Histone H3 hosts post-translational modifications responsible for nucleosome formation and gene regulation in eukaryotic cells. De novo synthesis of histone H3 occurs in the cytosol before being trafficked to the nucleus. A previous study had incubated crude histone fractions with mitochondrial extracts and performed SDS-Page with anti-H3 antibodies. The results indicated a binding affinity between histone H3 and the mitochondria. The experimental protocol involved incubation of histone H3 with purified mitochondria, and therefore may not accurately represent histone H3 activity in living cells. The purpose of this study is to visualize histone H3 and mitochondria to determine any histone H3 trafficking to mitochondria in intact cells. Transgenic Arabidopsis thaliana plants containing mCherry tagged histone H3 proteins have been cross bred with plants containing YFP tagged mitochondrial transit peptide from yeast cytochrome c oxidase IV. The source of the mCherry-histone plant line revealed its histone H3 was located in both the cytoplasm and nucleus of root cells. The research group that produced the YFP-mitochondria had found that the mitochondria was present in any given cell. Fluorescent images from our lab suggest the presence of tagged proteins within root cells and support the success of the fluorescent tags. Anthers of receiving flowers were removed during early development to prevent self-fertilization. Pollen grains of mature donor flowers were then introduced to isolated stigmata in order to perform the cross. After maturation, hybrid seeds will be collected for subsequent plant growth. Images of parent and offspring root cells will be used to determine the location of histone H3 and the mitochondria. We predict that after layering images of the same cell with different filters, we will find that histone H3 co-localizes with the mitochondria in living cells. The co-localization of histone H3 and mitochondria will provide more insight into the functions and structure of the histone H3 protein.
What will audience learn from your presentation?
- Fluorescent microscopy
- Plant growth and development
- Cross fertilization of A. thaliana
- Protein signaling and binding activity
- Using fluorescent tags to determine co-localization/binding activity