| dc.description.abstracteng | The establishment and maintenance of cell polarity is crucial for the function of many cell types in multicellular organisms. Especially in epithelial tissues, cell polarity is connected to the regulation of cell adhesion and regulated by a complex hierarchy of highly conserved proteins. These can be subdivided into three groups of genes, the bazooka and crumbs groups, which encode apically localizing proteins, and the discs large group that encodes laterally localizing tumor suppressor proteins. Among these classes of proteins, Bazooka (Baz), the Drosophila homolog of vertebrate Par-3, plays a predominant role as shown by genetic epistasis experiments.
In a yeast two-hybrid screen we identified the protein encoded by the annotated gene CG43427 which we named smallish (smash), as a new interaction partner of Baz. The gene product of smash possesses a C-terminal PDZ binding motif and a LIM domain close to the C-terminus. Endogenous Smash colocalizes with Baz apically in epithelial cells, a region harboring the adherens junctions (AJs). Co-immunoprecipitation of Baz and an N-terminally tagged version of Smash-PI (an isoform encoding for the last 889 amino acids of Smash) has confirmed that these proteins interact in vivo in embryos.
To analyze the function of smash during the development of Drosophila, we generated two different knockout alleles by transdeletion, one representing a null allele and the other a C-terminal truncation affecting the part of the protein carrying the LIM domain and the PDZ binding motif. We found that smash is not an essential gene, as homozygous mutants for both alleles are viable and fertile. The subcellular localization of polarity markers such as Baz were not affected upon smash knockout. On the other hand, overexpression of Smash using the UAS/Gal4 system and transgenes encoding for N-terminally GFP-tagged versions of Smash caused lethality in embryonic and larval stages. Rare eclosing escaper flies were decreased in body size. Overexpression of Smash in epithelial cells resulted in reduction of the apical surface area, indicating that Smash may function in apical constriction, a process important for morphogenetic rearrangements in epithelia. Overexpression of Smash during eye development caused a rough eye phenotype and reduction of eye size. Upon ubiquitous overexpression of Smash in embryos, many embryonic cuticles exhibited anterior and dorsal holes.
Following up on these findings, we showed that the non-receptor tyrosine kinase Src42A binds N-terminally GFP-tagged versions of Smash-PI in vitro in S2 cells and is furthermore able to phosphorylate GFP-Smash-PI. Endogenous Smash protein was found to be tyrosine phosphorylated in vivo in embryos as well. Domain deletion versions of Src42A still showed binding to Smash, indicating different binding mechanisims provided by the fact that tyrosine phosphorylation of Smash was only abolished upon deletion of the kinase domain.
A double mutant for Src64B, the second Src kinase encoded by the Drosophila genome, and smash is lethal. However, embryonic cuticles did not show defects and epithelial integrity appeared intact. | de |