Expression von verschiedenen Proteinversionen von Blown fuse sowie Skap2 im Embryo und die mögliche Rolle von Blown fuse in der Myoblastenfusion in Drosophila melanogaster

Die Myoblastenfusion in der Embryonalentwicklung von Drosophila verläuft in zwei Schritten. Das Blow-Protein ist essentiell für den Verlauf des zweiten und teilweise auch für den Verlauf des ersten Schrittes. blow2-Embryonen weisen einen sehr starken Fusionsdefekt auf. Durch Immunhistologie mit eine...

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Bibliographic Details
Main Author: Sickmann, Angela
Contributors: Renkawitz-Pohl, Renate (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2010
Online Access:PDF Full Text
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Table of Contents: Myoblast fusion in Drosophila proceeds in two distinct temporal phases. The Blow protein is essential for the second and partly for the first phase of fusion. blow2-embryos show a severe myoblast fusion defect. By immune histology with an anti-Blow-antibody directed against the N-Terminus of the protein it was possible to show that no remains of Blow were detectable in blow2-embryos. A sequencing of the allele showed that blow2 bears a deletion from the 2nd intron to the 2nd exon of blow, which leads to the degradation of the mRNA. Therefore blow2 is a null-allele. To gain further understanding of the fuction of Blow, blow-constructs were generated in which several features of the protein, found in an in-silico-analysis, were modified: Besides a PH-domain a putative phosphorylation- and interaction-signal was found at tyrosine 342, therefore Blow-versions were expressed which beared these features modified. Neither the expression of N-terminal truncated Blow nor Blow with a mutated Tyrosin 342 lead to a defect after ectopic expression in founder-cells. Also an expression of a construct with phosphomimetic modified tyrosin 342 or with deleted PH-domain showed no defects in myoblast fusion in the embryo. Surprisingly the expression of wildtype Blow did not rescue the blow2-phenotype. An analysis of blow-localisation in several fusion mutants showed that accumulation of Blow at the site of fusion is dependent on the adhesion of the fusion competent myoblast and of the actin reguator arp3. Despite the absence of adhesion molecules there are severeal Blow-foci at cell-cell contacts. Finally, the postulated Blow-ortholog Skap2 of zebrafisch was tested for functionality in the fly embryo. Neither a rescue of the blow2 phenotype, nor a defect of myoblast fusion after expression in the wildtype-background could be detected, meaning that a orthology of the two proteins remains spekulative.