Abstracts - Deutsche Zoologische Gesellschaft

Physiology Posters 201P PH.30 - ENA c-Src-limited, CaMKII-dependent apoptosis induces renal cystogenesis independentfrom Raf-1Daniel Weiß, Sarah Honisch, Winfried Beil, Michael FährmannInstitut für Pharmakologie OE5320, Medizinische Hochschule HannoverCysts are initial structures to develop renal tubules. A limited apoptosis is discussed to be a prerequisitefor cystogenesis. However, the precise molecular mechanism, and significance is unclear. Atrenal MDCKII cells, formation of cysts was analyzed for the dependence on post-receptor multifunctionalprotein kinases c-Src, Raf-1, and CaMKII. Activities of either c-Src or CaMKII in cystshave also been demonstrated by each specific CFP/YFP-FRET reporter in confocal, live cell imaging.We show that c-Src kinase has been necessary for cystogenesis since it was inhibited bydominant-negative c-Src. c-Src was predominantly active at basis and top of cysts. Overexpressionof dominant-negative Raf-1 301 or Raf-1 WT reduced formation of normal cysts but each stronglyincreased apoptosis. Overexpression of constitutively active CaMKII T286D increased apoptosis3-fold, and restricted cystogenesis. Co-overexpression of c-Src WT limited CaMKII-induced apoptosis,and partially abolished the CaMKII T286D-blocked cystogenesis. Restrained cystogenesis byoverexpression of dominant-negative Raf-1 301 was compensated by co-overexpression of CaM-KII T286D with either c-Src WT or constitutively active c-Src. In these cases, apoptosis was onlyincreased between 1.8- and 2-fold. Conclusively, in the case of lost Raf-1, our data describe that ac-Src-limited CaMKII-induced apoptosis is implicated with cystogenesis.P PH.31 - ENExpression and assembly properties of the recently identified “thread” keratins TKαand TKγ in the zebrafish Danio rerioSebastian Neumann, Michael SchaffeldInstitut für Zoologie, Johannes Gutenberg-Universität MainzKeratins form the two most complex groups of intermediate filament (IF) proteins, assembling intofilaments of type I/type II heterodimers. The thread keratins α and γ were first discovered in thehagfish slime, but since then have been considered a highly specialized group of IF-proteins, possiblyonly present in this ancient vertebrate group. Recently we discovered that both thread keratinsare also present in other vertebrates, including teleosts and amphibians and that TKα is indeed bestcharacterized as a type II keratin, whereas TKγ represents a rather ancient type I keratin. However,to-date little is known about thread keratin properties, expression and function in these animals.We therefore produced the respective recombinant proteins from zebrafish, (a) to investigate theirassembly properties, (b) to establish an overlay assay enabling us to identify the thread keratins incytoskeletal preparations and (c) to raise antibodies specific for both thread keratins in zebrafish totrace their expression patterns. Here we present our first results, which already indicate that TKαand TKγ do not show the typical behaviour known for other keratins, in that at least TKα is capableof forming homopolymeric, filamentous structures. Moreover, for the first time we have been able toidentify TKα at the protein level, indicating that it is present in different larval stages as well as inadult zebrafish. [We thank Prof. Jürgen Markl for his interest and support].