Gene expression in nitric oxide - dependent differentiation of Phhysarum polycephalum

The plasmodial slime mold Physarum polycephalum is a well established model system for studying cell cycle and differentiation processes. In previous work we have characterized nitric oxide synthase (NOS) and pteridine biosynthetic pathways from this organism. We could show that glucose deprivation, an essential prerequisite for rendering the plasmodium competent for sporulation, strongly induces NOS and identified NO/cGMP as crucial signalling molecules for light-induced formation of mature fruiting bodies (sporangia). Expression of lig1, an early sporulation-specific gene homologous to the cell-cycle and DNA-damage checkpoint gene hus1 identified in yeast and mammals, positively correlated with NOS expression and the ability to sporulate. We now plan to identify the targets of NO/cGMP signalling during sporulation, the signals responsible for induction of NOS in response to glucose deprivation during the starvation phase preceding sporulation and the genomic structures, promoters as well as signals influencing promoter activity and hence gene expression of the two NOS genes physnosa and physnosb identified in Physarum. These experiments include generation of Physarum strains with altered NOS expression (gene knock-out and overexpression), biochemical inhibition of enzymes involved in the NO/cGMP signalling cascade and the use of a sporulation-deficient Physarum strain already established. Various polymerase chain reaction (PCR) techniques and two-dimensional protein gel electrophoresis (proteom analysis) will be applied for profiling differentially expressed genes and proteins. Identified targets will be recombinantly expressed and further characterized. Genomic structures and promoters will be analysed by PCR techniques. Promoter activity in response to diverse signals will be monitored by reporter gene and gel shift assays. In animals, versatile biological functions of NO are well characterized. However, also in the intensely studied mammalian species the genes affected by NO/cGMP in the course of cell differentiation are still not identified in detail. Studying this issue in Physarum sporulation holds the potential to identify not yet understood signalling pathways and will thus extend our general understanding of the complex signalling cascades controlling cell differentiation.

In previous work we could show that obtaining sporulation competence is functionally linked to induction of nitric oxide synthase (NOS). In the currently ongoing project, we have characterized the two genes encoding inducible NOS in Physarum and their genomic structure. The proteins were overexpressed, biochemically characterized and N-terminal sequences required for activity were identified. We have furthermore generated NOS-knock-out plasmids and set up the techniques for transformation of amoebae with the aim to generate NOS-knock-out Physarum strains. Being the main target of NO in a variety of organisms we next turned our attention on a putative NO dependent guanylate cyclase. Soluble guanylate cyclase and its dependence on NO were studied in wild-type and sporulation- deficient strains. A new powerful tool for measuring changes in protein expression between different samples, 2-D Fluorescence Difference Gel Electrophoresis (DIGE), was set up and used to identify differentially expressed proteins during sporulation. Protein expression after 5d starvation prior the light pulse in the Physarum strain CS114 and CS310 and the impact of the light pulse on protein expression patterns in sporulation were investigated. Analysis of the spots and their identification is currently underway. Together with Wolfgang Marwan, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, and with Gernot Glöckner, Genome Analysis, Institute for Molecular Biotechnology, Jena, a transcriptom sequencing project was started which so far yielded more than 5000 different cDNA sequences expressed in starved macroplasmodia 6h after setting the light pulse. Currently, cDNA libraries from other time points are also sequenced. Building up a database with the Physarum transcriptom is required to annotate the differentially expressed proteins identified by DIGE. In addition to the transcriptom sequencing approach, the Physarum Genome Project could be initiated at the NIH (http://www.genome.gov/12511858 ) after a meeting of Physarum experts in our laboratory in Innsbruck in July 2004. Sequencing is carried out by the Genome Sequencing Center at Washington University in St. Louis, USA. The transcriptom database will also support finishing of the Physarum genome, a crucial prerequisite for studying signalling networks in Physarum by somatic complementation The event of differentiation and its regulation is one of the most principal questions in cell biology. Newly developed cancer concepts favor the hypothesis that cancer is a defect in differentiation rather than in cell multiplication. On the other hand, there is increasing evidence that cancers might derive from stem cells in which signalling pathways controlling self-renewal are affected. Understanding the switches that determine whether a cell is proliferating or differentiating is therefore of particular importance. Physarum has several characteristics which make it a useful model organism for addressing this question.