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Every organism contains a characteristic number of chromosomes that have to be segregated equally into
two daughter cells during mitosis. Any error during chromosome segregation results in daughter cells that
lost or gained a chromosome, a condition known as aneuploidy. Several studies from our laboratory and
across the world have previously shown that aneuploidy per se strongly affects cellular physiology.
However, these studies were limited mainly to the chromosomal gains due to the availability of several
model systems. Strikingly, no systemic study to evaluate the impact of chromosome loss in the human
cells has been performed so far. This is mainly due to the lack of model systems, as chromosome loss is
incompatible with survival and drastically reduces cellular fitness. During my PhD thesis, for the first time,
I used diverse omics and biochemical approaches to investigate the consequences of chromosome losses
in human somatic cells.
Using isogenic monosomic cells derived from the human cell line RPE1 lacking functional p53, we showed
that, similar to the cells with chromosome gains, monosomic cells proliferate slower than the parental
cells and exhibit genomic instability. Transcriptome and proteome analysis revealed that the expression
of genes encoded on the monosomic chromosomes was reduced, as expected, but the abundance was
partially compensated towards diploid levels by both transcriptional and post transcriptional mechanisms.
Furthermore, we showed that monosomy induces global gene expression changes that are distinct to
changes in response to chromosome gains. The most consistently deregulated pathways among the
monosomies were ribosomes and translation, which we validated using polysome profiling and analysis
of translation with puromycin incorporation experiments. We showed that these defects could be
attributed to the haploinsufficiency of ribosomal protein genes (RPGs) encoded on monosomic
chromosomes. Reintroduction of p53 into the monosomic cells uncovered that monosomy is incompatible
with p53 expression and that the monosomic cells expressing p53 are either eliminated or outgrown by
the p53 negative population. Given the RPG haploinsufficiency and ribosome biogenesis defects caused
by monosomy, we show an evidence that the p53 activation in monosomies could be caused by the
defects in ribosomes. These findings were further supported by computational analysis of cancer genomes
revealing those cancers with monosomic karyotype accumulated frequently p53 pathway mutations and
show reduced ribosomal functions.
Together, our findings provide a rationale as to why monosomy is embryonically lethal, but frequently
occurs in p53 deficient cancers.
Membrane proteins are of high pharmacological interest as they are involved in a variety of vital functions. However, to make them accessible to in vitro studies, they often need to be extracted from their natural lipid environment and stabilized with the aid of membrane-mimetic systems. Such membrane mimics can consist of diverse amphiphilic molecules. Small-molecule amphiphiles that can solubilize lipid bilayers, so-called detergents, have been invaluable tools for membrane-protein research in recent decades. Herein, novel small-molecule glyco-amphiphiles embodying three distinct design principles are introduced, and their biophysical and physicochemical properties are investigated. In doing so, the major aims consist in establishing new promising amphiphiles and in determining structure–efficacy relationships for their synthesis and application.
First, the software package D/STAIN was introduced to facilitate the analysis of demicellization curves obtained by isothermal titration calorimetry. The robustness of the underlying algorithm was demonstrated by analyzing demicellization curves representing large variations in amphiphile concentrations and thermodynamic parameters.
Second, the interactions of diastereomeric cyclopentane maltoside amphiphiles (CPMs) with lipid bilayers and membrane proteins were investigated. To this end, lipid model membranes, cellular membranes, and model membrane proteins were treated with different stereoisomer CPMs. These investigations pointed out the importance of stereochemical configuration in the solubilization of lipid bilayers, in the extraction of membrane proteins, and, ultimately, in the stabilization of the latter. Ultimately, CPM C12 could be identified as a particularly stabilizing agent.
Third, the influence of a polymerizable group attached to detergent-like amphiphiles was characterized regarding their micellization, micellar properties, and ability to solubilize lipid membranes. This revealed that such chemical modifications can have different degrees of impact regarding the investigated properties. In particular, micellization was influenced substantially, whereas the sizes of the resulting micelles varied slightly. The polymerizable amphiphiles were shown to solubilize artificial and natural lipid membranes and, consequently, to extract membrane proteins.
Last, the self-assembly of diglucoside amphiphiles bearing either a hydrocarbon or a lipophobic fluorocarbon chain to form native nanodiscs was investigated. It was shown that the presence of a fluorocarbon hydrophobic chain conveys superior stabilization properties onto the amphiphile and the resulting nanodiscs. Moreover, the kinetics of lipid exchange were fundamentally altered by the presence of the fluorocarbon amphiphiles in the nanodisc rim.
On a route from whole genome duplication to aneuploidy and cancer: consequences and adaptations
(2022)
Whole genome duplication (WGD) is commonly accepted as an intermediate state between healthy cells and aneuploid cancer cells. Usually, cells after WGD get removed from the replicating pool by p53-dependent cell cycle arrest or apoptosis. Cells, which are able to bypass these mechanisms exhibit chromosomal instability (CIN) and DNA damage, promoting the formation of highly aneuploid karyotypes. In general, WGD favors several detrimental consequences such as increased drug resistance, transformation and metastasis formation. Therefore, it is of special interest to investigate the limiting factors and consequences of tetraploid proliferation as well as the adaptations to WGD. In the past it has been difficult to study the consequences of such large-scale genomic changes and how cells adapt to tetraploidy in order to survive. Our lab established protocols to generate tetraploids as well as isolated post-tetraploid/aneuploid single cells clones derived from euploid parental cell lines after induction of cytokinesis failure. This system enables to study the consequences and adaptations of WGD in newly generated tetraploid cells and evolved post-tetraploid clones in comparison to their isogenic parental cell line.
Using newly generated tetraploids from HCT116 cells, we identified USP28 and SPINT2 as novel factors limiting the proliferation after WGD. Using mass spectrometry and immunoprecipitation, we revealed an interaction between USP28 and NuMA1 upon WGD, which affects centrosome coalescence of supernumerary centrosomes, an important process that enhances survival of tetraploids. Furthermore, we validated the occurrence of DNA damage in tetraploid cells and found that USP28 depletion diminished the DNA damage dependent checkpoint activation. SPINT2 influences the proliferation after WGD by regulating the transcription of CDKN1A via histone acetylation. Following proliferating tetraploid cells, we confirmed the activation of the DNA damage response (DDR) by immunoblotting and microscopic approaches. Furthermore, we show that the DDR in the arising post-tetraploid clones is reduced. Further experiments verified the appearance of severe mitotic aberrations, replication stress and accumulation of reactive oxygen species in newly generated tetraploids as well as in the aneuploid cancer cells contributing to the occurrence of DNA damage. Using various drug treatments, we observed an increased dependency on the spindle assembly checkpoint in aneuploid cancer cells compared to their diploid parental cell line. Additionally, siRNA knock down experiments revealed the kinesin motor protein KIF18A as an essential protein in aneuploid cells.
Taken together, the results point out cellular consequences of proliferation after tetraploidization as well as the cellular adaptations needed to cope with the increased amount of DNA.
The scaffolding protein family Fe65, composed of Fe65, Fe65L1, and Fe65L2, was identified as an interaction partner of the amyloid precursor protein (APP), which plays a key function in Alzheimer’s disease. All three Fe65 family members possess three highly conserved interaction domains, forming complexes with diverse binding partners that can be assigned to different cellular functions, such as transactivation of genes in the nucleus, modulation of calcium homeostasis and lipid metabolism, and regulation of the actin cytoskeleton. In this article, we rule out putative new intracellular signaling mechanisms of the APP-interacting protein Fe65 in the regulation of actin cytoskeleton dynamics in the context of various neuronal functions, such as cell migration, neurite outgrowth, and synaptic plasticity.
Multi-omics analysis as a tool to investigate causes and consequences of impaired genome integrity
(2022)
Impaired genome integrity has severe consequences for the viability of any cell. Unrepaired DNA lesions can lead to genomically unstable cells, which will often become predisposed for malignant growth and tumorigenesis, where genomic instability turns into a driving factor through the selection of more aggressive clones. Aneuploidy and polyploidy are both poorly tolerated in somatic cells, but frequently observed hallmarks of cancer. Keeping the genome intact requires the concentrated action of cellular metabolism, cell cycle and DNA damage response.
This study presents multi-omics analysis as a versatile tool to understand the various causes and consequences of impaired genome integrity. The possible computational approaches are demonstrated on three different datasets. First, an analysis of a collection of DNA repair experiments is shown, which features the creation of a high-fidelity dataset for the identification and characterization of DNA damage factors. Additionally, a web-application is presented that allows scientists without a computational background to interrogate this dataset. Further, the consequences of chromosome loss in human cells are analyzed by an integrated analysis of TMT labeled mass spectrometry and sequencing data. This analysis revealed heterogeneous cellular responses to chromosome losses that differ from chromosome gains. My analysis further revealed that cells possess both transcriptional and post-transcriptional mechanisms that compensate for the loss of genes encoded on a monosomic chromosome to alleviate the detrimental consequences of reduced gene expression. In my final project, I present a multi-omics analysis of data obtained from SILAC labeled mass spectrometry and dynamic transcriptome analysis of yeast cells of different ploidy, from haploidy to tetraploid. This analysis revealed that unlike cell volume, the proteome of a cell does not scale linearly with increasing ploidy. While the expression of most proteins followed this scaling, several proteins showed ploidy-dependent regulation that could not be explained by transcriptome expression. Hence, this ploidy-dependent regulation occurs mostly on a post-transcriptional level. The analysis uncovered that ribosomal and translation related proteins are downregulated with increasing ploidy, emphasizing a remodeling of the cellular proteome in response to increasing ploidy to ensure survival of cells after whole genome doubling. Altogether this study intends to show how state-of-the-art multi-omics analysis can uncover cellular responses to impaired genome integrity in a highly diverse field of research.
Like many other bacteria, the opportunistic pathogen P. aeruginosa encodes a broad network of enzymes that regulate the intracellular concentration of the second messenger c-di-GMP. One of these enzymes is the phosphodiesterase NbdA that consists of three domains: a membrane anchored, putative sensory MHYT domain, a non-functional diguanylate cyclase domain with degenerated GGDEF motif and an active PDE domain with EAL motif. Analysis of the nbdA open reading frame by 5’-RACE PCR revealed an erroneous annotation of nbdA in the Pseudomonas database with the ORF 170 bp shorter than previously predicted. The newly defined promoter region of nbdA contains recognition sites for the alternative sigma-factor RpoS as well as the transcription factor AmrZ. Promoter analysis within PAO1 wt as well as rpoS and amrZ mutant strains utilizing transcriptional fusions of the nbdA promoter to the reporter gene lacZ revealed transcriptional activation of nbdA by RpoS in stationary growth phase and transcriptional repression by AmrZ. Additionally, no influence of nitrite and neither exogenous nor endogenous NO on nbdA transcription could be shown in this study. However, deletion of the nitrite reductase gene nirS led to a strong increase of nbdA promoter activity which needs to be characterized further. Predicted secondary structures of the 5’-UTR of the nbdA mRNA indicated either an RNA thermometer function of the mRNA or post-transcriptional regulation of nbdA by the RNA binding proteins RsmA and RsmF. Nevertheless, translational studies using fusions of the 5’ UTR of nbdA to the reporter gene bgaB did not verify either of these hypotheses. In general, nbdA translational levels were very low and neither the production of the reporter BgaB nor genomically encoded NbdA could be detected on a western blot. Overproduction of NbdA variants induced many phenotypic changes in motility and biofilm formation. But strains overproducing variants containing the MHYT domain revealed greatly elongated cells and were impaired in surface growth, indicating a misbalance in the membrane protein homeostasis. Therefore, these phenotypes have to be interpreted very critically. Microscopic studies with fluorescently tagged NbdA revealed either a diffuse fluorescent signal of NbdA or the formation of fluorescent foci which were located mainly at the cell poles. Co-localization studies with the polar flagellum and the chemotaxis protein CheA showed that NbdA is not generally localizing to the flagellated cell pole. NbdA localization indicates the control of a specific local c-di-GMP pool in the cell which is most likely involved in MapZ mediated chemotactic flagellar motor switching.
The handling of oxygen sensitive samples and growth of obligate anaerobic organisms
requires the stringent exclusion of oxygen, which is omnipresent in our normal atmospheric
environment. Anaerobic workstations (aka. Glove boxes) enable the handling of
oxygen sensitive samples during complex procedures, or the long-term incubation of
anaerobic organisms. Depending on the application requirements, commercial workstations
can cost up to 60.000 €. Here we present the complete build instructions for a highly
adaptive, Arduino based, anaerobic workstation for microbial cultivation and sample handling,
with features normally found only in high cost commercial solutions. This build can
automatically regulate humidity, H2 levels (as oxygen reductant), log the environmental
data and purge the airlock. It is built as compact as possible to allow it to fit into regular
growth chambers for full environmental control. In our experiments, oxygen levels during
the continuous growth of oxygen producing cyanobacteria, stayed under 0.03 % for 21 days
without needing user intervention. The modular Arduino controller allows for the easy
incorporation of additional regulation parameters, such as CO2 concentration or air pressure.
This paper provides researchers with a low cost, entry level workstation for anaerobic
sample handling with the flexibility to match their specific experimental needs.
Botrytis cinerea is a world-wide occurring plant pathogen, causing pre- and post-harvest gray mold rot on a large number of fruit, vegetable, and flower crops. B. cinerea is closely related to Botrytis pseudocinerea, another broad host range species which often occurs in sympatry with B. cinerea, and to several host-specific species including Botrytis fabae and Botrytis calthae. B. cinerea populations have been shown to be genetically heterogeneous, and attempts have been made to correlate genetic markers to virulence and host adaptation. Here, we present the development of a multilocus sequence typing (MLST) scheme, with 10 genes selected for high variability and phylogenetic congruence, to evaluate the genetic diversity of B. cinerea, B. fabae, and B. pseudocinerea. Using PacBio-assisted simultaneous mass sequencing of PCR products, MLST analysis of about 100 strains from diverse geographical origins and years of isolation was performed, which resulted in high-resolution strain differentiation and robust species separation. Several B. cinerea strains formed an as yet unknown population, referred to as group B, which was well separated from all other B. cinerea strains. Furthermore, the gene cluster for biosynthesis of the phytotoxin botcinic acid was missing in B. cinerea B strains. B. cinerea strains from the monocot Iris pseudacorus were found to form a genetically distinct population, and contained an intact gene cluster for production of the red pigment bikaverin, which is usually degenerated in B. cinerea. Remarkably, these strains were much more aggressive on Iris than other B. cinerea strains, which is the first unequivocal example for host specialization in B. cinerea. Our data reveal new insights into the genetic diversity of B. cinerea and provide evidence for intraspecific differentiation and different degrees of host adaptation of this polyphagous necrotrophic pathogen.
Most mitochondrial proteins are synthesized in the cytosol and targeted to the mitochondrial
surface in a post-translational manner. The surface of the endoplasmic reticulum (ER) plays an
active role in this targeting reaction. ER-associated chaperones interact with certain mitochondrial
membrane protein precursors and transfer them onto receptor proteins of the mitochondrial surface
in a process termed ER-SURF. ATP-driven proteins in the membranes of mitochondria (Msp1, ATAD1)
and the ER (Spf1, P5A-ATPase) serve as extractors for the removal of mislocalized proteins. If the
re-routing to mitochondria fails, precursors can be degraded by ER or mitochondria-associated degradation
(ERAD or MAD respectively) in a proteasome-mediated reaction. This review summarizes the
current knowledge about the cooperation of the ER and mitochondria in the targeting and quality
control of mitochondrial precursor proteins.
To render membrane proteins amenable to in vitro functional and structural studies, they need to be extracted from cellular membranes and stabilised using membrane-mimetic systems. Amphiphilic copolymers gain considerable interest, because they are able to coextract
membrane proteins and their surrounding lipids from complex cellular membranes to form polymer-bounded nanodiscs. The latter harbour a native-like lipid-bilayer core stabilised by a copolymer rim. Accordingly, these membrane mimics are supposed to provide superior
stability to embedded membrane proteins as compared with conventional detergent micelles.
Herein, the formation of nanodiscs by the most commonly used styrene/maleic acid (SMA)copolymer, termed SMA(2:1), was elucidated in detail. To this end, the equilibrium solubilisation efficiencies towards model and cellular membranes were quantified and
compared with those of the more hydrophobic SMA(3:1) and the more hydrophilic diisobutylene/maleic acid (DIBMA) copolymers. It was shown that, from a thermodynamic viewpoint, SMA(2:1) is the most efficient membrane solubiliser in terms of lipid- and proteinextraction
yields. Solvent properties (pH, ionic strength) or membrane characteristics (lateral pressure, charge, or thickness) can affect the polymers’ solubilisation efficiency to a certain extent. In addition, the lipid transfer behaviour of SMA(2:1) nanodiscs was studied.
Notwithstanding their high effective negative charge, SMA(2:1) nanodiscs exchange phospholipids more rapidly among each other than vesicles or protein-bounded nanodiscs, thus rendering them highly dynamic nano-assemblies. Two alternative electroneutral polymers, namely SMA(2:1)-SB and DIBMA-SB, were introduced in this thesis. They were generated by polymer backbone modifications of SMA(2:1) and DIBMA, respectively. The derivatised polymers were shown to quantitatively solubilise model and biological membranes and, like DIBMA, only had a mild effect on lipidbilayer integrity. Along these lines, DIBMA-SB preserved membrane-protein complexes of distinct structural classes and extracted them from various cellular membranes. Importantly, the electroneutral polymers were amenable to protein/lipid interaction studies otherwise masked by unspecific interactions of their anionic counterparts with target lipids or proteins. Taken together, the in-depth characterisation of nanodiscs formed by anionic and electroneutral polymers allows for adjusting the nanodisc properties to specifically suit experimental requirements or address membrane-protein research questions.
Amino acids, apart from being building blocks of proteins, serve various cellular and metabolic functions1,2. Changes in amino acid handling have been observed in a wide range of human pathologies, including diabetes and various metabolic disorders (aminoacidopathies)3–5. Saccharomyces cerevisiae is used as a model to investigate how increase in amino acid content (in the form of amino acid dropout mix: AAM) in growth medium influences cell growth. Intriguingly, it was observed that increasing the concentration of AAM in the media (double or triple times; 2 X AAM and 3 X AAM respectively), severely affects the growth of auxotrophic but not of prototrophic yeast strains in presence of glucose as carbon substrate. Increased concentration of Ehrlich amino acids, which are degraded to fusel acidic/alcoholic compounds, induced the observed slow growth phenotype of BY4742. These phenotypes can be rescued by either re-establishing the functional leucine biosynthetic pathway in BY4742 (leucine auxotroph) or increasing leucine in proportion to the increased AAM. Interestingly, the amino acid dependent growth phenotypes are absent when cells grow in media containing non-fermentable carbon sources. Furthermore, the deletion of ILV2 or ILV3 (genes encoding enzymes involved in the leucine biosynthetic pathway) also rescues the growth phenotype of BY4742 on 2 X AAM and 3 X AAM growth media. It was found that Ilv3 is the potential switching point and links cellular growth to redox homeostasis. The possibility of leucine limitation per se or transport competition between different Ehrlich amino acids and leucine, as a cause for the observed phenotypes, is ruled out. Upregulation of the branched-chain amino acid pathway inhibits cell growth of BY4742 on 2 X AAM. Although we could not detect KIV, the α-keto acid intermediate formed by the Ilv3. It is proposed that KIV itself (or its unknown downstream product) leads to the onset of the observed phenotypes. Different studies suggest that oxidative stress (due to accumulation of branched-chain amino acids (BCaa) and their α-keto acids) contributes to the neurological damage of MSUD patients6–9. It was also observed that the trigger of the BCaa bio-synthesis pathway on 2 X AAM growth conditions also contributes to the significant oxidative stress in the cell. In conclusion, we propose that yeast can be used as a suitable model system to study how accumulation of BCaa and their α-keto acids are lead to oxidative stress that is potentially toxic to cells. Further, this knowledge and the underlying molecular mechanisms will enhance our understanding of MSUD in humans.
About 2.4 Ga ago the Great Oxygenation Event (GOE) started the permanent oxygenation of Earth’s anoxic atmosphere. The oxygen was most likely produced by oxygenic photosynthesis in Cyanobacteria. However, hints for local occurrences of Cyanobacterial life and free oxygen exists for at least 300 Ma prior to the GOE. Different hypotheses were proposed to explain this delay between the evolution of oxygen producers and the start of the GOE. For this thesis, theoretic predictions made by two of those hypotheses were tested in laboratory experiments using ancestral, basal clade Cyanobacteria grown under simulated Archean like conditions.
Cyanobacteria might have evolved in freshwater environments and subsequently had to adapt to the higher salinity of the Archean ocean. In turn, this would have delayed their global expansion required for the GOE. Experiments with the most primitive freshwater Cyanobacterium Gloeobacter violaceus PCC 7421, showed its ability to tolerate and slowly grow in brackish water, thereby providing a route for the evolution of open ocean dwelling, salt tolerant species. The Archean ocean may have presented another hurdle to Cyanobacterial expansion as it contained large amounts of Fe(II), which is presumed to be toxic to Cyanobacteria. This thesis shows that the localised activity of Cyanobacteria could have formed marine oxygen oases in shallow coastal regions. This would have negated the toxicity of Fe(II) and could have produced more net O2 then modern oxic systems. Additionally, the formation of green rust was observed, which seemed to have a toxic effect on Cyanobacterial growth and could be an important factor for the genesis of banded iron formations.
In conclusion, this thesis could show the viability of both, the “freshwater-origin” and “Fe(II)-toxicity”, hypothesis. Nevertheless, how long it took for Cyanobacteria to overcome the restrictions described above to expand into the open ocean is uncertain and needs to be further studied.
Characterization of a bacterial-like signal transduction phosphorelay in Methanosarcina acetivorans
(2021)
Signal transduction systems are of great importance for the adaptation of organisms to new conditions. These systems occur most frequently in bacteria and are well-understood thanks to research. It was not until 10 years after the discovery of two-component systems in bacteria that such a system was reported in archaea. This work provides new insights into signal transduction in archaea, through the characterization of a histidine kinase MA4377 and its multi-component system in the methanogenic archaeon Methanosarcina acetivorans. MA4377 is a hybrid kinase of bacterial origin and was probably integrated into M. acetivorans via horizontal gene transfer. Based on the fused receiver domains, MA4377 is classified as a hybrid kinase that regulates a cell response upon the perception of a signal through a multi-component system. These systems consist of four components, the first of which is the kinase. MA4377 has autophosphorylation activity and is phosphorylated at a conserved histidine residue (His497). While the kinase activity is independent of the redox state of the protein, the PAS domain is mandatory for autokinase activity. Using different protein variants, it could be shown that the two fused receiver domains are not involved in the phosphorelay. Rather, the single receiver domain MA4376 serves as the second component of the system. The signal is ultimately transmitted to a transcription factor (MA4375) of the Msr family. Factors of this family are involved in the regulation of methanogenesis, among other things. MA4377 could thus be involved in a multi-component system regulating methanogenesis. The receiver MA4376 plays a central role here since feedback regulation on the kinase was observed. Further investigations will show to what extent cross-regulation with other kinases takes place and in what way the receiver MA4376 plays a key role in this multi-component system.
The analysis of benthic bacterial community structure has emerged as a powerful alternative to traditional microscopy-based taxonomic approaches to monitor aquaculture disturbance in coastal environments. However, local bacterial diversity and community composition vary with season, biogeographic region, hydrology, sediment texture, and aquafarm-specific parameters. Therefore, without an understanding of the inherent variation contained within community complexes, bacterial diversity surveys conducted at individual farms, countries, or specific seasons may not be able to infer global universal pictures of bacterial community diversity and composition at different degrees of aquaculture disturbance. We have analyzed environmental DNA (eDNA) metabarcodes (V3–V4 region of the hypervariable SSU rRNA gene) of 138 samples of different farms located in different major salmon-producing countries. For these samples, we identified universal bacterial core taxa that indicate high, moderate, and low aquaculture impact, regardless of sampling season, sampled country, seafloor substrate type, or local farming and environmental conditions. We also discuss bacterial taxon groups that are specific for individual local conditions. We then link the metabolic properties of the identified bacterial taxon groups to benthic processes, which provides a better understanding of universal benthic ecosystem function(ing) of coastal aquaculture sites. Our results may further guide the continuing development of a practical and generic bacterial eDNA-based environmental monitoring approach.
Genome-based Approaches for Understanding Nutritional Iron Homeostasis in Chlamydomonas reinhardtii
(2021)
Iron is an essential nutrient for all life forms, including plants, but of limiting availability in many environments, affecting productivity of both food production and carbon capturing. Iron is essential because of its broad function as a catalyst of redox reactions and processes involving O2 chemistry in the catalytic centers of enzymes. Because of the nature of these reactions, excess amounts of the nutrient can be toxic, requiring a fine tuning of the cellular iron content, to both accommodate the essential demand and avoid detrimental effects simultaneously. A question of this project is how plant metabolism is modified in iron-deficient conditions, for which the green alga Chlamydomonas reinhardtii as a microbe is an excellent reference organism. The metabolic flexibility of C. reinhardtii, specifically the capacity for both heterotrophic (on acetate) and autotrophic (on CO2) growth, offers a unique opportunity to distinguish the impact of iron nutrition on photosynthetic versus respiratory metabolism. During steady-state photoheterotrophic Fe-limited growth, where the cells are provided with light, CO2, and acetate, but lack extracellular iron, cells maintain respiration while decreasing photosynthetic contribution to the energetics of the cell. This thesis analyzes the transition from photoautotrophic (light and CO2) to photoheterotrophic cultures in the context of Fe-nutrition by adding a reduced carbon source to phototrophic cultures and assessing the developing changes to the metabolism time-dependently, in various levels of readouts. Based on the transcriptome analysis, all major cellular processes and pathways respond to the availability of acetate, but Fe-limited cells specifically sacrifice photosynthetic capacity towards respiratory activity in the first 12h after the additional carbon source becomes available, allowing to gain mechanistic insights of transitioning between different ways of life, dependent on the nutritional makeup of the environment. Secondly, exposure to high extracellular iron amounts, its opportunities, and the mechanisms of avoiding deleterious effects as a result from it, had been under-investigated before the beginning of this thesis. Physiological and photosynthetic parameters, elemental analysis, transcriptomics, and a mutant depleted of functional acidic vacuoles, proposed to be involved in the storage for transition metals, were utilized to further the understanding of the processes. Altogether, the results presented in this thesis illustrate how C. reinhardtii can be successfully used as a model organism to study a large variety of aspects of cell and molecular biology, including dynamic acclimations to changing environments.
Synaptic transmission is controlled by re-uptake systems that reduce transmitter concentrations in the synaptic cleft and recycle the transmitter into presynaptic terminals. The re-uptake systems are thought to ensure cytosolic concentrations in the terminals that are sufficient for reloading empty synaptic vesicles (SVs). Genetic deletion of glycine transporter 2 (GlyT2) results in severely disrupted inhibitory neurotransmission and ultimately to death. Here we investigated the role of GlyT2 at inhibitory glycinergic synapses in the mammalian auditory brainstem. These synapses are tuned for resilience, reliability, and precision, even during sustained high-frequency stimulation when endocytosis and refilling of SVs probably contribute substantially to efficient replenishment of the readily releasable pool (RRP). Such robust synapses are formed between MNTB and LSO neurons (medial nucleus of the trapezoid body, lateral superior olive). By means of patch-clamp recordings, we assessed the synaptic performance in controls, in GlyT2 knockout mice (KOs), and upon acute pharmacological GlyT2 blockade. Via computational modeling, we calculated the reoccupation rate of empty release sites and RRP replenishment kinetics during 60-s challenge and 60-s recovery periods. Control MNTB-LSO inputs maintained high fidelity neurotransmission at 50 Hz for 60 s and recovered very efficiently from synaptic depression. During 'marathon-experiments' (30,600 stimuli in 20 min), RRP replenishment accumulated to 1,260-fold. In contrast, KO inputs featured severe impairments. For example, the input number was reduced to ~1 (vs. ~4 in controls), implying massive functional degeneration of the MNTB-LSO microcircuit and a role of GlyT2 during synapse maturation. Surprisingly, neurotransmission did not collapse completely in KOs as inputs still replenished their small RRP 80-fold upon 50 Hz | 60 s challenge. However, they totally failed to do so for extended periods. Upon acute pharmacological GlyT2 inactivation, synaptic performance remained robust, in stark contrast to KOs. RRP replenishment was 865-fold in marathon-experiments, only ~1/3 lower than in controls. Collectively, our empirical and modeling results demonstrate that GlyT2 re-uptake activity is not the dominant factor in the SV recycling pathway that imparts indefatigability to MNTB-LSO synapses. We postulate that additional glycine sources, possibly the antiporter Asc-1, contribute to RRP replenishment at these high-fidelity brainstem synapses.
Glycine constitutes the major neurotransmitter at inhibitory synapses of lower brain regions.
A rapid removal of glycine from the synaptic cleft and consequent recycling is crucial for
synaptic transmission in systems with high effort on temporal precision. This is mainly
achieved by glycine translocation via two glycine transporters (GlyTs), namely GlyT1 and
GlyT2. At inhibitory synapses, GlyT2 was found to be specifically expressed by neurons,
supplying the presynapse with glycine needed for vesicle filling. In contrast, GlyT1 is attributed
to astrocytes and primarily mediates the termination of synaptic transmission by glycine
removal from the synaptic cleft. Employing patch-clamp recordings from principal neurons of
the lateral superior olive (LSO) in acute brainstem slices of GlyT1b/c knockout (KO) mice and
wildtype (WT) littermates at postnatal day 20, I analyzed how postsynaptic responses are
changed in a GlyT1-depleted environment. During spontaneous vesicle release I found no
change of postsynaptic responses, contradicting my initial hypothesis of prolonged decay
times. Electrical stimulation of fibers of the medial nucleus of the trapezoid body (MNTB),
which are known to form fast, reliable and highly precise synapses with LSO principal neurons,
revealed that GlyT1 is involved in proper synaptic function during sustained, high frequent
synaptic transmission. Stimulation with 50 Hz led to a stronger decay time and latency
prolongation in GlyT1b/c KO, accelerating to 60% longer decay times and 30% longer latencies.
Additionally, a more pronounced frequency-dependent depression and fidelity decrease was
observed during stimulation with 200 Hz in GlyT1b/c KO, resulting in 67% smaller amplitudes
and only 25% of WT fidelity at the end of the challenge. Basic properties like readily releasable
pool, release probability, and quantal size (q) were not altered in GlyT1b/c KO, but
interestingly q decreased during 50 Hz and 100 Hz challenges to about 84%, which was not
observed in WT. I conclude that stronger accumulation of extracellular glycine due to GlyT1
loss leads to prolonged activation of postsynaptic glycine receptors (GlyRs). As a further
consequence, activation of presynaptic GlyRs in the vicinity of the synaptic cleft might be
enhanced, accompanied by a stronger occurrence of shunting inhibition. Furthermore, I
assume a GlyT1-dependent glycine shuttle, which is absent at GlyT1b/c KO synapses. This
could result in a diminished glycine supply to GlyT2 located at more distant sites, causing a
disturbed replenishment during periods with excess release of glycine. Conclusively, my study
reveals a contribution of astrocytes in fast and reliable synaptic transmission at the MNTB-LSO
synapse, which in turn is crucial for proper sound source localization.
Most of eukaryotes show signs of having sex or sexual recombination, and the other asexual eukaryotes have evidences of evolving from sexual ancestors. Meiotic recombination, or crossover are proved to have two pathways in eukaryotes, whose distribution was well studied in many model eukaryotes. However, the distribution of sex in specific lineage is debating. The distribution and evolution of meiotic recombination pathways in alveolates would provide us clues of lost/gaining of pathways in early eukaryotes and fill the gaps between protist and more complex multicellular organisms (fungi, animals and plants) .
In this dissertation, we designed a customized program with Python, which integrated Blastp and HMMER v3.0, to search for homologs of 51 meiotic genes (11 meiosis-specific and 40 meiosis-related genes ) in the whole genome sequences or EST data of five Ciliates, seven Apicomplexa, two Chromerida, one Perkinsus, one Dinoflagellates and Chrysophytes (golden algae). All candidate homologs were then verified by reciprocal Blastp search against the nonredundant protein sequence database of NCBI and phylogeny analysis of RAxML.
The gene inventory results shows that several eukaryotic meiosis-specific and meiosis-related genes are missing in every group. However, the presents of meiosis initiate protein Spo11 in some putative asexual lineage (Symbiodinium, Colpodean and Chrysophytes) suggest that they might be cryptically sexual. Within alveolates, Apicomplexa are capable of both pathways, while Ciliates and Dinoflagellates using a set of mitotic repair proteins for meiotic recombination. We speculated that the result of the abandonment of the pathway I might due to the abnormal chromosome structure of both Ciliates and Dinoflagellates. Phylogenetic analysis of the distribution of meiotic pathways within alveolates suggest that the reduction of meiotic pathway I in Ciliates and Dinoflagellates are independent. Considering that Apicomplexa are capable of both meiotic pathways, we would infer that those two pathways exist in the common ancestor of alveolates.
Potassium (K) is essential for the processes critical for plant performance, including photosynthesis, carbon assimilation, and response to stress. K also influences translocation of sugars in the phloem and regulates sucrose metabolism. Several plant species synthesize polyols and transport these sugar alcohols from source to sink tissues. Limited knowledge exists about the involvement of K in the above processes in polyol-translocating plants. We, therefore, studied K effects in Plantago major, a species that accumulates the polyol sorbitol to high concentrations. We grew P. major plants on soil substrate adjusted to low-, medium-, or high-potassium conditions. We found that biomass, seed yield, and leaf tissue K contents increased in a soil K-dependent manner. K gradually increased the photosynthetic efficiency and decreased the non-photochemical quenching. Concomitantly, sorbitol levels and sorbitol to sucrose ratio in leaves and phloem sap increased in a K-dependent manner. K supply also fostered plant cold acclimation. High soil K levels mitigated loss of water from leaves in the cold and supported cold-dependent sugar and sorbitol accumulation. We hypothesize that with increased K nutrition, P. major preferentially channels photosynthesis-derived electrons into sorbitol biosynthesis and that this increased sorbitol is supportive for sink development and as a protective solute, during abiotic stress
CRISPR/Cas has become the state-of-the-art technology for genetic manipulation in diverse
organisms, enabling targeted genetic changes to be performed with unprecedented efficiency. Here we report on the first establishment of robust CRISPR/Cas editing in the important necrotrophic plant pathogen Botrytis cinerea based on the introduction of optimized
Cas9-sgRNA ribonucleoprotein complexes (RNPs) into protoplasts. Editing yields were further improved by development of a novel strategy that combines RNP delivery with cotransformation of transiently stable vectors containing telomeres, which allowed temporary
selection and convenient screening for marker-free editing events. We demonstrate that
this approach provides superior editing rates compared to existing CRISPR/Cas-based
methods in filamentous fungi, including the model plant pathogen Magnaporthe oryzae.
Genome sequencing of edited strains revealed very few additional mutations and no evidence for RNP-mediated off-targeting. The high performance of telomere vector-mediated
editing was demonstrated by random mutagenesis of codon 272 of the sdhB gene, a major
determinant of resistance to succinate dehydrogenase inhibitor (SDHI) fungicides by in bulk
replacement of the codon 272 with codons encoding all 20 amino acids. All exchanges were
found at similar frequencies in the absence of selection but SDHI selection allowed the identification of novel amino acid substitutions which conferred differential resistance levels
towards different SDHI fungicides. The increased efficiency and easy handling of RNPbased cotransformation is expected to accelerate molecular research in B. cinerea and
other fungi.