The FoxA transcription factors have been implicated in many cellular processes, from the control of organ differentiation to the regulation of insulin responses (Kaestner, 2010;Wolfrum et al., 2003). The Drosophila FoxA homolog Fork head (Fkh) has been implicated in the survival and morphogenesis of the embryonic salivary gland (Myat and Andrew, 2000), as well as the regulation of expression of salivary gland-specific gene products (Fox et al., submitted). Recently, Fkh was shown to regulate expression of the predicted Rab-GAP encoded by tbc-1 (aka CG4552), though it has not been determined if this regulation is direct or indirect (Maruyama et al., 2011). Interestingly, the mammalian and C. elegans homologs of tbc-1 have been implicated in the innate immune response (Alper et al., 2008;De Arras et al., 2012). This finding suggests that Fkh, through tbc-1, may have an additional role in the salivary gland, that of responding to pathogenic assaults. Additionally, thi suggests that Rab-GAPs, which inhibit the GTPase activity of Rabs, may have a developmental role in the salivary gland. Our preliminary results show that knock-out of tbc-1 disrupts apical membrane morphology in the salivary gland.
In Aim 1 : we will fully characterize the loss and overexpression phenotypes of tbc-1 in the salivary gland and other relevant tissues. To accomplish this aim, we will determine what causes the apical membrane to be irregular in the salivary gland and ask if there is a migration defect in border cells, as has been suggested based on RNAi studies by another group (Laflamme et al., 2012). We will also determine if there are any changes in survival or the immune response machinery in adults and embryos exposed to different pathogens. Since the subcellular localization of tbc-1 can indicate which Rab or Rabs it affects, aim 2 addresses the subcellular localization of Tbc-1. To this end, we will develop an antibody to Tbc-1 and use it along with organelle-specific markers to determine where Tbc-1 localizes in the cells of the salivary gland. We will then begin to determine which Rab(s) are affected by Tbc-1 in Aim 3. For this, we will obtain fly lines for expressing the dominant negative (DN) and constitutively active (CA) forms of the Rabs. We will then express these specifically in the salivary gland to determine which of the altered Rab constructs can phenocopy the overexpression and knock-out phenotypes of tbc-1. Once we have determined this, we will also do a co-immunoprecipitation assay with Tbc-1 and the candidate Rab or Rabs to ask if there is a direct physical interaction. Overall, we expect to learn the role of Tbc-1 in he salivary gland and border cells, which may reveal a novel role for Fkh in the salivary gland through its regulation of the highly conserved Tbc-1 Rab-GAP.

Public Health Relevance

In this project we will study how a highly conserved protein, known as Tbc-1, affects organ development as well as the innate immune response, which is the first line of defense against bacteria and other pathogens. The mammalian and worm Tbc-1 genes have been shown to play a role in the innate immune response and my preliminary studies of tbc-1 loss-of-function mutations reveal defects in the apical membrane domain of the salivary gland cells that normally express this gene. In this work, I propose to learn: which membrane trafficking events are affected by tbc-1 loss and overexpression;to learn if Drosophila Tbc-1 gene also plays a role in innate immunity;to learn how the Tbc-1-regulated membrane trafficking events affect organ development and, if relevant, innate immunity;to identify the Rab(s) that Tbc-1, a Rab effector, regulates;and to ask if Tbc-1 and the candidate Rab(s) directly interact with one another.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Predoctoral Individual National Research Service Award (F31)
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NIDR Special Grants Review Committee (DSR)
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Frieden, Leslie A
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Johns Hopkins University
Anatomy/Cell Biology
Schools of Medicine
United States
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