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The transcriptional regulation of the cellular mechanisms involves many different components and different levels of control which together contribute to fine tune the response of cells to different environmental stimuli. In some responses, diverse signaling pathways can be controlled simultaneously. One of the most important cellular processes that seem to possess multiple levels of regulation is photosynthesis. A model organism for studying photosynthesis-related processes is the unicellular green algae Chlamydomonas reinhardtii, due to advantages related to culturing, genetic manipulation and availability of genome sequence. In the present study, we were interested in understanding the regulatory mechanisms underlying photosynthesis-related processes. To achieve this goal different molecular approaches were followed. In order to indentify protein transcriptional regulators we optimized a method for isolation of nuclei and performed nuclear proteome analysis using shotgun proteomics. This analysis permitted us to improve the genome annotation previously published and to discover conserved and enriched protein motifs among the nuclear proteins. In another approach, a quantitative RT-PCR platform was established for the analysis of gene expression of predicted transcription factor (TF) and other transcriptional regulator (TR) coding genes by transcript profiling. The gene expression profiles for more than one hundred genes were monitored in time series experiments under conditions of changes in light intensity (200 µE m-2 s-1 to 700 µE m-2 s-1), and changes in concentration of carbon dioxide (5% CO2 to 0.04% CO2). The results indicate that many TF and TR genes are regulated in both environmental conditions and groups of co-regulated genes were found. Our findings also suggest that some genes can be common intermediates of light and carbon responsive regulatory pathways. These approaches together gave us new insights about the regulation of photosynthesis and revealed new candidate regulatory genes, helping to decipher the gene regulatory networks in Chlamydomonas. Further experimental studies are necessary to clarify the function of the candidate regulatory genes and to elucidate how cells coordinately regulate the assimilation of carbon and light responses.
Subcellular compartmentation of primary carbon metabolism in mesophyll cells of Arabidopsis thaliana
(2011)
Metabolism in plant cells is highly compartmented, with many pathways involving reactions in more than one compartment. For example, during photosynthesis in leaf mesophyll cells, primary carbon fixation and starch synthesis take place in the chloroplast, whereas sucrose is synthesized in the cytosol and stored in the vacuole. These reactions are tightly regulated to keep a fine balance between the carbon pools of the different compartments and to fulfil the energy needs of the organelles. I applied a technique which fractionates the cells under non-aqueous conditions, whereby the metabolic state is frozen at the time of harvest and held in stasis throughout the fractionation procedure. With the combination of non-aqueous fractionation and mass spectrometry based metabolite measurements (LC-MS/MS, GC-MS) it was possible to investigate the intracellular distributions of the intermediates of photosynthetic carbon metabolism and its products in subsequent metabolic reactions. With the knowledge about the in vivo concentrations of these metabolites under steady state photosynthesis conditions it was possible to calculate the mass action ratio and change in Gibbs free energy in vivo for each reaction in the pathway, to determine which reactions are near equilibrium and which are far removed from equilibrium. The Km value and concentration of each enzyme were compared with the concentrations of its substrates in vivo to assess which reactions are substrate limited and so sensitive to changes in substrate concentration. Several intermediates of the Calvin-Benson cycle are substrates for other pathways, including dihydroxyacetone-phosphate (DHAP,sucrose synthesis), fructose 6-phosphate (Fru6P, starch synthesis), erythrose 4-phosphate (E4P,shikimate pathway) and ribose 5-phosphate (R5P, nucleotide synthesis). Several of the enzymes that metabolise these intermediates, and so lie at branch points in the pathway, are triose-phosphate isomerase (DHAP), transketolase (E4P, Fru6P), sedoheptulose-1,7-bisphosphate aldolase (E4P) and ribose-5-phosphate isomerase (R5P) are not saturated with their respective substrate as the metabolite concentration is lower than the respective Km value. In terms of metabolic control these are the steps that are most sensitive to changes in substrate availability, while the regulated irreversible reactions of fructose-1,6-bisphosphatase and sedoheptulose-1,7-bisphosphatase are relatively insensitive to changes in the concentrations of their substrates. In the pathway of sucrose synthesis it was shown that the concentration of the catalytic binding site of the cytosolic aldolase is lower than the substrate concentration of DHAP, and that the concentration of Suc6P is lower than the Km of sucrose-phosphatase for this substrate. Both the sucrose-phosphate synthase and sucrose-phosphatase reactions are far removed from equilibrium in vivo. In wild type A. thaliana Columbia-0 leaves, all of the ADPGlc was found to be localised in the chloroplasts. ADPglucose pyrophosphorylase is localised to the chloroplast and synthesises ADPGlc from ATP and Glc1P. This distribution argues strongly against the hypothesis proposed by Pozueta-Romero and colleagues that ADPGlc for starch synthesis is produced in the cytosol via ADP-mediated cleavage of sucrose by sucrose synthase. Based on this observation and other published data it was concluded that the generally accepted pathway of starch synthesis from ADPGlc produced by ADPglucose pyrophosphorylase in the chloroplasts is correct, and that the alternative pathway is untenable. Within the pathway of starch synthesis the concentration of ADPGlc was found to be well below the Km value of starch synthase for ADPGlc, indicating that the enzyme is substrate limited. A general finding in the comparison of the Calvin-Benson cycle with the synthesis pathways of sucrose and starch is that many enzymes in the Calvin Benson cycle have active binding site concentrations that are close to the metabolite concentrations, while for nearly all enzymes in the synthesis pathways the active binding site concentrations are much lower than the metabolite concentrations.
Regulation of potassium channels in plants : biophysical mechanisms and physiological implacations
(2011)
Mathematical modeling of biological phenomena has experienced increasing interest since new high-throughput technologies give access to growing amounts of molecular data. These modeling approaches are especially able to test hypotheses which are not yet experimentally accessible or guide an experimental setup. One particular attempt investigates the evolutionary dynamics responsible for today's composition of organisms. Computer simulations either propose an evolutionary mechanism and thus reproduce a recent finding or rebuild an evolutionary process in order to learn about its mechanism. The quest for evolutionary fingerprints in metabolic and gene-coexpression networks is the central topic of this cumulative thesis based on four published articles. An understanding of the actual origin of life will probably remain an insoluble problem. However, one can argue that after a first simple metabolism has evolved, the further evolution of metabolism occurred in parallel with the evolution of the sequences of the catalyzing enzymes. Indications of such a coevolution can be found when correlating the change in sequence between two enzymes with their distance on the metabolic network which is obtained from the KEGG database. We observe that there exists a small but significant correlation primarily on nearest neighbors. This indicates that enzymes catalyzing subsequent reactions tend to be descended from the same precursor. Since this correlation is relatively small one can at least assume that, if new enzymes are no "genetic children" of the previous enzymes, they certainly be descended from any of the already existing ones. Following this hypothesis, we introduce a model of enzyme-pathway coevolution. By iteratively adding enzymes, this model explores the metabolic network in a manner similar to diffusion. With implementation of an Gillespie-like algorithm we are able to introduce a tunable parameter that controls the weight of sequence similarity when choosing a new enzyme. Furthermore, this method also defines a time difference between successive evolutionary innovations in terms of a new enzyme. Overall, these simulations generate putative time-courses of the evolutionary walk on the metabolic network. By a time-series analysis, we find that the acquisition of new enzymes appears in bursts which are pronounced when the influence of the sequence similarity is higher. This behavior strongly resembles punctuated equilibrium which denotes the observation that new species tend to appear in bursts as well rather than in a gradual manner. Thus, our model helps to establish a better understanding of punctuated equilibrium giving a potential description at molecular level. From the time-courses we also extract a tentative order of new enzymes, metabolites, and even organisms. The consistence of this order with previous findings provides evidence for the validity of our approach. While the sequence of a gene is actually subject to mutations, its expression profile might also indirectly change through the evolutionary events in the cellular interplay. Gene coexpression data is simply accessible by microarray experiments and commonly illustrated using coexpression networks where genes are nodes and get linked once they show a significant coexpression. Since the large number of genes makes an illustration of the entire coexpression network difficult, clustering helps to show the network on a metalevel. Various clustering techniques already exist. However, we introduce a novel one which maintains control of the cluster sizes and thus assures proper visual inspection. An application of the method on Arabidopsis thaliana reveals that genes causing a severe phenotype often show a functional uniqueness in their network vicinity. This leads to 20 genes of so far unknown phenotype which are however suggested to be essential for plant growth. Of these, six indeed provoke such a severe phenotype, shown by mutant analysis. By an inspection of the degree distribution of the A.thaliana coexpression network, we identified two characteristics. The distribution deviates from the frequently observed power-law by a sharp truncation which follows after an over-representation of highly connected nodes. For a better understanding, we developed an evolutionary model which mimics the growth of a coexpression network by gene duplication which underlies a strong selection criterion, and slight mutational changes in the expression profile. Despite the simplicity of our assumption, we can reproduce the observed properties in A.thaliana as well as in E.coli and S.cerevisiae. The over-representation of high-degree nodes could be identified with mutually well connected genes of similar functional families: zinc fingers (PF00096), flagella, and ribosomes respectively. In conclusion, these four manuscripts demonstrate the usefulness of mathematical models and statistical tools as a source of new biological insight. While the clustering approach of gene coexpression data leads to the phenotypic characterization of so far unknown genes and thus supports genome annotation, our model approaches offer explanations for observed properties of the coexpression network and furthermore substantiate punctuated equilibrium as an evolutionary process by a deeper understanding of an underlying molecular mechanism.
A phagocyte-specific Irf8 gene enhancer establishes early conventional dendritic cell commitment
(2011)
Haematopoietic development is a complex process that is strictly hierarchically organized. Here, the phagocyte lineages are a very heterogeneous cell compartment with specialized functions in innate immunity and induction of adaptive immune responses. Their generation from a common precursor must be tightly controlled. Interference within lineage formation programs for example by mutation or change in expression levels of transcription factors (TF) is causative to leukaemia. However, the molecular mechanisms driving specification into distinct phagocytes remain poorly understood. In the present study I identify the transcription factor Interferon Regulatory Factor 8 (IRF8) as the specification factor of dendritic cell (DC) commitment in early phagocyte precursors. Employing an IRF8 reporter mouse, I showed the distinct Irf8 expression in haematopoietic lineage diversification and isolated a novel bone marrow resident progenitor which selectively differentiates into CD8α+ conventional dendritic cells (cDCs) in vivo. This progenitor strictly depends on Irf8 expression to properly establish its transcriptional DC program while suppressing a lineage-inappropriate neutrophile program. Moreover, I demonstrated that Irf8 expression during this cDC commitment-step depends on a newly discovered myeloid-specific cis-enhancer which is controlled by the haematopoietic transcription factors PU.1 and RUNX1. Interference with their binding leads to abrogation of Irf8 expression, subsequently to disturbed cell fate decisions, demonstrating the importance of these factors for proper phagocyte cell development. Collectively, these data delineate a transcriptional program establishing cDC fate choice with IRF8 in its center.
Functional analyses of microtubule and centrosome-associated proteins in Dictyostelium discoideum
(2011)
Understanding the role of microtubule-associated proteins is the key to understand the complex mechanisms regulating microtubule dynamics. This study employs the model system Dictyostelium discoideum to elucidate the role of the microtubule-associated protein TACC (Transforming acidic coiled-coil) in promoting microtubule growth and stability. Dictyostelium TACC was localized at the centrosome throughout the entire cell cycle. The protein was also detected at microtubule plus ends, however, unexpectedly only during interphase but not during mitosis. The same cell cycle-dependent localization pattern was observed for CP224, the Dictyostelium XMAP215 homologue. These ubiquitous MAPs have been found to interact with TACC proteins directly and are known to act as microtubule polymerases and nucleators. This work shows for the first time in vivo that both a TACC and XMAP215 family protein can differentially localize to microtubule plus ends during interphase and mitosis. RNAi knockdown mutants revealed that TACC promotes microtubule growth during interphase and is essential for proper formation of astral microtubules in mitosis. In many organisms, impaired microtubule stability upon TACC depletion was explained by the failure to efficiently recruit the TACC-binding XMAP215 protein to centrosomes or spindle poles. By contrast, fluorescence recovery after photobleaching (FRAP) analyses conducted in this study demonstrate that in Dictyostelium recruitment of CP224 to centrosomes or spindle poles is not perturbed in the absence of TACC. Instead, CP224 could no longer be detected at the tips of microtubules in TACC mutant cells. This finding demonstrates for the first time in vivo that a TACC protein is essential for the association of an XMAP215 protein with microtubule plus ends. The GFP-TACC strains generated in this work also turned out to be a valuable tool to study the unusual microtubule dynamics in Dictyostelium. Here, microtubules exhibit a high degree of lateral bending movements but, in contrast most other organisms, they do not obviously undergo any growth or shrinkage events during interphase. Despite of that they are affected by microtubuledepolymerizing drugs such as thiabendazole or nocodazol which are thought to act solely on dynamic microtubules. Employing 5D-fluorescence live cell microscopy and FRAP analyses this study suggests Dictyostelium microtubules to be dynamic only in the periphery, while they are stable at the centrosome. In the recent years, the identification of yet unknown components of the Dictyostelium centrosome has made tremendous progress. A proteomic approach previously conducted by our group disclosed several uncharacterized candidate proteins, which remained to be verified as genuine centrosomal components. The second part of this study focuses on the investigation of three such candidate proteins, Cenp68, CP103 and the putative spindle assembly checkpoint protein Mad1. While a GFP-CP103 fusion protein could clearly be localized to isolated centrosomes that are free of microtubules, Cenp68 and Mad1 were found to associate with the centromeres and kinetochores, respectively. The investigation of Cenp68 included the generation of a polyclonal anti-Cenp68 antibody, the screening for interacting proteins and the generation of knockout mutants which, however, did not display any obvious phenotype. Yet, Cenp68 has turned out as a very useful marker to study centromere dynamics during the entire cell cycle. During mitosis, GFP-Mad1 localization strongly resembled the behavior of other Mad1 proteins, suggesting the existence of a yet uncharacterized spindle assembly checkpoint in Dictyostelium.