The pictogram defines main topics of our currrent research.
Cell shape is certainly the most distinguishing histodiagnostic feature of differentiated cells and changes thereof are indicative of altered genetic programs as they occur physiologically during development and specification but also in pathological situations such as malignant tumour formation, which is coupled to dedifferentiation and migration. These processes necessiate pronounced restructuring of cells which is determined by the cytoskeleton. It consists of three major filament networks that are composed of actin, tubulin and intermediate filament proteins, respectively. Thus, an intricate relationship exists between regulated cytoplasmic cytoskeletal dynamics and cell migration/differentiation. Our own experiments in living cells producing fluorescent cytoskeletal filaments revealed coordinated and interdependent dynamic behaviour of all three filament systems albeit with different kinetics and distribution features and showed that signalling regulates their restructuring. Fluorescent fusion proteins will be used to label each of the three major cytoskeletal networks in gastric cancer cell lines, i.e. actin fusions for actin filaments, keratin 18 fusions for intermediate filaments, and tubulin fusions for microtubules. To facilitate detection in various combinations in the same cells, three different fluorescent proteins will be employed that can be individually detected. We will record the dynamic alterations of the different cytoskeletons under conditions that will enhance or inhibit motility. The goal is to define quantitative measures to describe multidimensional cytoskeletal dynamics in response to different stimuli.
Principal Investigators: Prof. Dr. Rudolf Leube and PD Dr. Reinhard Windoffer
Keratins form the major cytoskeleton of epithelial cells and behave at the same time as a highly dynamic protein scaffold. As such, they protect epithelia against mechanical stress but also play a major role in the regulation of cell growth, proliferation, stress pathways and organelle transport. The molecular mechanisms governing the localized assembly of keratins from heterodimeric subunits and their interaction with cell adhesion molecules and regulatory proteins are not well understood but are essential for an understanding of keratinopathies. Based on high resolution live cell imaging of cell transfectants we have recently developed a model for keratin assembly and the involvement of p38 MAP kinases that allows predictions on the in vivo properties of the keratin cytoskeleton. To evaluate these hypotheses, we propose to generate a knock-in allele coding for a hybrid fluorescent keratin 8. This will enable us to monitor keratin assembly and its MAP kinase-dependent regulation by direct fluorescence microscopy in pre- and postimplantation embryos, and in the long run, in postnatal mice. The contribution of other cytoskeletal components, desmosomal proteins and of certain kinases will be studied by genetic and pharmacological approaches in embryonal stem cell derivatives and mouse embryos. As a future perspective, fluorescent keratin 8 knock-in animals will be employed to elucidate the interdependence of desmosomes and adherens junctions with keratins, using mouse strains deficient in the relevant genes.
Principal investigators: Dr. Nicole Schwarz, Prof. Dr. Rudolf Leube
Funding: START Nachwuchsprogramm, DFG
Mutations of the desmosomal cadherin desmoglein 2 have been identified as a cause of dilated cardiomyopathy in human. Desmoglein 2 participates in the intercellular attachment of the force-generating and force-transmitting cytoskeleton. We have recently prepared desmoglein 2 mutant mice by homologous recombination that lack a part of its extracellular adhesive domains. These animals develop dialated cardiomyopathy, which is characterized by loss of cardiomyocytes and fibrotic substitution. The goal of the project is to find out how the mutated desmoglein 2 alters the active and passive mechanical properties of cardiomyocytes to result in fibrotic replacement and heart failure. Therefore, we will- measure the force transmission of cardiomyocytes to adjacent cardiomyocytes and to the extracellular matrix,- examine altered cell coupling using microscopical methods,- and search for markers of perturbed biomechanics based on transcriptomics.With the help of a mechanical stress regime we will investigate how these parameters change under stimulating conditions. The aim is to correlate disturbances of the force-generating and force-transmitting systems to pathologically relevant molecular pathways.
Prof. Dr. Rudolf Leube and PD Dr. Claudia Krusche
in cooperation with:
Prof. Dr. Rudolf Merkel and Dr. Bernd Hoffmann (Institute of Complex Systems, FZ Jülich)
Funding: IZKF RWTH Aachen University
Intermediate filaments are major components of the cytoskeleton acting as mechanical stabilizers and as modulators of cellular differentiation and proliferation. The molecular mechanisms, which regulate intermediate filament dynamics and their 3D architecture in a true tissue context, have not been elucidated. To do this, we use the model organism Caenorhabditis elegans. With the help of transgenic strains we have performed a chemical mutagenesis screen and isolated mutants with altered intermediate filament organization. The goal of the current project is to characterize these mutants and to isolate further mutants in order to work out the molecular pathways that determine intermediate filament network dynamics. We also want to find out how the specific intermediate filament network architecture contributes to intestinal cell polarity and function. The expected results will be of fundamental importance for the understanding of the cytoplasmic intermediate filament cytoskeleton and its alterations in the context of epithelial differentiation and its manifold disturbances.
Prof. Dr. Rudolf Leube and Prof. Dr. Olaf Bossinger
The dynamic relationship between cell adhesion, differentiation and proliferation is of paramount importance for tissue homeostasis. Desmosomes are prominent adhesion sites in epithelial tissues mediating mechanical stability by anchoring the keratin intermediate filament cytoskeleton. In addition, it has been suggested that desmosomal components contribute to cell differentiation and participate in growth regulation. To test the various desmosomal functions in vivo, we have generated transgenic mice that allow temporally-defined and cell type-restricted depletion of the desmosome-specific cell-cell adhesion molecule desmoglein 2 either in enterocytes or hepatocytes. The goal of the project is to understand how the loss of desmoglein 2 affects desmosome formation and, more importantly, how compromised desmosomal adhesion influences the properties and dynamic behaviour of cell types with either low or high proliferative activity (i.e., enterocytes and hepatocytes, respectively) in health and disease. To this end, we will compare clinical features, histopathology, cell adhesion, cytoskeletal organisation, growth, and gene transcription in mice lacking desmoglein 2 in the intestine and liver. In addition, using chemical and genetic tumor models we will determine how the altered phenotypes affect tissue reactivity, tumor growth, tumor differentiation, and malignant transformation.
Principal investigator: Prof. Dr. Rudolf Leube
Plasticity of the cytoskeleton supports many basic cell functions. Very little is known, how this is accomplished in the case of the intermediate filament cytoskeleton. Based on recordings of epithelial cells producing fluorescent keratin intermediate filaments, we have recently proposed that a perpetual cycle of assembly and disassembly is a major property of the keratin system allowing precise adaptation of the network to altering requirements. It was further suggested that the cycle consists of distinct steps, each of which is subject to regulation. The goal of the project is to analyze these steps and to study their modulation in the context of cellular function. Specifically, we will examine how keratin isotype, cell type, motility and keratin phosphorylation affect the turnover cycle. Network dynamics will be monitored by confocal time-lapse fluorescence microscopy of cells synthesizing wild type and mutant fluorescent keratins. To enable the precise description and quantification of the dynamic 3D network organization, the characterization of assembly intermediates, and the study of their transitions in the resulting large multi-dimensional data sets we will develop image analysis algorithms – especially for 3D motion and segmentation. These analyses will provide the basis for modeling keratin dynamics. The long term goal of the project is to understand how modulation of the keratin filament turnover cycle affects epithelial cell mechanics and physiology.
Prof. Dr. Rudolf Leube and PD Reinhard Windoffer
A basic requirement for human embryo implantation, which comprises penetration of endometrial epithelial cells (EEC) by trophoblast cells, is the appropriate preparation of the endometrium. Its cyclic differentiation leads to a short receptive period called window of implantation (WOI). About 30% of all pregnancies are lost during the early implantation phase. Insights in early mechanisms of human implantation may therefore improve the success rates of assisted reproductive technologies (ART).Our goal is to unravel basic mechanisms of early human implantation by using a newly established 3D cell culture confrontation system. Previously, in a study on human endometrial tissue we observed an altered distribution of adhering junctions along lateral membranes of glandular EEC during the WOI that indicates a change in EEC polarity. In the 3D cell culture confrontation system gland-like endometrial cell line spheroids with a junction distribution similar to EEC during the WOI were more strongly invaded by the extravillous trophoblast cell line AC-1M88 than spheroids that were derived from highly polarised endometrial cell lines.In the present project we will focus on the investigation of changes in epithelial junctional complexes and the associated cytoskeleton to elucidate mechanisms of trophoblast invasion. Using the established 2D and 3D culture systems as well as primary cells both spatial and temporal aspects of trophoblast-endometrial interaction will be analysed in detail by means of immunohistology and live cell imaging. By use of light sheet microscopy we expect a particularly high resolution of the invasive processes. A further aim is to analyse the influence of 17beta-estradiol and progesterone on endometrial epithelial receptivity and on the invasiveness of trophoblast cells.We expect that the results will provide new insights into the crucial mechanisms of human implantation and thus may help to improve the outcome of ART in the future.