Untersuchung zur chemotaktischen Signalkaskade bei Dictyostelium discoideum

Abstract

The isolation and characterization of the phenotype of two indepent cellpolarity REMI mutants ("Ganko", "Enigma") were described in this work. A putative PMCA gene was identified in Dictyostelium and also described.The first cellpolarity mutant "Ganko" received the REMI vector insertion in the 3´UTR of actin 8, 283 bp downstream of first and 277 bp after the second polyadenylation signal. The mutants showed an alteration in assembling a normal F-actin network which enables the mutant to chemotax up a shallow cAMP gradient (50 µM) within the performed selection assay. Furthermore, the cells were able to squeeze through 1. 2 µm filter pores. In the standart chemotaxis assay with a glasscapillary filled with 100 µM cAMP, "Ganko" showed no alteration with respect to the wildtype. Performing the same assay with 10 µM cAMP more mutants as wildtype cells reached the glasscapillary. At this cAMP concentration the "Ganko" cells showed an abberant chemotaxis behaviour since they developed more lateral pseudopodia with respect to the wildtype. Measuring F-actin accumulation after cAMP stimulation either with 10 µM cAMP or 1 µM cAMP the mutant showed less actin assembly as compared to the wildtype. The localization of F-actin was the same as in wildtype cells, but the F-actin network was diffusively distributed in the pseudopodia of the mutant. Other alterations e. g. growth rate, development, cell volumes, endocytosis, cell shape, ligth-scattering oscillations or dark field waves were not observed.In summary, I concluded that the 3´UTR reagion that is affeceted is necessary for proper posttransciptional processing and production of the actin 8 mRNA in the cells.The second REMI- mutant "Enigma" was also able to chemotax through 1. 2 µm filter pores and also moved in a verry short time towards a cAMP gradient.F-actin was also affected as more filopods were visible in F-actin staining of the mutant. Futhermore, the mutant spread more surface as the wildtype and established more pseudopods under standart chemotaxis assay conditions. Also endocytosis was affected and "Enigma" showed a developmental defect in the mound stadium resulting in the breaking of the aggregation streams in a parcticulary fashion. The mutant needed approximatly 30 h for full development. Alterations in e.g. growth rate, cell volume, ligth-scattering oscillation and dark field waves were not observed.Lack of sequence homology of the mutated genetic sequence in the mutant disables the further molecular genetic characterization of "Enigma".By an informatic approach I have identified a putativ plasmamembran calcium ATPase (dPMCA) from Dictyostelium discoideum. The putative PMCA was cloned from a cDNA bank and amplified from genomic DNA. The PMCA is present as single copy in the genome and is expressed throughout development. The gene of the PMCA is localized at the chromosome 5.