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Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense.
Human herpesvirus-6 (HHV-6) exists in latent form either as a nuclear episome or integrated into human chromosomes in more than 90% of healthy individuals without causing clinical symptoms. Immunosuppression and stress conditions can reactivate HHV-6 replication, associated with clinical complications and even death. We have previously shown that co-infection of Chlamydia trachomatis and HHV-6 promotes chlamydial persistence and increases viral uptake in an in vitro cell culture model. Here we investigated C. trachomatis-induced HHV-6 activation in cell lines and fresh blood samples from patients having Chromosomally integrated HHV-6 (CiHHV-6). We observed activation of latent HHV-6 DNA replication in CiHHV-6 cell lines and fresh blood cells without formation of viral particles. Interestingly, we detected HHV-6 DNA in blood as well as cervical swabs from C. trachomatis-infected women. Low virus titers correlated with high C. trachomatis load and vice versa, demonstrating a potentially significant interaction of these pathogens in blood cells and in the cervix of infected patients. Our data suggest a thus far underestimated interference of HHV-6 and C. trachomatis with a likely impact on the disease outcome as consequence of co-infection.
We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.
We review fluorescent probes that can be photoswitched or photoactivated and are suited for single-molecule localization based super-resolution microscopy. We exploit the underlying photochemical mechanisms that allow photoswitching of many synthetic organic fluorophores in the presence of reducing agents, and study the impact of these on the photoswitching properties of various photoactivatable or photoconvertible fluorescent proteins. We have identified mEos2 as a fluorescent protein that exhibits reversible photoswitching under various imaging buffer conditions and present strategies to characterize reversible photoswitching. Finally, we discuss opportunities to combine fluorescent proteins with organic fluorophores for dual-color photoswitching microscopy.
In Brassicaceae, tissue damage triggers the mustard oil bomb i.e., activates the degradation of glucosinolates by myrosinases leading to a rapid accumulation of isothiocyanates at the site of damage. Isothiocyanates are reactive electrophilic species (RES) known to covalently bind to thiols in proteins and glutathione, a process that is not only toxic to herbivores and microbes but can also cause cell death of healthy plant tissues. Previously, it has been shown that subtoxic isothiocyanate concentrations can induce transcriptional reprogramming in intact plant cells. Glutathione depletion by RES leading to breakdown of the redox potential has been proposed as a central and common RES signal transduction mechanism. Using transcriptome analyses, we show that after exposure of Arabidopsis seedlings (grown in liquid culture) to subtoxic concentrations of sulforaphane hundreds of genes were regulated without depletion of the cellular glutathione pool. Heat shock genes were among the most highly up-regulated genes and this response was found to be dependent on the canonical heat shock factors A1 (HSFA1). HSFA1-deficient plants were more sensitive to isothiocyanates than wild type plants. Moreover, pretreatment of Arabidopsis seedlings with subtoxic concentrations of isothiocyanates increased resistance against exposure to toxic levels of isothiocyanates and, hence, may reduce the autotoxicity of the mustard oil bomb by inducing cell protection mechanisms.
A fundamental question in current biology concerns the translational mechanisms leading from genetic variability to phenotypes. Technologies have evolved to the extent that they can efficiently and economically determine an individual’s genomic composition, while at the same time big data on clinical profiles and diagnostics have substantially accumulated. Genome-wide association studies linking genomic loci to certain traits, however, remain limited in their capacity to explain the cellular mechanisms that underlie the given association. For most associations, gene expression has been blamed; yet given that transcript and protein abundance oftentimes do not correlate, that finding does not necessarily decrypt the underlying mechanism. Thus, the integration of further information is crucial to establish a model that could prove more accurate in predicting genotypic effects on the human organism.
In this work we describe the so-called proteotype as a feature of the cell that could provide a substantial link between genotype and phenotype. Rather than looking at the proteome as a set of independent molecules, we demonstrate a consistent modular architecture of the proteome that is driven by molecular cooperativity. Functional modules, especially protein complexes, can be further interrogated for differences between individuals and tackled as imprints of genetic and environmental variability. We also show that subtle stoichiometric changes of protein modules could have broader effects on the cellular system, such as the transport of specific molecular cargos.
The presented work also delineates to what extent temporal events and processes influence the stoichiometry of protein complexes and functional modules. The re-wiring of the glycolytic pathway for example is illustrated as a potential cause for an increased Warburg effect during the ageing of the human bone marrow. On top of analyzing protein abundances we also interrogate proteome dynamics in terms of stability and solubility transitions during the short temporal progression of the cell cycle. One of our main observations in the thesis encompass the delineation of protein complexes into respective sub-complexes according to distinct stability patterns during the cell cycle. This has never been demonstrated before, and is functionally relevant for our understanding of the dis- and assembly of large protein modules.
The insights presented in this work imply that the proteome is more than the sum of its parts, and primarily driven by variability in entire protein ensembles and their cooperative nature. Analyzing protein complexes and functional modules as molecular reflections of genetic and environmental variations could indeed prove to be a stepping stone in closing the gap between genotype and phenotype and customizing clinical treatments in the future.
The anti-silencing function protein 1 (Asf1) is a chaperone that forms a complex with histones H3 and H4 facilitating dimer deposition and removal from chromatin. Most eukaryotes possess two different Asf1 chaperones but their specific functions are still unknown. Trypanosomes, a group of early-diverged eukaryotes, also have two, but more divergent Asf1 paralogs than Asf1 of higher eukaryotes. To unravel possible different functions, we characterized the two Asf1 proteins in Trypanosoma brucei. Asf1A is mainly localized in the cytosol but translocates to the nucleus in S phase. In contrast, Asf1B is predominantly localized in the nucleus, as described for other organisms. Cytosolic Asf1 knockdown results in accumulation of cells in early S phase of the cell cycle, whereas nuclear Asf1 knockdown arrests cells in S/G2 phase. Overexpression of cytosolic Asf1 increases the levels of histone H3 and H4 acetylation. In contrast to cytosolic Asf1, overexpression of nuclear Asf1 causes less pronounced growth defects in parasites exposed to genotoxic agents, prompting a function in chromatin remodeling in response to DNA damage. Only the cytosolic Asf1 interacts with recombinant H3/H4 dimers in vitro. These findings denote the early appearance in evolution of distinguishable functions for the two Asf1 chaperons in trypanosomes.
The Popeye domain containing (Popdc) gene family of membrane proteins is predominantly expressed in striated and smooth muscle tissues and has been shown to act as novel cAMP-binding proteins. In mice, loss of Popdc1 and Popdc2, respectively, affects sinus node function in the postnatal heart in an age and stress-dependent manner. In this thesis, I examined gene expression pattern and function of the Popdc gene family during zebrafish development with an emphasis on popdc2. Expression of the zebrafish popdc2 was exclusively present in cardiac and skeletal muscle during cardiac development, whereas popdc3 was expressed in striated muscle tissue and in distinct regions of the brain. In order to study the function of these genes, an antisense morpholino-based knockdown approach was used. Knockdown of popdc2 resulted in aberrant development of facial and tail musculature. In the heart, popdc2 morphants displayed irregular ventricular contractions with 2:1 and 3:1 ventricular pauses. Recordings of calcium transients using a transgenic indicator line Tg(cmlc2:gCaMP)s878 and selective plane illumination microscopy (SPIM) revealed the presence of an atrioventricular (AV) block in popdc2 morphants as well as a complete heart block. Interestingly, preliminary data revealed that popdc3 morphants developed a similar phenotype. In order to find a morphological correlate for the observed AV conduction defect, I studied the structure of the AV canal in popdc2 morphants using confocal analysis of hearts of the transgenic line Tg(cmlc2:eGFP-ras)s883, which outlines individual cardiac myocytes with the help of membrane-localized GFP. However, no evidence for morphological alterations was obtained. To ensure that the observed arrhythmia phenotype in the popdc2 morphant was based on a myocardial defect and not caused by defective valve development, live imaging was performed revealing properly formed valves. Thus, in agreement with the data obtained in knockout mice, popdc2 and popdc3 genes in zebrafish are involved in the regulation of cardiac electrical activity. However, both genes are not required for cardiac pacemaking, but they play essential roles in AV conduction. In order to elucidate the biological importance of cAMP-binding, wild type Popdc1 as well as mutants with a significant reduction in binding affinity for cAMP in vitro were overexpressed in zebrafish embryos. Expression of wild type Popdc1 led to a cardiac insufficiency phenotype characterized by pericardial edema and venous blood retention. Strikingly, the ability of the Popdc1 mutants to induce a cardiac phenotype correlated with the binding affinity for cAMP. These data suggest that cAMP-binding represents an important biological property of the Popdc protein family.
Flagellar motility and chemotaxis are essential virulence traits required for the ability of Helicobacter pylori to colonize the gastric mucosa. The flagellar regulatory network and the complex chemotaxis system of H. pylori are fundamentally different from other bacteria, despite many similarities. In H. pylori expression of the flagella is controlled by a complex regulatory cascade involving the two-component system FlgR-HP244, the sigma factors 54 and 28 and the anti-sigma 28 factor FlgM. Thus far, the input signal for histidine kinase HP244, which activates the transcriptional regulator FlgR, which triggers sigma factor 54-dependent transcription of the flagellar class 2 genes, is not known. Based on a yeast two-hybrid screen a highly significant protein-protein interaction between the H. pylori protein HP137 and both the histidine kinase HP244 and the flagellar hook protein HP908 (FlgE´) has been reported recently (Rain et al., 2001). So far, no function could be assigned to HP137. Interestingly, the interaction between HP137 and histidine kinase HP244 was observed in the characteristic block N sequence motif of the C-terminal ATP-binding kinase domain. In this work a potential role of HP137 in a feedback regulatory mechanism controlling the activity of histidine kinase HP244 in the flagellar regulation of H. pylori was investigated. Although the substitution of the gene encoding HP137 by a kanamycin cassette resulted in non-motile bacteria, the failure to restore motility by the reintroduction of hp137 in cis into the mutant strain, and the observation that HP137 has no significant effect on the activity of histidine kinase HP244 in vitro indicated that HP137 is not directly involved in flagellar regulation. Therefore, it was demonstrated that HP137 does not participate in the regulation of flagellar gene expression, neither in H. pylori nor in the closely related bacterium C. jejuni. Chemotactic signal transduction in H. pylori differs from the enterobacterial paradigm in several respects. In addition to a CheY response regulator protein (CheY1) H. pylori contains a CheY-like receiver domain (CheY2) which is C-terminally fused to the histidine kinase CheA. Furthermore, the genome of H. pylori encodes three CheV proteins consisting of an N-terminal CheW-like domain and a C-terminal receiver domain, while there are no orthologues of the chemotaxis genes cheB, cheR, and cheZ. To obtain insight into the mechanism controlling the chemotactic response of H. pylori the phosphotransfer reactions between the purified two-component signalling modules were investigated in vitro. Using in vitro phosphorylation assays it was shown that both H. pylori histidine kinases CheAY2 and CheA´ lacking the CheY-like domain (CheY2) act as ATP-dependent autokinases. Similar to other CheA proteins CheA´ shows a kinetic of phosphorylation represented by an exponential time course, while the kinetics of phosphorylation of CheAY2 is characterized by a short exponential time course followed by the hydrolysis of CheAY2~P. Therefore, it was demonstrated that the presence of the CheY2-like receiver domain influences the stability of the phosphorylated P1 domain of the CheA part of the bifunctional protein. Furthermore, it was proven that both CheY1 and CheY2 are phosphorylated by CheAY2 and CheA´~P and that the three CheV proteins mediate the dephosphorylation of CheA´~P, although with a clearly reduced efficiency as compared to CheY1 and CheY2. Moreover, CheA´ is capable of donating its phospho group to the CheY1 protein from C. jejuni and to CheY protein from E. coli. Retrophosphorylation experiments indicated that CheY1~P is able to transfer the phosphate group back to the HK CheAY2 and the receiver domain present in the bifunctional CheAY2 protein acts as a phosphate sink fine tuning the activity of the freely diffusible CheY1 protein, which is thought to interact with the flagellar motor. Hence, in this work evidence of a complex phosphorelay in the chemotaxis system was obtained which has similarities to other systems with multiple CheY proteins. The role of the CheV proteins remain unclear at the moment, but they might be engaged in a further fine regulation of the phosphate flow in this complex chemotaxis system and the independent function of the two domains CheA´ and CheY2 is not sufficient for normal chemotactic signalling in vivo.
The unicellular pathogen Trypanosoma brucei is the causative agent of African
trypanosomiasis, an endemic disease prevalent in sub-Saharan Africa. Trypanosoma brucei alternates between a mammalian host and the tsetse fly vector. The extracellular parasite survives in the mammalian bloodstream by periodically exchanging their ˈvariant surface glycoproteinˈ (VSG) coat to evade the host immune response. This antigenic variation is achieved through monoallelic expression of one VSG variant from subtelomeric ˈbloodstream
form expression sitesˈ (BES) at a given timepoint. During the differentiation from the bloodstream form (BSF) to the procyclic form (PCF) in the tsetse fly midgut, the stage specific surface protein is transcriptionally silenced and replaced by procyclins. Due to their subtelomeric localization on the chromosomes, VSG transcription and silencing is partly regulated by homologues of the mammalian telomere complex such as TbTRF, TbTIF2 and TbRAP1 as well as by ˈtelomere-associated proteinsˈ (TelAPs) like TelAP1. To gain more insights into transcription regulation of VSG genes, the identification and characterization of other TelAPs is critical and has not yet been achieved. In a previous study, two biochemical approaches were used to identify other novel TelAPs. By using ˈco-immunoprecipitationˈ (co-IP) to enrich possible interaction partners of TbTRF and by affinity chromatography using telomeric repeat oligonucleotides, a listing of TelAP candidates has been conducted. With this approach TelAP1 was identified as a novel component of the telomere complex, involved in the kinetics of transcriptional BES silencing during BSF to PCF differentiation. To gain further insights into the telomere complex composition, other previously enriched proteins were characterized through a screening process using RNA interference to deplete potential candidates. VSG expression profile changes and overall proteomic changes after depletion were analyzed by mass spectrometry. With this method, one can gain insights into the functions of the proteins and their involvement in VSG expression site regulation. To validate the interaction of proteins enriched by co-IP with TbTRF and TelAP1 and to identify novel interaction proteins, I performed reciprocal affinity purifications of the four most promising candidates (TelAP2, TelAP3, PPL2 and PolIE) and additionally confirmed colocalization of two candidates with TbTRF via immunofluorescence (TelAP2, TelAP3). TelAP3 colocalizes with TbTRF and potentially interacts with TbTRF, TbTIF2, TelAP1 and TelAP2, as well as with two translesion polymerases PPL2 and PolIE in BSF. PPL2 and PolIE seem to be in close contact to each other at the telomeric ends and fulfill different roles as only PolIE is involved in VSG regulation while PPL2 is not. TelAP2 was previously characterized to be associated with telomeres by partially colocalizing with TbTRF and cells show a VSG derepression phenotype when the protein was depleted. Here I show that TelAP2 interacts with the telomere-binding proteins TbTRF and TbTIF2 as well as with the telomere-associated protein TelAP1 in BSF and that TelAP2 depletion results in a loss of TelAP1 colocalization with TbTRF in BSF.
In conclusion, this study demonstrates that characterizing potential TelAPs is effective in gaining insights into the telomeric complex's composition and its role in VSG regulation in Trypanosoma brucei. Understanding these interactions could potentially lead to new therapeutic targets for combatting African trypanosomiasis.
African trypanosomiasis is a disease endemic to sub-Saharan Africa. It affects humans as well as wild and domestic animals. The human form of the disease is known as sleeping sickness and the animal form as nagana, which are usually fatal if left untreated. The cause of African trypanosomiasis is the unicellular parasite Trypanosoma brucei. During its life cycle, Trypanosoma brucei shuttles between a mammalian host and the tsetse fly vector. In the mammalian host the parasite multiplies as bloodstream form (BSF) extracellularly in the bloodstream or the lymphatic system. Survival of BSF parasites relies on immune evasion by antigenic variation of surface proteins because its extracellular lifestyle leads to direct exposure to immune responses. At any given time each BSF cell expresses a single type of variant surface glycoprotein (VSG) on its surface from a large repertoire. The active VSG is transcribed from one of 15 specialized subtelomeric domains, termed bloodstream expression sites (BESs). The remaining 14 BESs are silenced. This monoallelic expression and periodic switching of the expressed VSG enables to escape the immune response and to establish a persistent infection in the mammalian host. During developmental differentiation from BSF to the insect vector-resident procyclic form (PCF), the active BES is transcriptionally silenced to stop VSG transcription. Thus, all 15 BESs are inactive in the PCF cells as surface protein expression is developmentally regulated.
Previous reports have shown that the telomere complex components TbTRF, TbRAP1 and TbTIF2 are involved in VSG transcriptional regulation. However, the precise nature of their contribution remains unclear. In addition, no information is available about the role of telomeres in the initiation and regulation of developmental BES silencing. To gain insights into the regulatory mechanisms of telomeres on VSG transcription and developmental repression it is therefore essential to identify the complete composition of the trypanosome telomere complex.
To this end, we used two complementary biochemical approaches and quantitative label-free interactomics to determine the composition of telomere protein complexes in T. brucei. Firstly, using a telomeric pull-down assay we found 17 potential telomere-binding proteins including the known telomere-binding proteins TbTRF and TbTIF2. Secondly, by performing a co-immunoprecipitation experiment to elucidate TbTRF interactions we co-purified five proteins. All of these five proteins were also enriched with telomeric DNA in the pull-down assay.
To validate these data, I characterized one of the proteins found in both experiments (TelBP1). In BSF cells, TelBP1 co-localizes with TbTRF and interacts with already described telomere-binding proteins such as TbTRF, TbTIF2 and TbRAP1 indicating that TelBP1 is a novel component of the telomere complex in trypanosomes. Interestingly, protein interaction studies in PCF cells suggested a different telomere complex composition compared to BSF cells. In contrast to known members of the telomere complex, TelBP1 is dispensable for cell viability indicating that its function might be uncoupled from the known telomere-binding proteins. Overexpression of TelBP1 had also no effect on cell viability, but led to the discovery of two additional shorter isoforms of TelBP1. However, their source and function remained elusive.
Although TelBP1 is not essential for cell viability, western blot analysis revealed a 4-fold upregulation of TelBP1 in the BSF stage compared to the PCF stage supporting the concept of a dynamic telomere complex composition. We observed that TelBP1 influences the kinetics of transcriptional BES silencing during developmental transition from BSF to PCF. Deletion of TelBP1 caused faster BES silencing compared to wild-type parasites.
Taken together, TelBP1 function illustrates that developmental BES silencing is a fine-tuned process, which involves stage-specific changes in telomere complex formation.
In this thesis we have used Drosophila melanogaster as a model organism to investigate proteins and their putative interacting partners that are directly or indirectly involved in the release of neurotransmitters at the synapse. We have used molecular techniques to investigate conserved synaptic proteins, synapsin and synapse associated protein of 47 kD (SAP47), and a putative interaction partner of SAP47, tubulin binding chaperone E-like (TBCEL). SAP47 and synapsins are highly conserved synaptic vesicle associated proteins in Drosophila melanogaster. To further investigate the role and function of Sap47 and Syn genes, we had earlier generated the null mutants by P-element mutagenesis (Funk et al., 2004; Godenschwege et al., 2004). Western blots and ELISA of brain homogenates from Sap47156 null mutants showed the presence of up-regulated phospho-synapsin in comparison to wild-type (CS) and the presence of up-regulated phospho-synapsin was partially abolished when a pan-neuronal rescue of SAP47 was performed by the Gal4- UAS technique. Thus, the results suggest a qualitative and quantitative modulation of synapsin by SAP47. At the transcript level, we did not observe any difference in content of Syn transcript in Sap47156 and wild-type CS flies. The question of a direct molecular interaction between SAP47 and synapsin was investigated by co-immunoprecipitation (Co-IP) experiments and we did not find any stable interactions under the several IP conditions we tested. The possibility of Sap47 as a modifier of Syn at the genetic level was investigated by generating and testing homozygous double null mutants of Sap47 and Syn. The Syn97, Sap47156 double mutants are viable but have a reduced life span and decreased locomotion when compared to CS. In 2D-PAGE analysis of synapsins we identified trains of spots corresponding to synapsins, suggesting that synapsin has several isoforms and each one of them is posttranslationally modified. In an analysis by Blue native-SDS-PAGE (BN-SDS-2D- PAGE) and Western blot we observed synapsin and SAP47 signals to be present at 700-900 kDa and 200-250 kDa, respectively, suggesting that they are part of large but different complexes. We also report the possibility of Drosophila synapsin forming homo- and heteromultimers, which has also been reported for synapsins of vertebrates. In parallel to the above experiments, phosphorylation of synapsins in Drosophila was studied by IP techniques followed by 1D-SDS gel electrophoresis and mass spectrometry (in collaboration with S. Heo and G. Lubec). We identified and verified 5 unique phosphorylation sites in Drosophila synapsin from our MS analysis. Apart from phosphorylation modifications we identified several other PTMs which have not been verified. The significance of these phosphorylations and other identified PTMs needs to be investigated further and their implications for synapsin function and Drosophila behavior has to be elucidated by further experiments. In a collaborative project with S. Kneitz and N. Nuwal, we investigated the effects of Sap156 and Syn97 mutations by performing a whole Drosophila transcriptome microarray analysis of the individual null mutants and the double mutants (V2 and V3). We obtained several candidates which were significantly altered in the mutants. These genes need to be investigated further to elucidate their interactions with Sap47 and Syn. In another project, we investigated the role and function of Drosophila tubulin- binding chaperone E-like (Tbcel, CG12214). The TBCEL protein has high homology to vertebrate TBCE-like (or E-like) which has high sequence similarity to tubulin-binding chaperone E (TBCE) (hence the name TBCE-Like). We generated an anti-TBCEL polyclonal antiserum (in collaboration with G. Krohne). According to flybase, the Tbcel gene has only one exon and codes for two different transcripts by alternative transcription start sites. The longer transcript RB is present only in males whereas the shorter transcript RA is present only in females. In order to study the gene function we performed P- element jump-out mutagenesis to generate deletion mutants. We used the NP4786 (NP) stock which has a P(GawB) insertion in the 5’ UTR of the Tbcel gene. NP4786 flies are homozygous lethal due to a second-site lethality as the flies are viable over a deficiency (Df) chromosome (a deletion of genomic region spanning the Tbcel gene and other upstream and downstream genes). We performed the P-element mutagenesis twice. In the first trial we obtained only revertants and the second experiment is still in progress. In the second attempt, jump-out was performed over the deficiency chromosome to prevent homologous chromosome mediated double stranded DNA repair. During the second mutagenesis an insertion stock G18151 became available. These flies had a P-element insertion in the open reading frame (ORF) of the Tbcel gene but was homozygous viable. Western blots of fresh tissue homogenates of NP/Df and G18151 flies probed with anti-TBCEL antiserum showed no TBCEL signal, suggesting that these flies are Tbcel null mutants. We used these flies for further immunohistochemical analyses and found that TBCEL is specifically expressed in the cytoplasm of cyst cells of the testes and is associated with the tubulin of spermatid tails in wild-type CS, whereas in NP/Df and G18151 flies the TBCEL staining in the cyst cells was absent and there was a disruption of actin investment cones. We also found enrichment of TBCEL staining around the actin investment cone. These results are also supported by the observation that the enhancer trap expression of the NP4786 line is localised to the cyst cells, similar to TBCEL expression. Also, male fertility of NP/Df and G18151 flies was tested and they were found to be sterile with few escapers. Thus, these results suggest that TBCEL is involved in Drosophila spermatogenesis with a possible role in the spermatid elongation and individualisation process.
Genes involved in sex determination and differentiation have been identified in mice, humans, chickens, reptiles, amphibians and teleost fishes. However, little is known of their functional conservation, and it is unclear whether there is a common set of genes shared by all vertebrates. Coelacanths, basal Sarcopterygians and unique "living fossils", could help establish an inventory of the ancestral genes involved in these important developmental processes and provide insights into their components. In this study 33 genes from the genome of Latimeria chalumnae and from the liver and testis transcriptomes of Latimeria menadoensis, implicated in sex determination and differentiation, were identified and characterized and their expression levels measured. Interesting findings were obtained for GSDF, previously identified only in teleosts and now characterized for the first time in the sarcopterygian lineage; FGF9, which is not found in teleosts; and DMRT1, whose expression in adult gonads has recently been related to maintenance of sexual identity. The gene repertoire and testis-specific gene expression documented in coelacanths demonstrate a greater similarity to modern fishes and point to unexpected changes in the gene regulatory network governing sexual development.
Multiple myeloma (MM) is a disease of terminally differentiated B-cells which accumulate in the bone marrow leading to bone lesions, hematopoietic insufficiency and hypercalcemia. Genetically, MM is characterized by a great heterogeneity. A recent next-generation sequencing approach resulted in the identification of a signaling network with an accumulation of mutations in receptor-tyrosine kinases (RTKs), adhesion molecules and downstream effectors. A deep-sequencing amplicon approach of the coding DNA sequence of the six RTKs EPHA2, EGFR, ERBB3, IGF1R, NTRK1 and NTRK2 was conducted in a patient cohort (75 MM samples and 68 corresponding normal samples) of the “Deutsche Studiengruppe Multiples Myelom (DSMM)” to further elucidate the role of RTKs in MM. As an initial approach the detected mutations were correlated with cytogenetic abnormalities and clinical data in the course of this thesis. RTK mutations were present in 13% of MM patients of the DSMM XI trial and accumulated in the ligand-binding and tyrosine-kinase domain. The newly identified mutations were associated with an adverse patient survival, but not with any cytogenetic abnormality common in MM. Especially rare patient-specific SNPs (single nucleotide polymorphism) had a negative impact on patient survival. For a more comprehensive understanding of the role of rare RTK SNPs in MM, a second amplicon sequencing approach was performed in a patient cohort of the DSMM XII trial that included 75 tumor and 184 normal samples. This approach identified a total of 23 different mutations in the six RTKs EPHA2, EGFR, ERBB3, IGF1R, NTRK1 and NTRK2 affecting 24 patients. These mutations could furthermore be divided into 20 rare SNPs and 3 SNVs (single nucleotide variant). In contrast to the first study, the rare SNPs were significantly associated with the adverse prognostic factor del17p.
IGF1R was among the most commonly mutated RTKs in the first amplicon sequencing approach and is known to play an important role in diverse cellular processes such as cell proliferation and survival. To study the role of IGF1R mutations in the hard-to-transfect MM cells, stable IGF1R-knockdown MM cell lines were established. One of the knockdown cell lines (L363-C/C9) as well as a IGF1R-WT MM cell line (AMO1) were subsequently used for the stable overexpression of WT IGF1R and mutant IGF1R (N1129S, D1146N). Overall, an impact on the MAPK and PI3K/AKT signaling pathways was observed upon the IGF1R knockdown as well as upon WT and mutant IGF1R overexpression. The resulting signaling pattern, however, differed between different MM cell lines used in this thesis as well as in a parallel performed master thesis which further demonstrates the great heterogeneity described in MM.
Taken together, the conducted sequencing and functional studies illustrate the importance of RTKs and especially of IGF1R and its mutants in the pathogenesis of MM. Moreover, the results support the potential role of IGF1R as a therapeutic target for a subset of MM patients with mutated IGF1R and/or IGF1R overexpression.
Glioblastoma multiforme (GBM) is one of the most frequent and malignant forms of brain cancer in adults. The prognosis is poor with a median survival time of 12-15 months. There is a broad range of alternative treatment options studied in preclinical and clinical trials for GBM. One alternative treatment option is oncolytic virotherapy, defined as the use of replication‐competent viruses that selectively infect and destroy cancer cells while leaving, non‐transformed cells unharmed. Vaccinia virus (VACV) is one favorable candidate. Although oncolytic viruses can kill tumor cells grown in vitro with high efficiency, they often exhibit reduced replication capacity in vivo suggesting that physiological aspects of the tumor microenvironment decrease the virus’ therapeutic potential. The percentage and composition of immune cells varies between cancer types and patients and is investigated as a biomarker in several studies. Making oncolytic virotherapy successful for GBM, it is necessary to understand the individual tumor biology, the interaction with the microenvironment and immune system.
It was demonstrated that the attenuated VACV wild-type (wt) isolate LIVP 1.1.1 replicate and lyse the murine GL261 glioma cell line in vitro. In the following, the replication efficacy was characterized in a comparative approach in vivo. Immunocompetent C57BL/6 (wt) mice and immunodeficient mouse strains of different genetic background C57BL/6 athymic and Balb/c athymic mice were used. In addition, subcutaneous and intracranial locations were compared. The results revealed viral replication exclusively in Balb/c athymic mice with subcutaneous tumors but in none of the other models.
In the following, the tumor microenvironment of the subcutaneous tumor models at the time of infection was performed. The study showed that implantation of the same tumor cells in different mouse strains resulted in a different tumor microenvironment with a distinct composition of immune cells. Highest differences were detected between immunodeficient and immunocompetent mice. The study showed major differences in the expression of MHCII with strongest expression in C57BL/6 wt and weakest in Balb/c athymic tumors. In the following, the influence of the phenotypic change associated with the upregulation of MHCII on GL261 tumor cells on viral replication was analyzed. Comparison of C57BL/6 wt and C57BL/6 IFN-γ knockout mice revealed endogenous IFN-γ levels to upregulate MHCII on GL261 tumor cells and to reduce viral replication in C57BL/6 wt mice. Analysis of single cell suspensions of tumor homogenates of C57BL/6 and Balb/c athymic mice showed that the IFN-γ-mediated anti-tumor effect was a reversible effect. Furthermore, reasons for inhibition of virus replication in orthotopic glioma models were elucidated. By immunohistochemical analysis it was shown that intratumoral amounts of Iba1+ microglia and GFAP+ astrocytes in Gl261 gliomas was independent from intratumoral VACV injection. Based on these findings virus infection in glioma, microglia and astrocytes was compared and analyzed in cell culture. In contrast to the GL261 glioma cells, replication was barely detectable in BV-2 microglia and IMA2.1 astrocytic cells. Co-culture experiments revealed that microglia compete for virus uptake in cell culture. It was further shown that BV-2 cells showed apoptotic characteristics after VACV infection while GL261 cells showed signs of necrotic cell death. Additionally, in BV-2 cells with M1-phenotype a further reduction of viral replication and inhibition of cell lysis was detected. Infection of IMA 2.1 cells was independent of the M1/M2-phenotype. Application of BV-2 microglia with M1-phenotype onto organotypic slice cultures with implanted GL261 tumors resulted in reduced infection of BV-2 cells with LIVP 1.1.1, whereas GL261 cells were significantly infected.
Taken together, the analyzed GL261 tumors were imprinted by the immunologic and genetic background in which they grow. The experimental approach applied in this thesis can be used as suitable model which reflects the principles of personalized medicine
In an additional project, based on gene expression data and bioinformatic analyses, the biological role and function of the anti-apoptotic factor AVEN was analyzed with regard to oncolytic VACV therapy. Besides a comparison of the replication efficacy of GLV-1h68 and VACV-mediated cell killing of four human tumor cell lines, it was shown that AVEN was expressed in all analyzed cells. Further, shown for HT-29 and 1936-MEL, the knockdown of AVEN by siRNA in cell culture resulted in an increase of apoptotic characteristics and a decrease of VACV infection. These findings provide essential insights for future virus development.
Characterization of motility and erythrocyte adherence as virulence factors in African trypanosomes
(2018)
Pathogens causing African animal trypanosomiasis (AAT), the major livestock disease in sub-Saharan Africa, belong to the salivarian group of the African trypanosomes, which are transmitted by the bite of the tsetse fly (Glossina spec.). T. vivax, T. congolense and T. brucei brucei are major pathogens of cattle in particular, causing nagana, with dramatic socio-economic consequences for the affected regions. The parasites additionally have a huge reservoir of other livestock and wild animal hosts. T. brucei, the species which also includes the subspecies pathogenic to humans causing sleeping sickness, has been extensively studied as the cultivatable model trypanosome. But less is known about the other salivarian species, which are not routinely held in culture, if at all possible. A hallmark of trypanosomal lifestyle is the protozoan flagellates incessant motility, which enables them to populate an enormous range of habitats in very diverse hosts. We were now able to characterize, for the first time with high spatiotemporal resolution microscopy, the swimming behaviour and mechanism of the most relevant salivarian species isolated directly from blood. We show the influence of viscosity on the motility of bloodstream form (BSF) cells and simulate their movement between erythrocytes, giving a clear picture of how all analyzed species move under varying environmental conditions. We show that although the basic mechanism of flagellar motility applies to all analyzed species, there are clear morphological differences that produce different reactions to the physical environment. We could define specific conditions for highly increased swimming persistence and speed for compared to the behaviour in standard culture. These results have important implications for the parasites survival strategies in the host, e.g. regarding the capacity for antibody clearance. Although we show all species to effectively remove antibodies from the cell surface, T. congolense differed markedly in its motility behaviour, which gives rise to interesting questions about this species behaviour in the bloodstream. Most of the T. congolense parasites (and to a lesser extent T. vivax) adhere to sheep erythrocytes. Further in vitro studies showed that T. congolense and T. vivax adhered to rabbit, goat, pig and cattle erythrocytes- but binding behaviour was absent in murine blood. Notably, both T. brucei and T. evansi lacked adherence to all studied host erythrocytes. Generally, attachment to blood cells caused reduction of swimming velocities. Judging from its cell architecture, as well as the motility studies in higher media viscosity and in micropillar arrays, T. congolense is not adapted to swim at high speeds in the mammalian bloodstream. Low swimming speeds could allow these purely intravascular parasites to remain bound to the host erythrocytes.
Characterization of memories and ignorant (S6KII) mutants in operant conditioning in the heat-box
(2002)
Learning and memory processes of operant conditioning in the heat-box were analysed. Age, sex, and larval desity were not critical parameters influencing memory, while low or high activity levels of flies were negatively correlated with their performance. In a search for conditioning parameters leading to high retention scores, intermittent training was shown to give better results than continuous training. As the memory test is the immediate continuation of the conditioning phase just omitting reinforcement, we obtain a memory which consists of two components: a spatial preference for one side of the chamber and a stay-where-you-are effect in which the side preference is contaminated by the persistence of heat avoidance. Intermittent training strengthens the latter. In the next part, memory retention was investigated. Flies were trained in one chamber and tested in a second one after a brief reminder training. With this direct transfer, memory scores reflect an associative learning process in the first chamber. To investigate memory retention after extended time periods, indirect transfer experiments were performed. The fly was transferred to a different environment between training and test phases. With this procedure an after-effect of the training was still observed two hours later. Surprisingly, exposure to the chamber without conditioning also lead to a memory effect in the indirect transfer experiment. This exposure effect revealed a dispositional change that facilitates operant learning during the reminder training. The various memory effects are independent of the mushroom bodies. The transfer experiments and yoked controls proved that the heat-box records an associative memory. Even two hours after the operant conditioning procedure, the fly remembers that its position in the chamber controls temperature. The cAMP signaling cascade is involved in heat-box learning. Thus, amnesiac, rutabaga, and dunce mutants have an impaired learning / memory. Searching for, yet unknown, genes and signaling cascades involved in operant conditioning, a Drosophila melanogaster mutant screen with 1221 viable X-chromosome P-element lines was performed. 29 lines with consistently reduced heat avoidance/ learning or memory scores were isolated. Among those, three lines have the p[lacW] located in the amnesiac ORF, confirming that with the chosen candidate criteria the heat-box is a useful tool to screen for learning and /or memory mutants. The mutant line ignP1 (8522), which is defective in the gene encoding p90 ribosomal S6 kinase (S6KII), was investigated. The P-insertion of line ignP1 is the first Drosophila mutation in the ignorant (S6KII) gene. It has the transposon inserted in the first exon. Mutant males are characterized by low training performance, while females perform well in the standard experiment. Several deletion mutants of the ignorant gene have been generated. In precise jumpouts the phenotype was reverted. Imprecise jumpouts with a partial loss of the coding region were defective in operant conditioning. Surprisingly, null mutants showed wild-type behavior. This might indicate an indirect effect of the mutated ignorant gene on learning processes. In classical odor avoidance conditioning, ignorant null mutants showed a defect in the 3-min, 30-min, and 3-hr memory, while the precise jumpout of the transposon resulted in a reversion of the behavioral phenotype. Deviating results from operant and classical conditioning indicate different roles for S6KII in the two types of learning.
Characterization of a novel putative factor involved in host adaptation in Trypanosoma brucei
(2016)
Trypanosomes are masters of adaptation to different host environments
during their complex life cycle. Large-scale proteomic approaches provide information on changes at
the cellular level in a systematic way. However, a detailed work on single components is necessary
to understand the adaptation mechanisms on a molecular level. Here we have performed a detailed
characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation
factor (Tb927.11.2400) identified previously in a SILAC-based comparative proteome study.
Tb927.11.2400 shares 38% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form
(PCF) stage specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin like
(TbFlabarinL) and demonstrate that it is a result of a gene duplication event, which occurred in
African trypanosomes. TbFlabarinL is not essential for growth of the parasites under cell culture
conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We
generated a TbFlabarinL-specific antibody and showed that it localizes in the flagellum. The
co-immunoprecipitation experiment together with a biochemical cell fractionation indicated a dual
association of TbFlabarinL with the flagellar
membrane and the components of the paraflagellar rod.
Monolayer or suspension cell cultures are of only limited value as experimental models for human cancer. Therefore, more sophisticated, three-dimensional culture systems like spheroid cultures or histocultures are used, which more closely mimic the tumor in individual patients compared to monolayer or suspension cultures. As tissue culture or tissue engineering requires more sophisticated culture, specialized in vitro techniques may also improve experimental tumor models. In the present work, a new miniaturized hollow-fiber bioreactor system for mammalian cell culture in small volumes (up to 3 ml) is characterized with regard to transport characteristics and growth of leukemic cell lines (chapter 2). Cell and medium compartment are separated by dialysis membranes and oxygenation is accomplished using oxygenation membranes. Due to a transparent housing, cells can be observed by microscopy during culture. The leukemic cell lines CCRF-CEM, HL-60 and REH were cultivated up to densities of 3.5 x 107/ml without medium change or manipulation of the cells. Growth and viability of the cells in the bioreactor were the same or better, and the viable cell count was always higher compared to culture in Transwellâ plates. As shown using CCRF-CEM cells, growth in the bioreactor was strongly influenced and could be controlled by the medium flow rate. As a consequence, consumption of glucose and generation of lactate varied with the flow rate. Influx of low molecular weight substances in the cell compartment could be regulated by variation of the concentration in the medium compartment. Thus, time dependent concentration profiles (e.g. pharmacokinetic profiles of drugs) can be realized as illustrated using glucose as a model compound. Depending on the molecular size cut-off of the membranes used, added growth factors like GM-CSF and IL-3 as well as factors secreted from the cells are retained in the cell compartment for up to one week. Second, a method for monitoring cell proliferation the hollow-fiber bioreactor by use of the Alamar BlueTM dye was developed (chapter 3). Alamar BlueTM is a non-fluorescent compound which yields a fluorescent product after reduction e.g. by living cells. In contrast to the MTT-assay, the Alamar BlueTM-assay does not lead to cell death. However, when not removed from the cells, the Alamar BlueTM dye shows a reversible, time- and concentration-dependent growth inhibition as observed for leukemic cell lines. When applied in the medium compartment of a hollow-fiber bioreactor system, the dye is delivered to the cells across the hollow-fiber membrane, reduced by the cells and released from the cell into the medium compartment back again. Thus, fluorescence intensity can be measured in medium samples reflecting growth of the cells in the cell compartment. This procedure offers several advantages. First, exposure of the cells to the dye can be reduced compared to conventional culture in plates. Second, handling steps are minimized since no sample of the cells needs to be taken for readout. Moreover, for the exchange of medium, a centrifugation step can be avoided and the cells can be cultivated further. Third, the method allows to discriminate between cell densities of 105, 106 and 107 of proliferating HL-60 cells cultivated in the cell compartment of the bioreactor. Measurement of fluorescence in the medium compartment is more sensitive compared to glucose or lactate measurement for cell counts below 106 cells/ml, in particular. In conclusion, the Alamar BlueTM-assay combined with the hollow-fiber bioreactor offers distinct advantages for the non-invasive monitoring of cell viability and proliferation in a closed system. In chapter 4 the use of the hollow-fiber bioreactor as a tool for toxicity testing was investigated, as current models for toxicity as well as efficacy testing of drugs in vitro allow only limited conclusions with regard to the in vivo situation. Examples of the drawbacks of current test systems are the lack of realistic in vitro tumor models and difficulties to model drug pharmacokinetics. The bioreactor proved to be pyrogen free and is steam-sterilizable. Leukemic cell lines like HL-60 and primary cells such as PHA-stimulated lymphocytes can be grown up to high densities of 1-3 x 107 and analyzed during growth in the bioreactor by light-microscopy. The cytostatic drug Ara-C shows a dose-dependent growth inhibition of HL-60 cells and a dose-response curve similar to controls in culture plates. The bioreactor system is highly flexible since several systems can be run in parallel, soluble drugs can be delivered continuously via a perfusion membrane and gaseous compounds via an oxygenation membrane which also allows to control pO2 and pH (via pCO2) during culture in the cell compartment. The modular concept of the bioreactor system allows realization of a variety of different design properties, which may lead to an improved in vitro system for toxicity testing by more closely resembling the in vivo situation. Whereas several distinct advantages of the new system have been demonstrated, more work has to be done to promote in vitro systems in toxicity testing and drug development further and to reduce the need for animal tests.