In Vivo Analysis of Eggshell Assembly in Drosophila

Marquette University, Milwaukee WI

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): The formation of functional three dimensional structures is a fundamental process that occurs in all cells and developing organisms. Much of our knowledge of how secreted proteins organize into functional architectures comes from studies on the assembly of vertebrate extracellular matrices. While the basic components of several tissue-specific extracellular matrices are known, there is little knowledge of the molecular interactions that drive the assembly of these structures in vitro or in vivo. The Drosophila eggshell is a specialized extracellular matrix that forms between the oocyte and overlaying follicle cells during the latter stages of oogenesis. The eggshell is a large, proteinaceous, structure that is amenable to genetic, biochemical, molecular, and morphological analyses. Our research is directed towards elucidating the molecular mechanisms and interactions that underlie its assembly. By understanding the molecular strategies and protein motifs that are utilized in its construction, we can begin to appreciate how complex extracellular architectures form in the right place, at the right time, and with the right shape. We propose to continue our studies on the dec-1 gene, a gene that produces several distinct proteins that are essential for eggshell assembly. Genetic and biochemical data suggest that two dec-1 derivatives, s25 and s80, function in oligomeric complexes. Using a variety of biochemical methods we propose to isolate dec-1 containing complexes, identify proteins that interact with these derivatives, determine the sequences that are required for the interactions, and test the effects of mutations that disrupt the interactions on eggshell assembly. The effects of the mutations on eggshell function, assembly, and protein localization will be assessed by a variety of means including ultrastructural analyses, Western blot analyses, immunoelectron and immunofluorescence microscopy. Conversely, we propose to test the effects of engineered dec-1 mutations on eggshell assembly, dec-1 complex formation, and protein interactions. Dec-1 proteins with partially processed N-termini cause dose dependent negative effects in wild type flies, including female sterility and collapse of the eggshell. We propose to elucidate the molecular basis of these negative effects.