Abstract:
The migration of embryonic macrophages in the fruit fly is an ideal system to investigate the regulation and the mechanisms of cell movement in vivo. The macrophages originate from the cephalic mesoderm and perform a complex migration throughout the entire embryo. The Drosophila PDZ G-nucleotide exchange factor (PDZ-GEF) Dzy was identified as an essential component in macrophages for proper migration and cell shape regulation. In mutants lacking dzy function, macrophages have smaller cellular protrusions and migrate less efficiently. In contrast, macrophages overexpressing dzy are vastly enlarged and form multiple long protrusions. Additionally, dzy is known to play a role in adult morphogenesis. Homozygous dzy mutants are lethal, but "escaper" flies are occasionally observed, showing a characteristic phenotype with bent downward wings, rough eyes and distorted genitalia. The Dzy protein contains several conserved domains found in PDZ-GEFs and is expressed in three isoforms (dzyA, dzyB and dzyC) due to alternative splicing of the mRNA. While all domains typical of PDZ-GEFs are equally present in all isoforms, the variable regions have three proline-rich motifs (PRMs), of which DzyA and DzyB have all three and DzyC only one. Here we explore the function of the N-terminal PDZ domain and of the different isoforms which are distinguished by their unique C-termini and the presence or absence of two proline-rich motifs (PRMs). Both structural features, the PDZ domain and PRMs, are often involved in protein-protein interactions. Here we tested the relevance of the PDZ domain and PRMs for the function of Dzy in macrophage migration. We investigated the impact of the ΔPDZ form and the different splice forms on the migration of macrophages by analysing their overexpression phenotypes. Furthermore, we examined the ability of the various forms to provide full wild-type dzy function. Our findings on the function of dzy revealed that dzyC is the only splice form capable of causing cell shape changes when expressed in macrophages. Furthermore, we found that the dzyC splice form is also sufficient to partially rescue the phenotype of homozygous dzy mutant adult escapers. The only difference between dzyC and the other two splice forms (dzyA and dzyB) is the presence or absence of exon 5 in the different splice forms. Therefore, the domain encoded by exon 5 is the key component in regulating the activity of the different splice forms. We hypothesised that the PRMs encoded by exon 5 interact intramolecularly with the Dzy PDZ domain and have a function-inhibiting effect. This suggests a novel PDZ-GEF regulatory mechanism which is dependent on alternative splicing. Thus, extending this model is a promising endeavour to elucidate the molecular mechanisms that cause and control the process of cell migration in all organisms.
The migration of embryonic macrophages in the fruit fly is an ideal system to investigate the regulation and the mechanisms of cell movement in vivo. The macrophages originate from the cephalic mesoderm and perform a complex migration throughout the entire embryo. The Drosophila PDZ G-nucleotide exchange factor (PDZ-GEF) Dzy was identified as an essential component in macrophages for proper migration and cell shape regulation. In mutants lacking dzy function, macrophages have smaller cellular protrusions and migrate less efficiently. In contrast, macrophages overexpressing dzy are vastly enlarged and form multiple long protrusions. Additionally, dzy is known to play a role in adult morphogenesis. Homozygous dzy mutants are lethal, but "escaper" flies are occasionally observed, showing a characteristic phenotype with bent downward wings, rough eyes and distorted genitalia. The Dzy protein contains several conserved domains found in PDZ-GEFs and is expressed in three isoforms (dzyA, dzyB and dzyC) due to alternative splicing of the mRNA. While all domains typical of PDZ-GEFs are equally present in all isoforms, the variable regions have three proline-rich motifs (PRMs), of which DzyA and DzyB have all three and DzyC only one. Here we explore the function of the N-terminal PDZ domain and of the different isoforms which are distinguished by their unique C-termini and the presence or absence of two proline-rich motifs (PRMs). Both structural features, the PDZ domain and PRMs, are often involved in protein-protein interactions. Here we tested the relevance of the PDZ domain and PRMs for the function of Dzy in macrophage migration. We investigated the impact of the ΔPDZ form and the different splice forms on the migration of macrophages by analysing their overexpression phenotypes. Furthermore, we examined the ability of the various forms to provide full wild-type dzy function. Our findings on the function of dzy revealed that dzyC is the only splice form capable of causing cell shape changes when expressed in macrophages. Furthermore, we found that the dzyC splice form is also sufficient to partially rescue the phenotype of homozygous dzy mutant adult escapers. The only difference between dzyC and the other two splice forms (dzyA and dzyB) is the presence or absence of exon 5 in the different splice forms. Therefore, the domain encoded by exon 5 is the key component in regulating the activity of the different splice forms. We hypothesised that the PRMs encoded by exon 5 interact intramolecularly with the Dzy PDZ domain and have a function-inhibiting effect. This suggests a novel PDZ-GEF regulatory mechanism which is dependent on alternative splicing. Thus, extending this model is a promising endeavour to elucidate the molecular mechanisms that cause and control the process of cell migration in all organisms.
Dizzy, Drosophila
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