Award Date

8-2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Biological Sciences

Department

Life Sciences

First Committee Member

Andrew J. Andres, Chair

Second Committee Member

Frank van Breukelen

Third Committee Member

Allen Gibbs

Fourth Committee Member

Steven deBelle

Graduate Faculty Representative

Ron Gary

Number of Pages

206

Abstract

Drosophila melanogaster provides an ideal model organism to test genetic and molecular biological mechanisms within the context of a living animal. For over one hundred years Drosophila continues to produce a boundless extent of informative and important scientific data providing crucial insight into development, disease progression and genetic interactions. A century as a model organism allowed for the development of an abundance of unique genetic and molecular tools allowing researchers to tease apart cellular mechanisms with very little limitation. From the whole adult body to tissue function to molecular networks, if a biological question arises it most likely can be answered using the fruit fly.

The Drosophila larval salivary gland is an organ that not only takes advantage of the many biological tools available, but also has innate properties favoring it as a model tissue to investigate nuclear hormone activity. In the final larval stage (third instar) of Drosophila, the salivary gland responds to the steroid hormone, 20-hydroxyecdysone (20E) by synthesizing and secreting a glycosylated polypeptide glue mix (glue).

In the mid-third instar, a low titer of 20E initiates a complex of gene expression that targets the glue genes resulting in the synthesis of glue. Using fluorescent markers tagged to the glue, this event is easily monitored using confocal microscopy. The first part of my dissertation describes a model in which glue synthesis is a 20E-coordinated event and that surprisingly did not act through a canonical nuclear hormone receptor. Prior to my research, it was known that 20E-activated genes act through a nuclear hormone receptor comprised of a heterodimer between two proteins, ecdysone receptor (EcR) and ultraspirical (USP). However, I show that 20E-regulated glue synthesis is USP independent. Included in this section of my dissertation are experiments designed to test this model of glue synthesis that is independent of USP and to investigate the potential for another co-receptor functioning with EcR to initiate glue synthesis.

Following the logic that another receptor is functioning with EcR in 20E-mediated glue synthesis, I investigated other candidate receptors. I found the nuclear receptor, DHR96, to be required for glue synthesis and performed experiments that suggest DHR96 may work with EcR to initiate glue synthesis in response to a 20E signal.

The remaining sections of my dissertation are focused on the latter half of the third instar. This stage of the larval life cycle is the late-third instar and is marked in the salivary gland by the secretion of mature glue granules into the lumen of the gland in response to a large pre-metamorphic pulse of 20E. During this period, 20E initiates the expression of proteins that are mostly transcription factors; however, a gene was identified that had sequence similarity to both calmodulin and myosin light chains. This gene, E63-1, has been shown to trigger precocious glue secretion in glands overexpressing E63-1. However, E63-1 mutants secrete glue normally. Interestingly, calmodulin has been shown to prevent glue secretion in experiments using calmodulin inhibitors. These data provoked a model that E63-1 and calmodulin are both needed for secretion to be successful. The goal of chapter 4 was to test this model using available molecular and genetic tools.

Finally, I analyze a secretion phenotype caused by the overexpression of GDP dissociation inhibitor (GDI) that produces rubble-like glue granules, which do not secrete. Part of this analysis involved describing the localization and morphology of wildtype cells using fluorescently tagged markers. When I compared the markers from the wildtype cells to those overexpressing GDI, I found a number of cellular disruptions. GDI overexpression caused an extension of ER membrane, a failure of membrane recycling and a failure for the glue granules to acidify.

Thus, through the use of molecular and genetic tools, I characterize 20E-signaling events in the third instar salivary gland, and more importantly, I lay the foundation to test novel models of 20E signaling and intracellular trafficking mechanisms.

Keywords

20-hydroxyecdysone; Calmodulin; Drosophila melanogaster; Fruit-flies; Nuclear receptors (Biochemistry); Salivary glands; Steroid hormones

Disciplines

Genetics | Molecular Biology

Language

English


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