsoil may influence abundance, activity, and function of microorganisms in deeper soil layers. With respect to substrate quality it is thought that bacteria are the dominant users of high quality substrates and more labile components whereas fungi are more important for the degradation of low quality and more recalcitrant substrates (i.e. cellulose, lignin). Therefore, this thesis was designed to increase our understanding of C turnover and the influence of both availability and quality of substrates on microorganisms in an agricultural soil. In the first and second studies, a recently established C3-C4 plant exchange field experiment was used to investigate the C flow from belowground (root) and aboveground (shoot litter) resources into the belowground food web. Maize plants were cultivated to introduce a C4 signal into the soil both by plant growth (belowground / root channel) and also by applying shoot litter (aboveground litter channel). To separate C flow from the shoot litter versus the root channel, maize litter was applied on wheat cultivated plots, while on half of the maize planted plots no maize litter was returned. Wheat cultivated plots without additional maize litter application served as a reference for the calculation of incorporated maize-C into different soil pools. Soil samplings took place in two consecutive years in summer, autumn and winter. Three depths were considered (0-10 cm: topsoil, 40-50 cm: rooted zone beneath the plough layer, 60-70 cm: unrooted zone). In the third study a microcosm experiment with substrates of different recalcitrance and complexity was carried out to identify primary decomposers of different plant litter materials (leaves and roots) during early stages of decomposition (duration of 32 days) and to follow the C flow into the next higher trophic level (protozoa).Bodenmikroorganismen spielen eine zentrale Rolle im Abbau von organischer Substanz und beeinflussen somit Nährstoffkreisläufe und die Funktion von Ökosystemen. Die Verfügbarkeit und Qualität von Ressourcen bestimmt die Aktivität, das Wachstum und die Identität der entsprechenden Substratnutzer. In Agrarökosystemen ist die Verfügbarkeit von Ressourcen zum Beispiel abhängig von Kulturpflanzenart, Bodenbearbeitung, Jahreszeit und Bodentiefe. In der Tiefe ist die Substratverfügbarkeit und somit auch die mikrobielle Biomasse verringert. Aber es ist immer noch wenig bekannt über den C-Umsatz im Unterboden und wie und ob Prozesse im Oberboden die Abundanz, Aktivität und Funktion von Mikroorganismen im Unterboden beeinflussen. In Bezug auf die Substratqualität wird davon ausgegangen, dass Bakterien vor allem am Abbau hoch qualitativer und leicht abzubauender Substrate beteiligt sind, während Pilze eine wichtigere Rolle beim Abbau von qualitativ minderwertigeren und schwerer abzubauenden Substraten (z.B. Cellulose, Lignin) spielen. Die vorliegende Arbeit wurde durchgeführt, um das Verständnis des C-Umsatzes und den Einfluss von Verfügbarkeit und Qualität von Substraten auf Mikroorganismen in einer Agrarfläche zu verbessern. In der ersten und zweiten Studie wurde ein Feldversuch genutzt, der angelegt wurde, um mit Hilfe eines C3-C4 Pflanzenwechsels den C-Fluss von unterirdischen (Wurzel) und oberirdischen (Streu) Ressourcen ins unterirdische Nahrungsnetz zu untersuchen. Maispflanzen wurden angebaut, um das C4-bürtige Signal in den Boden einzuführen. Dies geschah einerseits durch Pflanzenwachstum (unterirdisch / Wurzelkanal) und andererseits durch das Rückführen von Streu auf die Flächen (oberirdischer Streukanal). Um den C-Fluss des Wurzel- und Streu-Kanals zu trennen, wurde Streu auf Flächen aufgebracht, auf denen Weizen angebaut wurde, während auf der Hälfte der Flächen, auf denen Mais angebaut wurde, keine Streu zurückgeführt wurde. Weizenflächen auf denen keine zusätzliche Maisstreu aufgebracht wurde, wurden als Referenz für die Berechnung des C-Eintrags in die unterschiedlichen Bodenpools genutzt. Bodenproben wurden in 2 aufeinanderfolgenden Jahren im Sommer, Herbst und Winter genommen. Es wurden 3 unterschiedliche Bodentiefen betrachtet (0-10 cm: Oberboden, 40-50 cm: durchwurzelte Zone unter dem Pflughorizont, 60-70 cm: nicht durchwurzelte Zone). In der dritten Studie wurde ein Mikrokosmenexperiment mit Substraten unterschiedlicher Qualität und Komplexität durchgeführt, um primäre Nutzer von unterschiedlichem Pflanzenmaterial (Blätter und Wurzeln) während der frühen Abbauphase (32 Tage) zu identifizieren und den C-Fluss in eine nächst höhere trophische Ebene (Protozoen) zu untersuchen

Comparative proteomics of the two T. brucei PABPs suggests that PABP2 controls bulk mRNA

<div><p>Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both <i>T</i>. <i>brucei</i> PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes.</p></div

Multispecies reconstructions uncover widespread conservation, and lineage-specific elaborations in eukaryotic mRNA metabolism.

The degree of conservation and evolution of cytoplasmic mRNA metabolism pathways across the eukaryotes remains incompletely resolved. In this study, we describe a comprehensive genome and transcriptome-wide analysis of proteins involved in mRNA maturation, translation, and mRNA decay across representative organisms from the six eukaryotic super-groups. We demonstrate that eukaryotes share common pathways for mRNA metabolism that were almost certainly present in the last eukaryotic common ancestor, and show for the first time a correlation between intron density and a selective absence of some Exon Junction Complex (EJC) components in eukaryotes. In addition, we identify pathways that have diversified in individual lineages, with a specific focus on the unique gene gains and losses in members of the Excavata and SAR groups that contribute to their unique gene expression pathways compared to other organisms

Is mRNA decapping by ApaH like phosphatases present in eukaryotes beyond the Kinetoplastida?

BACKGROUND: ApaH like phosphatases (ALPHs) originate from the bacterial ApaH protein and have been identified in all eukaryotic super-groups. Only two of these proteins have been functionally characterised. We have shown that the ApaH like phosphatase ALPH1 from the Kinetoplastid Trypanosoma brucei is the mRNA decapping enzyme of the parasite. In eukaryotes, Dcp2 is the major mRNA decapping enzyme and mRNA decapping by ALPHs is unprecedented, but the bacterial ApaH protein was recently found decapping non-conventional caps of bacterial mRNAs. These findings prompted us to explore whether mRNA decapping by ALPHs is restricted to Kinetoplastida or could be more widespread among eukaryotes. RESULTS: We screened 827 eukaryotic proteomes with a newly developed Python-based algorithm for the presence of ALPHs and used the data to characterize the phylogenetic distribution, conserved features, additional domains and predicted intracellular localisation of this protein family. For most organisms, we found ALPH proteins to be either absent (495/827 organisms) or to have non-cytoplasmic localisation predictions (73% of all ALPHs), excluding a function in mRNA decapping. Although, non-cytoplasmic ALPH proteins had in vitro mRNA decapping activity. Only 71 non-Kinetoplastida have ALPH proteins with predicted cytoplasmic localisations. However, in contrast to Kinetoplastida, these organisms also possess a homologue of Dcp2 and in contrast to ALPH1 of Kinetoplastida, these ALPH proteins are very short and consist of the catalytic domain only. CONCLUSIONS: ALPH was present in the last common ancestor of eukaryotes, but most eukaryotes have either lost the enzyme, or use it exclusively outside the cytoplasm. The acceptance of mRNA as a substrate indicates that ALPHs, like bacterial ApaH, have a wide substrate range: the need to protect mRNAs from unregulated degradation is one possible explanation for the selection against the presence of cytoplasmic ALPH proteins in most eukaryotes. Kinetoplastida succeeded to exploit ALPH as their only or major mRNA decapping enzyme. 71 eukaryotic organisms outside the Kinetoplastid lineage have short ALPH proteins with cytoplasmic localisation predictions: whether these proteins are used as decapping enzymes in addition to Dcp2 or else have adapted to not accept mRNAs as a substrate, remains to be explored

Parallel monitoring of RNA abundance, localisation and compactness with correlative single molecule FISH on LR White embedded samples

Single mRNA molecules are frequently detected by single molecule fluorescence in situ hybridization (smFISH) using branched DNA technology. While providing strong and background-reduced signals, the method is inefficient in detecting mRNAs within dense structures, in monitoring mRNA compactness and in quantifying abundant mRNAs. To overcome these limitations, we have hybridized slices of high pressure frozen, freeze-substituted and LR White embedded cells (LR White smFISH). mRNA detection is physically restricted to the surface of the resin. This enables single molecule detection of RNAs with accuracy comparable to RNA sequencing, irrespective of their abundance, while at the same time providing spatial information on RNA localization that can be complemented with immunofluorescence and electron microscopy, as well as array tomography. Moreover, LR White embedding restricts the number of available probe pair recognition sites for each mRNA to a small subset. As a consequence, differences in signal intensities between RNA populations reflect differences in RNA structures, and we show that the method can be employed to determine mRNA compactness. We apply the method to answer some outstanding questions related to trans-splicing, RNA granules and mitochondrial RNA editing in single-cellular trypanosomes and we show an example of differential gene expression in the metazoan Caenorhabditis elegans