This R21 grant application grant application addresses the goals stated in the Research Objectives. Specifically, the intent is to establish a robust platform in Drosophila tissues to characterize changes occurring in mitochondria (activity, protein composition), and in the genome (nuclear-encoded mitochondrial genes and others), in response to exposure to environmental mitochondrial stressors, such as toxic metals. The initial studies will focus on the gut, as it is the tissue where metals are first absorbed. Results from these studies will allow one to engineer flies carrying biomarkers of environmental mitochondrial toxicants that can be used to dissect the molecular mechanisms underlying cross-talk and signaling between mitochondria and the nucleus. The grant application is composed of three specific aims:
Specific Aim 1) Characterize changes in mitochondrial activities and proteome constitution in response to environmental mitochondrial toxicants in the adult Drosophila gut. Mitochondrial activities, including citrate synthase activity and O2 consumption rate, as well as mitochondrial proteome constitution, will be measured at different timepoints following toxic metals feedings. For the proteomic studies, a novel protein labeling technique will be exploited, based on an engineered ascorbate peroxidase (APEX), that was recently adapted for studies in Drosophila live tissues;
Specific Aim 2) Characterize the transcriptional changes occurring in the Drosophila gut in response to environmental mitochondrial toxicants. RNA-Seq of gut will be examined at different timepoints to characterize the transcriptional changes occurring in this tissue following feeding of toxic metals. Further, using the recently developed COMPLEAT tool, mitochondrial protein complexes that are preferentially affected by environmental mitochondrial toxicants will be determined;
and Specific Aim 3) Generate biomarkers of environmental mitochondrial stress. Genes will be selected that are induced by environmental mitochondrial stressors to engineer, using CRISPR, GFP sensors that can be used to dissect the mitochondrial toxicant response. Following the R21 phase, we envision expanding this project into an R33 where we will: 1) Study additional environmental mitochondrial stressors to establish signatures of specific toxicants; 2) Expand to other tissues (brain, muscle, and adipose tissues) to gain a general understanding of the diversity of responses of different cell types; and 3) Use engineered flies carrying biomarkers of environmental mitochondrial toxicants to dissect the molecular mechanisms underlying cross-talk and signaling between mitochondria and the nucleus.

Public Health Relevance

The proposed research will characterize changes occurring in Drosophila mitochondria and in the genome in response to exposure to environmental mitochondrial stressors, such as toxic metals. Results from these studies will allow us to engineer flies carrying biomarkers of environmental mitochondrial toxicants.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21ES025615-02
Application #
9058544
Study Section
Special Emphasis Panel (ZES1)
Program Officer
Shaughnessy, Daniel
Project Start
2015-05-01
Project End
2017-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Type
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
Mohr, Stephanie E; Rudd, Kirstin; Hu, Yanhui et al. (2018) Zinc Detoxification: A Functional Genomics and Transcriptomics Analysis in Drosophila melanogaster Cultured Cells. G3 (Bethesda) 8:631-641
Chen, Chiao-Lin; Perrimon, Norbert (2017) Proximity-dependent labeling methods for proteomic profiling in living cells. Wiley Interdiscip Rev Dev Biol 6:
Chen, Chiao-Lin; Hu, Yanhui; Udeshi, Namrata D et al. (2015) Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase. Proc Natl Acad Sci U S A 112:12093-8