Probing Heme Trafficking Factors via Organellar Contact Points Using Genetically Encoded Fluorescent Heme Sensors

Saini, Arushi
Reddi, Amit R.
Heme is an important protein cofactor and signaling molecule that plays diverse roles in biological systems. The hydrophobicity and cytotoxicity of heme necessitates that it is transported and trafficked in a regulated manner. However, the molecules and mechanisms responsible for mediating heme trafficking remain poorly understood. Until recently, the tools to study heme in vivo did not exist, but the emergence of genetically encoded fluorescent sensors has enabled comprehensive real time analysis of heme in model organisms such as Saccharomyces cerevisiae. This study showcases a new a protocol that allows investigation of heme trafficking from its site of synthesis in the matrix side of the mitochondrial inner membrane to the outer matrix, cytosol, and nucleus over time. The method allows for the simultaneous examination of heme re-population in three cellular compartments after chemically depleting it. The study revealed that mitochondrial contact points play central roles in regulating heme availability and illuminates novel approaches to heme trafficking. These methods have the potential to be adapted to more inclusive compartmental analyses and enable a better understanding of heme trafficking which can empower innovative approaches to study infectious diseases, neurodegenerative disorders, and anemias associated with perturbations in heme cellular dynamics.
Undergraduate Thesis