Graduate Programme in Molecular and Cell Biology
Research Groups in the Graduate Programme in Molecular and Cell Biology :
All activities of the Graduate Programme are in English.
Theses may be submitted in English and no German knowledge is required for registration for a Ph. D. at the University. However, it is advisable for foreign students to learn some German to deal with daily life outside the University.
The Graduiertenkolleg Programme begins with a three-month rotation period, during which you carry out one-month research projects in three labs. After this, you go to one of these labs for three years, to carry out your full PhD project. In outline, we operate this scheme as follows: before you arrive, you are asked to list five labs in which you would like to work, and using these lists, we do our best to place you in the labs in which you are most interested. At the end of the rotation period, both students and supervisors hand in lists of first, second and third choices of labs or students, and we aim to ensure that as many students as possible will go to the lab of their first choice, while also taking supervisors' choices into consideration.
All Graduiertenkolleg students are expected to attend "Progress Report" meetings every month. Two students per session give a 20 min talk about their work (or introduce their project), and every student is expected to give a talk during the year.
One student has the role of Spokesperson. This position rotates
every two months, and every student is expected to take a turn.
The Spokesperson's roles include:
First year report
All students write a report at the end of their first year. This should consist of an introduction to the project, aim of the project, results so far, problems that have arisen, and discussion of future plans. The report should be 3 - 10 pages long.
It will be read by the student's supervisor, and by one other project leader in the Graduiertenkolleg programme, who will then discuss it with the student and, if necessary, and give criticism and advice on the written report, the experimental work or any other matters.
Graduiertenkolleg students are encouraged to invite scientists
of international standing as seminar speakers to Cologne. The
student who has invited the speaker acts as host during the speaker's
visit, and is therefore responsible for arranging the following
(details of how to make these arrangements are kept with the
Spokesperson, and secretarial help is available):
Travel money is available for attending meetings and conferences. Travel to conferences is usually only funded if the student presents a poster or gives a talk. In order for other members of the student's lab to benefit from the visit, the student is expected to give a report upon returning, in which the most important new information from the meeting is summarized.
Some useful Cologne links:
Concerts, Museums, cultural events:
Current Graduate Students and their Projects
Project: Odorant receptor gene expression and regulation in the olfactory system of the zebrafish, Danio rerio
The differential gene regulation of a large family of highly diversified odorant receptor (OR) genes is essential for the discriminatory power of the olfactory system. Each olfactory sensory neuron expresses only one or very few OR genes. Cells of the same receptor phenotype are sparsely scattered within restricted expression domains on the sensory surface.
Investigating the developmental time course of expression by quantitative in situ hybridization, we show that expresssion of ten zebrafish OR genes begins asynchronously within the first three days of embryonic development and follows a stereotyped temporal pattern. From the earliest time intervals, observed individual OR genes are expressed in sparse subpopulations of cells. Penetrance of expression varies dramatically between genes. A more detailed analysis of onset kinetics suggests at least two levels of gene regulation: first, the onset of competence for expression at different time points and second, a random selection of the OR to be expressed from the pool of genes competent for expression.
To gain further insight into the actual mechanism of OR gene expression, we set out to search for regulatory elements. Screening of a zebrafish cosmid library revealed zebrafish OR genes to be organized in clusters. Mapping of these clusters is currently underway. Transgenic reporter gene expression (GFP) in embryonic zebrafish will allow us to analyse the intergenic regions of suitable clusters for presence of regulatory elements. Reporter gene expression will be compared with the temporal and spatial expression patterns described above for zebrafish OR genes to identify cis elements governing OR gene expression.
Project: Molecules involved in FGF signaling during Drosophila embryogenesis
In Drosophila, cell migration and rearrangement are required for the formation of the mesoderm and the tracheae. In turn, the motility of these cells is controlled by the FGF signal transduction pathway. To understand the mechanisms by which components of the FGF signal transduction pathway cause cell movement and differentiation, I have conducted a yeast two hybrid screen to identify further components of this pathway.
There are two known FGF receptors in Drosophila, heartless (involved in mesoderm formation) and breathless (involved in the branching of the tracheae). Both require the cytoplasmic protein Dof for the transduction of the FGF signal. Although it has been shown that Dof functions upstream of the small GTPase ras, the molecules that link the FGF receptor to Dof and likewise Dof to ras are unknown. I have conducted a yeast two hybrid screen for interactors with Dof. In order to establish whether the candidates from the two hybrid screen are true interactors with Dof, I am analysing their putative role in mesoderm and tracheae morphogenesis. To quickly narrow down the number of candidates, I have looked at the tracheae and mesoderm of embryos from deficiencies that lack a candidate gene. Defiencies are flies that have fairly large regions of their genome deleted. However, deficiencies delete more than just the gene I am interested in, so I am also using RNA interference to disrupt the gene function of individual candidates. I inject double stranded RNA of a candidate gene into early embryos and determine whether heart cell precursors still form (heart cells are mesoderm derivatives). Lastly, I am studying the regions of the Dof molecule that are responsible for its interaction with the candidate genes. For this purpose I am testing smaller regions of the Dof molecule in the yeast two hybrid system to find out whether binding to the candidate proteins still occurs.
Project: Pathfinding of olfactory sensory neurons: involvement of eph receptors and ephrins
The functioning of the central nervous system depends upon the organization of a very precise pattern of neural connectivity formed during development. Many CNS neurons are able to establish topologically ordered maps in their target regions. Mechanisms that govern this process on a cellular level include the coordinated action of guidance molecules that are specifically distributed on the axons and their target regions.
In the olfactory system, the projection of olfactory receptor neurons to the olfactory bulb is non-topological. The characteristic feature is the convergence of same-function neurons into glomeruli, globular neuropil consisting of receptor neuron terminals and dendrites of mitral cells, the projection neurons. Glomerular position and function seem to be stereotyped. Because of the continuous turnover of olfactory receptor neurons throughout life, we expect the guidance molecules to be present not only during development but also to remain present in the adult olfactory bulb.
I am investigating whether molecules implicated in the establishment of neuronal maps in other sensory systems might have similar functions in the olfactory system. Eph receptor tyrosine kinases and their ligands are thought to be important in the guidance of axons through repulsive interactions. I could show that often receptors are expressed complementary to their corresponding ligands in the olfactory epithelium and the olfactory bulb. Furthermore, a layer-specific expression in the olfactory bulb suggests a role of these genes in establishing and/or maintaining intrabulbar connectivity.
Project: Studies on the regulation of the silent bgl operon
The expression of the bgl operon in wild type Escherichia coli is repressed. This negative regulation is known to occur via at least two phenomena. The bgl promoter is flanked by DNA silencing elements known as bgl silencers, and is silenced by a presumed nucleoprotein complex which prevents the binding of CAP protein and RNA polymerase. Additionally, rho-independent transcriptional terminators prevent the RNA polymerase from transcribing the entire operon. For expression, at least the following events must occur. First the promoter must be derepressed e.g. by a cis mutation in the bgl silencer, which disrupts the formation of the repressing nucleoprotein complex. Also the first gene bglG of the operon must be expressed, so that its product, the BglG protein, that acts as a specific antiterminator, prevents formation of the transcriptional terminator hairpins and allows further transcription. I am studying the components of the proposed silencing nucleoprotein complex. This involves their genetic and biochemical characterization and functional studies relating to the mechanism by which they regulate the bgl operon expression.
Project: Molecular characterisation of the secretory pathway in the green alga Scherffelia dubia
The cells of the flagellate green alga S. dubia are covered by structures of distinct size and shape, termed scales. Biogenesis of scales has become a unique model system for the analysis of Golgi function (1). Following deflagellation, cells regenerate flagella and flagellar scales within four hours. During flagellar regeneration, scales can be detected intracellularly by electron microscopy. Scales are synthesized in the Golgi apparatus (GA) and are thought to move through the GA by cisternal progression, since they are never found inside the numerous transition vesicles which accompany the Golgi stack.
My task is to isolate markers involved in the secretory pathway of S. dubia. These proteins will be then used for antibody production. The antibodies will be used as molecular tools in immunofluorescence and immunogold experiments to study the secretory pathway, leading to a better understanding of the intra-Golgi transport mechanism.
(1) Becker B, Marin B, Melkonian M (1994) Protoplasma 181:233-244
Project: Conditional gene inactivation of the ubiquitin ligase Itch and analysis of its function in the immune system.
A novel murine model of auto-immunity has recently been discovered during the analysis of a18H mice. The a18H mice (on a C57Bl/6J background) develop chronic interstitial inflammation of the lung and skin as well as hyperplasia of the spleen and lymph nodes, leading to death of the mice at the age of four to six month. Analysis of the genetic defect leading to the development of the auto-immune-like syndrome in a18H mice revealed an inversion between the agouti locus and a gene encoding a novel E3 ligase named Itch. Most likely, the development of auto-immune disease is due to the deletion of Itch. By conditional gene targeting of itch, using the cre- loxP-mediated cell type specific gene targeting, we aim to identify which cell type is primarily involved, and the molecular mechanism behind the expected development of auto-immune disease in Itch-deleted mice. We also aim to identify the substrates of the E3 ligase itch.
Project: Characterization of DLIM1, a LIM domain-containing protein from Dictyostelium discoideum
DLIM1, a double LIM domain-containing protein from the model organism Dictyostelium discoideum, is a member of the Cysteine Rich Protein (CRP) family of LIM domain proteins. LIM domains are characterized by the presence of a cysteine-rich sequence motif with the consensus amino acid sequence CX2CX16-23HX2C-X2-CX2CX16-21CX2(C/H/D). Preliminary structural studies predict that each LIM domain has two finger-like structures, and coordinates two atoms of zinc in a tetrahedral fashion, via the conserved cysteine and histidine residues of the LIM consensus. Although a number of LIM domain proteins have been implicated in mediating specific protein-protein interactions within the cytoskeleton and are involved in control of gene expression and cell differentiation, the function of the CRP family of LIM domain proteins has not been fully established. They may have a structural or regulatory role with actin in the cytoskeleton, since they interact with actin-binding proteins, and are localized at focal contacts and within stress fibers in mammalian cells.
To understand the cellular dynamics of DLIM1, we have used a green fluorescent protein (GFP)-tagged version of DLIM1. The confocal fluorescence microscopic studies of the GFP-DLIM1-expressing cells show a preferential localization of the DLIM1 protein at the cell periphery and at regions of intercellular contacts. The pattern of GFP-DLIM1 fluorescence was found to coincide with that of actin staining. Hence, we decided to study the subcellular distribution of DLIM1 during the dynamic processes of the cell cortex in which filamentous actin plays a major role: phagocytosis, pinocytosis, exocytosis and extension of pseudopodia. We report that GFP-DLIM1 is strongly enriched in phagocytic cups and macropinosomes and is gradually released within 1 min after formation of the phagosome or the macropinosome. In aggregation-competent cells, which acquire a typical elongated shape with a well-defined front and tail, high levels of GFP-DLIM1 accumulated at the leading edge. GFP-DLIM1 was also observed to play a role in exocytosis. Our results suggest that DLIM1 plays an important regulatory role in the rapid reorganization of the cytoskeleton. The two LIM domains in DLIM1 predispose DLIM1 for multiple protein-protein interactions at the cytoskeleton.
I have also performed N- and C-terminal deletion studies to identify the region of DLIM1 which is responsible for its involvement in dynamic processes and its colocalization with actin. Our studies suggest that either LIM domain of DLIM1 is sufficient for both these roles. Recently, I have generated DLIM1 knockout mutant cells which will further help us in understanding the role of DLIM1.
Since it would be interesting to identify the binding partners of DLIM1, we decided to do immunoprecipitation studies using DLIM1-specific antisera. Hence, DLIM1 protein was expressed and purified as a glutathione S-transferase (GST)-fusion protein. Recently, I have generated DLIM1-specific monoclonal antibodies which I am now using in immunoprecipitation studies.
Grover YP, Khurana B, Gulati BR, Patnayak DP, Pandey R (1997) Prevalence of bovine rotavirus infection in organized dairy farms at Ambala and Meerut during 1995-96. Indian J Virol 13:117-118
Khurana B, Mukherjee S, Pandey R (1998) Surveillance and genotyping of rotavirus from diarrhoeic and non-diarrhoeic bovine calves. Poster presented and abstract published in "Second ICGEB Virology Symposium",New Delhi, India, Nov 1998
Khurana B, Taruna K, Noegel AA (1999) DLIM1 and DLIM2 are two LIM domain containing proteins involved in phagocytosis, pinocytosis and motility in Dictyostelium. Poster presented and abstract published in "First Munich Symposium on Cell Dynamics - from Molecular Structure to Cellular Motility", Martinsried, Munich, Germany, Oct 1999
Project: Functional expression of Zebrafish odorant receptors
We use Zebrafish as a simple model to study the peripheral neuronal mechanisms of sensing a cup of Espresso. My project involves overexpression of Zebrafish odorant receptors, in order to look for their ligands using optical imaging.
Project: Significance of the laminin-nidogen interaction for the formation and stability of the basement membrane
Our lab is interested in the basement membrane (BM), a thin layer of extracellular matrix. It separates epithelial and endothelial cells from the underlying connective tissue and surrounds muscle, fat and Swann cells. The BM has many different functions. It acts as a diffusion barrier in kidney and plays a role in cell adhesion, cell migration, wound healing and presumably in cell differentiation.
The BM consists of many different proteins, e.g. the collagens, laminins, heparan sulfate proteoglycan, fibulins and nidogens. We aim to understand which of these numerous components are essential for building up the BM. Based on data from in vitro binding assays and protein purifications, a model has been derived in which the BM consists of two major meshworks, the laminin-1 and the Collagen type IV network, which are supposedly linked by nidogen-1.
In this context, I am investigating the significance of the laminin-nidogen interaction for the establishment and stability of the BM. I use the F9 embryoid body system as a model for BM formation. This system resembles an early stage of mouse embryonic development,in which the BM separates two different cell types, mesenchymal from epithelial cells. By overexpressing the nidogen-binding site on the laminin molecule we hope to interfere with BM formation, and to be able to analyse possible effects of BM disruption on epithelial differentiation.
Project: Analysis of the immune system of pkcd-/- mice
TexidoG, Su I, Mecklenbräuker I, Saijo K, Malek SN, Desiderio S, Rajewsky K, Tarakhovsky A (2000) The B-cell specific src-family kinase Blk is dispensable for B-cell development and activation. Mol Cell Biol 20:1227-1233
Miyake K, Su I, Ogata H, Nagai Y, Akashi S, Mecklenbräuker I, Rajewsky K, Kimoto M, Tarakhovsky A (2000) The toll-like receptor protein RP105 is essential for LPS signaling in B cells. J Exp Med (in press)
Project: Ablation of Nidogen-1 gene in mouse
Nidogen-1 (entactin-1) is a ubiquitous basement membrane protein. In vitro studies have suggested that nidogen-1 can bind other major basement membrane proteins collagen IV, laminin-1 and perlecan. Antibody perturbation experiment blocking laminin-nidogen interaction resulted in impaired branching morphogenesis in cultured embryonic lungs and kidneys and induced a distortion of the basement membranes in these organs. Therefore, it has been proposed that nidogen-1 stabilizes the basement membrane by connecting two interacting networks of collagen IV and laminin and is important for morphogenesis during embryonic development. To define the role of nidogen-1 in vivo, we produced a null mutation of the NID-1 gene in embryonic stem cells and used these to derive mouse lines. Homozygous animals produce neither nidogen-1 mRNA nor protein. Surprisingly, these animals do not show any overt abnormality and are fertile. Electron micrographs show no alteration in basement membrane structure in knockout animals. However, immunostaining shows a different deposition pattern of nidogen-2 (second member of nidogen family sharing 46% homology with nidogen-1) in heart and skeletal muscle basement membranes in absence of nidogen-1. A recent publication has indicated a possible role of nidogen-1 in axonal guidance. We are currently investigating our mice for any possible neurological phenotype(s).
Project: Isolation of axon guidance molecules within the olfactory bulb of Zebrafish
Olfactory receptor neurons expressing the same olfactory receptor converge into glomeruli within the olfactory bulb. We expected to find axon guidance molecules among differentially expressed genes. Differentially expressed molecules are identified by using subtractive suppression PCR.
Project: The dimerization interface of Lac repressor of E. coli
The LacR/GalR family of prokaryotic regulating proteins is highly conserved in its amino acid sequence. The structural relationship is also documented by the comparison of the x-ray structure of LacR, the repressor of the lac operon, with the crystal structure of PurR, the repressor of the pur operon. Most of the members of this group of related proteins function as dimers: only a few, for example the Lac repressor, work as tetramers. At the beginning of this study the question arose of how these proteins manage their specific dimerization and how they avoid interactions with other closely related proteins. There must be a specific mechanism of dimerization. While the amino acid sequences of these proteins are highly conserved in some regions, for example the DNA binding motif (a helix-turn-helix motif), the amino acid sequences of individual dimerization interfaces are very different. The only factor which is conserved in this region is a high hydrophobicity.
The Lac repressor consists of 360 amino acids which are grouped into three domains: the DNA-binding headpiece, which contains the amino acids 1 to 60; the core, with the inducer binding region and the dimerization interface, which consists of the amino acids 61 to 330; and the tetramerization region, including the amino acids 331 to 360. The study of the crystal structure of Lac repressor and more than 4000 single mutants (amino acids 2 to 328 were exchanged with amber codons and then tested with 12 or 13 suppressor strains) point out the residues which may participate in the mechanism of dimerization (Suckow et al. 1996, J Mol Biol 261: 509 - 523). These residues S221, A222, M223, F226, L251, M254, R255, T258, E259, D278, S280, C281, Y282, I283 and P285 are positioned in three helices of the core. It is impossible to conclude that all of these residues take part in dimerization only on the basis of this information. Their position, their distances from one another in the crystal and their behaviour as single mutants indicate that they most probably are involved in dimerization. The residues A222, M223, F226, L251, R255, E259, D278, C281, Y282 and I283 point towards the other monomer, while the remaining six side chains are orientated towards the core of the same monomer.
During the examination of numerous mutants within the dimerisation interface of Lac repressor, we found that substitution of one amino acid, D278 to leucine, is sufficient to change the specificity of dimerization. Analysis of this single substitution indicates that D278L mutant Lac repressor represses like wild-type. However, it no longer forms heterodimers with wild-type Lac repressor.
One further approach for understanding the mechanism of dimerization of Lac repressor is to create specific heterodimers by introducing differently charged residues in the dimerization interface of Lac repressor.
The second approach is to create a dimeric protein by specific exchanges in the potential interface of a monomeric protein. For this purpose we choose the ribose binding protein (RBP) which is closely related in its amino acid sequence as well as in the structure of the core to LacR repressor.
Spott S, Dong F, Kisters-Woike B, Müller-Hill B (2000) Dimerisation mutants of Lac repressor. II. A single amino acid substitution, D278L, changes the specificity of dimerization. J Mol Biol 296:673-684
Project: Role of guanylate binding proteins in the many cellular responses to IFN-gamma.
IFN-gamma is a cytokine secreted mainly by activated T lymphocytes, natural killer cells and macrophages. We have identified genes that are differentially expressed upon IFN-gamma induction in mouse. They are grouped into two structural sub-families, GKS and GMS, based on the amino acid sequence in the first canonical GTP binding motif. I am working on IIGP, a member of the GKS subfamily.
I have expressed and purified IIGP in a prokaryotic expression system, and have characterised it biochemically as a GTPase. I have also crystallised the protein and the structure will be solved soon (in collaboration with MPI, Dortmund). Now I am trying to look for the molecular neighbours of IIGP by a cell-biological approach. The aim is to understand the complex responses of IFN-Gamma.
Ana Berta Sousa
Project: Unravelling the molecular basis of MHC-gene diversification
It has been proposed that the extreme polymorphism observed in class I and II genes of the Major Histocompatibility Complex (MHC) arises from selection pressures exerted by infectious pathogens. Our interest concentrates on the molecular mechanisms generating the variants which are ultimately offered to natural selection.
Segmental exchange between MHC genes in the germline could explain the patterns of allelic variation observed in both human and mouse populations. The best evidence for the occurrence of microconversion in MHC genes comes from the documented instances of spontaneous class I gene mutation in the laboratory mouse. The major target for the recorded events was the H-2Kb gene, with an overall frequency in the region of 1 in 104 gametes. The aim of our work is to establish an H-2Kb gene conversion detector with a view to ascertaining the adaptive significance and molecular properties of the underlying mutational mechanism.
Project: Study and analysis of molecules involved in mesoderm
One of the fundamental problems in Drosophila development
is how a single layer of cells becomes multiply layered and finally
forms a complex organism. It is clear that the process is driven
by cell movements and proliferation, and some of the molecules
involved in the intrinsic and extrinsic signals that are responsible
for the process have already been identified. However the signal
pathways that direct cell migration are far from completely understood.
In my project, I will identify and analyze molecules involved
in mesoderm cell migration by two approaches. One is to examine
the subcellular localization of different cytoskeleton molecules
during mesoderm formation. The other is to identify more molecules
involved in mesoderm cell migration by an EMS mutagenesis screen.