RESEARCH GROUPS

1: Konstantinos Anastassiadis - Engineering Stem Cells Group

konstantinos.anastassiadis@biotec.tu-dresden.de            +49-351-463-40127

We are interested in exploiting the mechanisms that control self- renewal and differentiation of mouse embryonic stem cells. These cells originate from the Inner Cell Mass (ICM) of the blastocyst and give rise to all tissues of the embryo including the germline. ES cells are a powerful experimental system to understand the mechanisms that underlie developmental processes.Embryonic stem cells can differentiate into all 3 germ layers in culture. During differentiation the pluripotent ES cells give rise to progenitor cells that in their turn give rise to terminally differentiated cells. The process of differentiation is guided by the various factors that are added to the culture media and is not yet well understood. It is important to understand this process in order to guide and control it. For this reason we have developed (and continue to develop) various genetic experimental tools. One approach is to conditionally immortalize using tetracycline controlled SV40 large T-Antigen the various cell types that arise during ES cell differentiation. The expression of Large T-Antigen is induced during differentiation followed by cellular cloning. During the induction period the cells proliferate. After ending the induction the cells can be terminally differentiated and characterized with the help of existing markers. The goal is to expand homogenous populations of cells that undergo differentiation and compare their expression profile and epigenetic status.

In other words, we have developed a system that enables conditional immortalization of cell populations that arise during ES cell differentiation. This technical advance opens a door to new mechanistic studies on gene expression hierarchies in differentiation.

2: Andreas Beyer - Cellular Networks & Systems Biology

andreas.beyer @biotec.tu-dresden.de  +49-351-463-40080

A growing number of technologies allow for the genome-scale measurement of biological properties such as protein and mRNA concentrations or phenotypic changes (e.g. response to RNAi knock-downs). The genome-wide nature of the available data facilitates a systems perspective: It becomes possible to go beyond individual genes or pathways and to study regulatory processes of the entire system ‘cell’. However, up to now the potential is by far not being fully exploited. We develop computational tools to aid the processing and interpretation of large-scale biological data. Our group adopts a network perspective by studying relationships between proteins and other biomolecules (e.g. DNA, RNA) in silico to reveal the regulatory context of relevant genes.

3: Michael Brand - Patterning and Regeneration of the Vertebrate

Brainmichael.brand@biotec.tu-dresden.de  +49-351-463-40345 

A fundamental problem in neurobiology is how the multitude of different cells and their connections are generated from their precursors, or stem cells. We have studied extensively how embryonic neural precursor cells at the border between midbrain and hindbrain (MHB) act as organizers of cell fate onto the surrounding cells, which eventually form the midbrain and cerebellum. We also study which signals determine where the MHB organizer forms initially. Fgf8 is absolutely required for MHB organizing activity. For instance, zebrafish acerebellar mutants have no functional Fgf8, and hence lack a cerebellum and proper polarity in the midbrain. In genetic, cell biological and biophysical studies, we are unraveling how secreted Fgf signals exert their function at the MHB and in other embryonic organizer cell populations.More recently we have probed for a possible role of organizer- associated signaling molecules also in the adult brain. We find that in contrast to mammals, adult zebrafish brains retain an amazing number of active neural stem cells at all times, and in very discreet spatial domains. Given the well known ability of teleost brains to repair damage, and the lack thereof in mammalian brains, stem cell based regeneration studies in fish may provide clues which mechanisms need to be activated to stimulate CNS regeneration also in mammalian brains. Indeed numerous new neurons of different subtypes are produced in the adult zebrafish brain, providing an ideal genetically and experimentally tracktable system for understanding brain repair processes.

4: Denis Corbeil-Tissue Engineering from Prominin-1/CD133+ stem and progenitor cells

denis.corbeil @biotec.tu-dresden.de           +49-351-463-40118

The focus of our research is to understand the first step of tissue formation, which relies on the cell biological basis of stem cell proliferation and differentiation. We particularly concentrate on stem cells that express the marker prominin-1 (CD133).Previously, we have reported the molecular and cell biological characterization of prominin-1, a pentaspan membrane glycoprotein (Fig. 1). This cholesterol-binding protein is specifically associated with plasma membrane protrusions, irrespective of the cell type, by a molecular mechanism that involves a membrane lipid microdomain. We have identified also a second prominin molecule (referred to as prominin-2) that exhibits a similar, but not identical, tissue distribution and subcellular localization to prominin-1. Moreover, several splice variants of prominin-1 have been identified and characterized. They show a broad range of expression, from myelin to the tail of spermatozoa. Importantly, prominin-1 is expressed in several stem cells originating from various sources, including the neural and hematopoietic system, and prominin-1 is now used for stem cell isolation. Likewise, certain epitopes, e.g. AC133, of prominin-1 might be used as markers of cancer stem cells.The physiological function of these pentaspan membrane glycoproteins, which are conserved through metazoan evolution, remains to be established. Nevertheless, the general preference of these proteins for plasma membrane protrusions – including the membrane evaginations at the based of the outer segment of photoreceptor cells – and the identification of mutations in the human PROM-1 gene that cause retinal degeneration, have led to the hypothesis that prominin-1 acts as an organizer of plasma membrane protrusions.

5: Bernard Hoflack-Regulation of membrane traffic during osteoclast

differentiationbernard.hoflack @biotec.tu-dresden.de +49-351-463-40235

Our group has been interested in understanding basic processes of lysosome biogenesis, an essential process for homeostasis of eukaryotic cells. We focus on 1) the mechanisms by which soluble and membrane proteins as well as lipids are sorted from the secretory pathway for subsequent transport to the endosomal/lysosomal system, 2) how these processes are regulated, especially during cell differentiation as observed with osteoclasts, which acquire the property of building-up an extracellular lysosome in order to digest bone. This prompted us to become interested in the biology of bone remodeling. Much of our progress came from proteomic screens performed on in vitro reconstitution systems recapitulating key steps of lysosome biogenesis, in particular the selective interaction of the AP-1 and AP-3 coats with membranes. We have now identified the two sorting machineries involved, composed each of ≈50 different proteins that belong not only to sorting devices like coat components, but also to devices involved in actin polymerization and membrane fusion. This highlights the complexity of protein-protein interactions required for coordinating protein sorting and transport. Our progress also came from proteomic and genomic screens performed on osteoclasts and their precursors. These screens allowed us to identify a large number of proteins, in particular effectors of small GTPases, that are good candidates for establishing the cell polarity of osteoclasts and for remodeling their membrane traffic and actin dynamics, three essential processes required for efficient bone degradation. The role of interesting candidates is now investigated using RNA interference and appropriate functional assays monitoring cell polarity, membrane traffic or actin dymanics in mature osteoclasts. 

6: Daniel J. Müller-Characterizing molecular interactions driving the function of cells and cellular machineries

daniel.mueller @biotec.tu-dresden.de                   +49-351-463-40330

Molecular interactions drive all processes in life. They determine the molecular crosstalk and build the basic language of biological processes. In water-soluble and membrane proteins molecular interactions fold the polypeptide into the functional protein, stabilize the structure, or lead to protein misfolding. These molecular forces determine protein-protein interactions, switching on and off ion channels, ligand-binding, the functional states of receptors, and the supramolecular assembly of molecular machines to functional units. Because of this enormous importance it is one pertinent demand in life sciences to characterize how these interactions drive biological processes and thus to decipher fundamentals of the biological language. To do so, we have pioneered two bionanotechnological methods, single-molecule atomic force microscopy (AFM) and single-molecule force spectroscopy (SMFS), which allows detecting inter- and intramolecular interactions of native membrane proteins. Recent extensions of both methods allow to image cells at nanometer resolution and to study interactions of single cells at molecular resolution using single-cell force spectroscopy (SCFS).

7: Francis Stewart- Epigenetic regulation and genomic engineering

Our work focuses on two complementary aspects of genomics,                                                        
(i) mechanisms of epigenetic regulation in eukaryotic chromatin and
(ii) technologies of genetic engineering.

EPIGENETIC REGULATION IN CHROMATIN.

Although the complete DNA sequence of an organism encodes the primary information, additional information is added by epigenetic regulation. In eukaryotic chromatin, epigenetic regulation is conveyed by covalent modifications of DNA (DNA methylation) and histone tails (acetylation, phosphorylation, methylation, ubiquitinylation). Much attention worldwide is now focused on the histone tails and the proposition that patterns of covalent modifications serve as an epigenetic code. Our approach to unravelling epigenetic mechanisms and hierarchies is based on complementary uses of the yeast, S. cerevisiae and the mouse as experimental systems. We apply advanced reverse genetic strategies, some of which were developed by us, to analyze select classes of epigenetic regulators in both organisms. In yeast, we are using protein-tagging and mass spectrometry to characterize complexes containing epigenetic regulators. Amongst other complexes that we have identified in the proteomic environment of chromatin, we have recently identified a new histone methyltransferase activity for lysine 4 of histone 3.

In mice, we are studying two candidate histone methyltransferases by knock-out and conditional strategies using Cre/lox, as well applying proteomic approaches to characterize the complexes. A future aspect of our mouse work is directed towards use of ES cell differentiation in culture as a model for epigenetic decisions and stem cell manipulations.

GENOMIC ENGINEERING

We have developed several aspects of genetic engineering technology using site specific and homologous recombination. We aim at more fluent manipulation of mammalian cells, particularly ES cells and in mice. Most recent work involves exploration and implementation of a novel homologous recombination system that we discovered in E.coli phages. This permits fluent engineering of BACs in E.coli, and may offer new routes for directly engineering eukaryotic cells.