Cytoplastic NAD+ biosynthesis affects gonad development in Caenorhabditis elegans

Open Access
- Author:
- Shu, Muya
- Graduate Program:
- Biochemistry and Molecular Biology
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- March 21, 2016
- Committee Members:
- Wendy Hanna Rose, Thesis Advisor/Co-Advisor
- Keywords:
- C. elegans
NAD+
NMATs
development
Glycolysis. - Abstract:
- In recent years, nicotinamide adenine dinucleotide (NAD+) has gained renewed attention from scientists. This is because NAD+ serves not only as a cofactor but also as a substrate for numerous enzymes that are widely dispersed in cellular signaling reactions. These signaling pathways affect many aspects of organisms, like aging, development, circadian rhythm and axon pathology. However, the mechanism that NAD+ homeostasis influences development is still elusive. In my project, I used the C. elegans pnc-1(pk9506) mutant as a model to understand why blocked NAD+ salvage biosynthesis delays gonad development. Our lab has found that blocking salvage NAD+ biosynthesis affects gonad developmental progression in worms. My project was to investigate the mechanism linking NAD+ production to gonad development in C. elegans. Because the block in salvage synthesis had already been linked to a deficit in glycolysis, I hypothesized that disrupted glycolysis caused by NAD+ deficiency leads to the gonad developmental defects. I supplied the pnc-1(pk9605) mutants with late glycolytic intermediates 3-phosphoglycerate (3PG) and phosphoenolpyruvate (PEP), and found that these intermediates increased the percentage of normal gonad development in the pnc-1 population, which supported the hypothesis that disrupted glycolysis causes the gonad developmental defects. Cells have relatively separated NAD+ pools between mitochondria, nucleus and cytoplasm, and individual NMNAT genes independently regulate these NAD+ pools to meet specific requirements of NAD+ in cells. I would like to use NMNAT function to investigate the compartment-specific requirements for NAD+ biosynthesis in C. elegans gonad development. However, it is unclear which of the two C. elegans NMNAT genes, nmat-1 and nmat-2, corresponds to which compartment-specific gene. To determine the subcellular localization of the two C. elegans NMATs. I fused nmat-1 and nmat-2 to CFP under the myo-3 promoter and examined their subcellular localization in muscle cells. The subcellular localization experiment results showed that NMAT1 is a mitochondrial protein and NMAT2 is a Golgi/cytoplasmic protein. To further explore the function of NMNAT, I applied the new genomic engineering CRISPR/Cas9 system to create an allele of nmat-1. Now, I am screening for homozygous nmat-1 mutants. This work will provide us the insights on metabolic products function in animal’s development and also will shed light on an intimate connection between metabolism and development in human health and disease.