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Funder: Julius-Maximilians-Universität Würzburg (3088), 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.

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.

Additional file 1: Figure S1. Phylogenetic tree of ALPH in eukaryotes.

The aim of this study was to investigate the use of inexpensive and easy-to-use hydrogel &ldquo, marble&rdquo, electrodes for the recording of electrical potentials of the human visual cortex using visual evoked potentials (VEPs) as example. Top hat-shaped holders for the marble electrodes were developed with an electrode cap to acquire the signals. In 12 healthy volunteers, we compared the VEPs obtained with conventional gold-cup electrodes to those obtained with marble electrodes. Checkerboards of two check sizes&mdash, 0.8&deg, and 0.25&deg, &mdash, were presented. Despite the higher impedance of the marble electrodes, the line noise could be completely removed by averaging 64 single traces, and VEPs could be recorded. Linear mixed-effect models using electrode type, stimulus, and recording duration revealed a statistically significant effect of the electrode type on only VEP N75 peak latency (mean &plusmn, SEM: 1.0 &plusmn, 1.2 ms) and amplitude (mean &plusmn, SEM: 0.8 &plusmn, 0.9 &micro, V) The mean amplitudes of the delta, theta, alpha, beta, and gamma frequency bands of marble electrodes were statistically significantly different and, on average, 25% higher than those of gold-cup electrodes. However, the mean amplitudes showed a statistically significant strong correlation (Pearson&rsquo, s r = 0.8). We therefore demonstrate the potential of the inexpensive and efficient hydrogel electrode to replace conventional gold-cup electrodes for the recording of VEPs and possibly other recordings from the human cortex.

The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes.

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 T. brucei 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., Author summary Poly(A)-binding proteins (PABPs) bind to the poly(A) tails of eukaryotic mRNAs and function in regulating mRNA fate. Many eukaryotes have several PABP paralogs and the current view is that each PABP binds a specific subset of mRNAs. Trypanosoma brucei has two PABPs, and to understand the differential functionality of these paralogs we identified interacting proteins for each. We found unique interactors for both PABPs, and significant differences between the two interaction cohorts. Our data indicate that the two PABP paralogs of trypanosomes have very distinct roles in mediating mRNA fate.

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.

For persistent infections of the mammalian host, African trypanosomes limit their population size by quorum sensing of the parasite-excreted stumpy induction factor (SIF), which induces development to the tsetse-infective stumpy stage. We found that besides this cell density-dependent mechanism, there exists a second path to the stumpy stage that is linked to antigenic variation, the main instrument of parasite virulence. The expression of a second variant surface glycoprotein (VSG) leads to transcriptional attenuation of the VSG expression site (ES) and immediate development to tsetse fly infective stumpy parasites. This path is independent of SIF and solely controlled by the transcriptional status of the ES. In pleomorphic trypanosomes varying degrees of ES-attenuation result in phenotypic plasticity. While full ES-attenuation causes irreversible stumpy development, milder attenuation may open a time window for rescuing an unsuccessful antigenic switch, a scenario that so far has not been considered as important for parasite survival., Author summary African trypanosomes escape the mammalian host’s immune system by antigenic variation of their variant surface glycoprotein (VSG) coat. VSGs are expressed from a specialized region in the genome, the expression site (ES), that contains essential expression site associated genes (ESAGs). So far, it was assumed that only successful antigenic switches to an intact expression site are viable. Here we show that unsuccessful VSG switches are not a dead-end, but may rather contribute to the persistence of the trypanosomes at the population level. We have simulated an unsuccessful VSG switch in pleomorphic trypanosomes by expression of a second VSG from a locus without ESAGs. The parasites responded with surprising phenotypic plasticity. All parasites immediately exchanged the surface coat and reduced the abundance of ES-derived transcripts. However, depending on the degree of ES-attenuation, the transgenic trypanosomes either resumed growth, or stopped proliferation. We show that the growth-arrested populations synchronously differentiate to the stumpy life cycle stage and become infective for the tsetse fly. This occurs at low cell densities and in the absence of the quorum sensing factor SIF. Thus, unsuccessful VSG switches are not lethal and cell density-dependent quorum sensing is not the only path to the tsetse fly competence.

The detection of mRNAs undergoing transcription or decay is challenging, because both processes are fast. However, the relative proportion of an mRNA in synthesis or decay increases with mRNA size and decreases with mRNA half-life. Based on this rationale, I have exploited a 22 200 nucleotide-long, short-lived endogenous mRNA as a reporter for mRNA metabolism in trypanosomes. The extreme 5΄ and 3΄ ends were labeled with red- and green-fluorescent Affymetrix® single mRNA FISH probes, respectively. In the resulting fluorescence images, yellow spots represent intact mRNAs; red spots are mRNAs in transcription or 3΄-5΄ decay, and green spots are mRNAs in 5΄-3΄ degradation. Most red spots were nuclear and insensitive to transcriptional inhibition and thus likely transcription intermediates. Most green spots were cytoplasmic, confirming that the majority of cytoplasmic decay in trypanosomes is 5΄-3΄. The system showed the expected changes at inhibition of transcription or translation and RNAi depletion of the trypanosome homologue to the 5΄-3΄ exoribonuclease Xrn1. The method allows to monitor changes in mRNA metabolism both on cellular and on population/tissue wide levels, but also to study the subcellular localization of mRNA transcription and decay pathways. I show that the system is applicable to mammalian cells.

Eukaryotic cells have several mRNA quality control checkpoints to avoid the production of aberrant proteins. Intron-containing mRNAs are actively degraded by the nuclear exosome, prevented from nuclear exit and, if these systems fail, degraded by the cytoplasmic NMD machinery. Trypanosomes have only two introns. However, they process mRNA5 from long polycistronic precursors by trans-splicing and polycistronic mRNA molecules frequently arise from any missed splice site. Here, we show that RNAi depletion of the trypanosome exosome, but not of the cytoplasmic 5'-3' exoribonuclease XRNA or the NMD helicase UPF1, causes accumulation of oligocistronic mRNA5. We have also revisited the localization of the trypanosome exosome by expressing eYFP-fusion proteins of the exosome subunits RRP44 and RRP6. Both proteins are significantly enriched in the nucleus. Together with published data, our data suggest a major nuclear function of the trypanosome exosome in rRNA, snoRNA and mRNA quality control. (C) 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.orgilicensesiby/4.0/)., This work was funded by the DFG (Kr4017/1-1 and Kr4017/1-2 to SK), by the Wellcome trust (grant number 085956/Z/08/Z to MC) and by the Spanish Ministerio de Economia e Innovacion (BFU2014-55193-P to AE). Markus Engstler (University of Wurzburg) is acknowledged for mentoring and providing lab space and research infrastructure. Anti-BiP was kindly provided by Jay Bangs (University of Wisconsin-Madison, Madison, US).

RNP granules are ribonucleoprotein assemblies that regulate the post-transcriptional fate of mRNAs in all eukaryotes. Their exact function remains poorly understood, one reason for this is that RNP granule purification has not yet been achieved. We have exploited a unique feature of trypanosomes to prepare a cellular fraction highly enriched in starvation stress granules. First, granules remain trapped within the cage-like, subpellicular microtubule array of the trypanosome cytoskeleton while soluble proteins are washed away. Second, the microtubules are depolymerized and the granules are released. RNA sequencing combined with single molecule mRNA FISH identified the short and highly abundant mRNAs encoding ribosomal mRNAs as being excluded from granules. By mass spectrometry we have identified 463 stress granule candidate proteins. For 17/49 proteins tested by eYFP tagging we have confirmed the localization to granules, including one phosphatase, one methyltransferase and two proteins with a function in trypanosome life-cycle regulation. The novel method presented here enables the unbiased identification of novel RNP granule components, paving the way towards an understanding of RNP granule function.

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.

Mit der Einführung der Filtek Silorane steht dem Markt der konservativen Zahnheilkunde ein neuartiges Füllungsmaterial zur Verfügung, welches besonders mit seiner verringerten Volumenschrumpfung beworben wird. Aufgrund ihrer deutlich anderen Matrixchemie gegenüber den konventionellen, vielfach untersuchten Methacrylaten unterscheidet sich auch der Adhäsionsmechanismus der stark hydrophoben Silorane. Herkömmliche Methacrylatadhäsive reichen laut Hersteller nicht aus, um einen suffizienten Verbund zu erzielen. Benötigt wird vielmehr ein speziell entwickeltes Silorane Systeme Adhesive. Ziel der in-vitro Studie war es, mögliche Alternativen beim Bonding siloranbasierter Komposite auf ihre Scherhaftfestigkeit hin zu untersuchen. Auf je 320 Schmelz- und Dentinprüfkörpern aus menschlichen Molaren wurden die Scherhaftkräfte gemessen, die bei der Adhäsion von Filtek Siloranen (3M Espe) oder Hermes Flow (3M Espe) einem siloranbasierten, niedrigviskösen Komposit als Füllungsmaterialien auftraten. Die aufgetretenen Bruchmodi wurden lichtmikroskopisch bestimmt. Als Haftvermittler dienten drei erprobte konventionelle Methacrylatadhäsive: Optibond FL (Kerr), Adper Scotchbond Multi Purpose (3M Espe) und Clearfil SE Bond (Kuraray). Anwendungskombinationen waren: deren Primer alleine oder in Verbindung mit dem dazugehörigen Bonding oder in Kombination mit dem Bonding des Silorane System Adhesive. Außerdem wurde Hermes Flow als Haftvermitller in Zusammenhang mit den Methacrylatadhäsiven getestet. Es konnte gezeigt werden, dass keines der konventionellen Adhäsivsysteme alleine in der Lage war, auf beiden Zahnhartsubstanzen durchgehend höhere oder gleichwertige Haftwerte für siloranbasierte Komposite zu liefern als das Silorane System Adhesive. Alle drei getesteten Produkte konnten allerdings eine Verbesserung gegenüber dem empfohlenen Adhäsiv erreichen, wenn sie zusammen mit dem Bonding dieses speziellen Systems angewendet wurden. Als besonders erfolgsversprechend im Hinblick auf die gemessenen Haftkräfte erwies sich der Versuch, Clearfil SE Bond mit dem Silorane System Adhesive Bond zu kombinieren. Hermes Flow als Haftvermittler hingegen erreichte keine suffiziente Verbindung zu den Zahnhartsubstanzen., Objective: The so called Filtek Siloranes are a new, low shrinking dental composite with a chemical matrix different to commonly used methacrylate composites. This in vitro study investigates the effect of different methacrylate and silorane based adhesives on the adhesion of Filtek Siloranes to dental hard tissues. Study design: Shearbondstrength was measured for 640 specimens based on enamel and dentin of human molars. Filtek Siloranes and Hermes Flow, a silorane composite of low viscosity, were adhesive bonded with three clinical well tried methacrylat adhesives: Optibond FL (Kerr), Adper Scotchbond Multi Purpose (3M Espe) und Clearfil SE Bond (Kuraray). There were tested alternating combinations of methacrylate bonding components with and without Silorane Systeme Adhesive Bond and Hermes Flow used as bonding. The fracture modus was analyzed under the light microscope. Results: For silorane based composites none of the tested conventional adhesive systems supplied higher or equal shearbondstrength on both dental hard tissues on its own than Silorane Systeme Adhesive did. On the other hand all three improved adhesion compared to Silorane Systeme Adhesive if combined with Silorane Systeme Adhesive Bond. Conclusion: Particular promising for the adhesive bonding of silorane based composites appeared the combination of Clearfil SE Bond with Silorane Systeme Adhesive Bond. Hermes Flow reached no noteworthy adhesion used as bonding.

Susanne Kramer Graz, Univ., Masterarb., 2009

Lit.opg.

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.