|Fakultät für Biologie und Vorklinische Medizin|
|Institute für Anatomie|
|Cell Polarity and Cell Proliferation Control|
|Junior Prof. Dr. vet. med. Dr. rer. nat. Michael Krahn|
In summary, the major focus of our research group is the establishment and maintenance of cell polarity in Drosophila and vertebrate systems with a particular emphasis on the link between cell polarity and cell proliferation control / tumourigenesis.
Due to its limited genetic variety and the availability of numerous genetic tools, we mainly use Drosophila as model organism for our investigations and screens. However, in all projects we also test if our findings derived from Drosophila are conserved in vertebrates. Therefore, we use mammalian cell culture systems as well as mice models as in vivo system.
LKB1 is a master kinase, which is associated with the Peutz-Jeghers-Syndrome in men. Patients suffering from this rare dominant autosomal inherited cancer syndrome disease develop benign gastrointestinal harmatomas and show a higher risk for intestinal and extraintestinal cancer. Deletion of the lkb1 locus is frequently found in various cancer specimen and -cell lines. Concomitantly, LKB1 can induce cell polarization independently of cell-cell contacts, suggesting a crucial role in cell polarity, although the detailed mechanism underlying this phenomenon is not yet described.
We aim to elucidate the role of LKB1 in the regulation of cell polarity on the one hand and its impact on cell proliferation control on the other hand. We recently identified several potential interaction partners of LKB1 in a mass spectroscopy-approach and are currently validating some upstream regulators of LKB1 as well as downstream targets.
One different overall future goal of my group is to elucidate the correlation between cell polarity and cell proliferation / cell cycle control in the physiological state and disease. Notably, many key players which are involved in cell polarity are also implicated in cell transformation and the onset of cancer (for instance Lethal giant larvae (Lgl), Discs large (Dlg), Scribble (Scrb), aPKC), although the mechanistic basis of this correlation is up to now only poorly understood. Furthermore, most of the described polarity determinants are not exclusively found at the cortex but also localize to the nucleus (e.g. Par3, Par6, aPKC, Pals1/Stardust, Lgl, Dlg, Scrb, Par1, LKB1), although the functional impact of this localization remains elusive in most cases.
In order to approach this question by a systematic set-up, we aim to analyze the function of certain regulators of cell polarity in cell transformation and cell proliferation control. We are going to explore the mechanisms by which these proteins are targeted from the cortex into the nucleus and how this dual-localization is regulated in the context of cell polarity, cell growth and cell differentiation. In a first step we will use Drosophila and mammalian cell culture to identify nuclear import and export signals in proteins, which are known to play a crucial role in cell polarity. Subsequently we will establish transgenic cell lines and -flies and optionally knock-in mice, which lack the endogenous protein but express only a version of the polarity marker, which is not capable to enter the nucleus. From phenotypic and functional analyses of these modified knockout systems, we hope to obtain information about the function of certain cell polarity determinants in the nucleus. In parallel, nuclear-localizing polarity proteins will be tested for nuclear-specific functions such as activation or repression of transcription. Thereby we hope to obtain new insights into the mechanisms regulating the interplay between cell polarization/cell differentiation and cell proliferation/tumour control.
A different focus of our group is the analysis of different protein complexes during the establishment of cell polarity. Although many key regulators have been described over the years, many underlying mechanisms are still unknown and some links between the diverse protein complexes, which have been proven genetically or biochemically, have to be analyzed in vivo.
Therefore we aim to investigate the composition of different polarity complexes during the establishment of epithelial polarity using biochemical as well as cell biological approaches. In particular, we currently implement Fluorescence Resonance Energy Transfer (FRET) with GFP- and RFP-tagged as well as antibody labelled proteins to describe functional association of proteins in vivo during different polarization steps. In addition, we apply immune-electron-microscopy to further visualize the subcellular localization of different proteins in a better spatial resolution.
Another goal is to elucidate the role of PATJ in ectodermal epithelial polarization. In vertebrates, loss of PATJ has been described to impair the formation of Tight Junctions – however, no functional explanation is given for this phenotype. In Drosophila, due to a lack of a suitable null allele, the function of PATJ in cell polarity still remains elusive in many regards. We have established a null allele for PATJ and currently perform a biochemical and functional-structural analysis of the protein.
In vertebrates, blood filtration and secretion is accomplished within the same organ – the kidney. In Drosophila, the filtrating cell, the so called “nephrocytes” are separated from the secreting cells - the first are located within the body cavity, the latter are integrated in the “Malpighian tubules”. Whereas many studies have been performed describing several aspects of the development of the Malpighian tubules, little is known about the nephrocytes. Therefore, we are currently characterizing the role of several cell polarity determinants in this cell type. In a second step we aim to identify new genes involved in the development and function of nephrocytes by screening about 75% of the Drosophila genes (around 12 000 genes) via RNA-interference. Promising candidates will first be verified in Drosophila using mutant alleles and in a further step tested whether its function in Drosophila nephrocytes is conserved in mouse podocytes.