UNIVERSITY OF COLOGNE

Synopsis

Redox reactions are at the center of most cellular processes: they are at the mechanistic heart of metabolic pathways, they contribute to proteostasis e.g. by the introduction and removal of disulfide bonds, and they drive the production of reactive oxygen species (ROS), which - with their Janus-faced character of being on the one hand toxic and on the other essential for signaling - impact heavily on cellular physiology. A number of diseases have been directly linked with dysregulated redox homeostasis, including cancer, neurological disorders, cardiovascular diseases, obesity and metabolic diseases, as well as aging. 

In this practical course, you will explore concepts of redox biochemistry, its connection to metabolism and modern tools to analyse redox processes in cells and in vitro. The course comprises three different approaches:

1. Saccharomyces cerevisiae – baker’s yeast: A highly versatile, genetically malleable system that allows us to interrogate components of the cellular redox machinery with straightforward and fast experimental techniques. Working with yeast provides you with the ability to follow your own research interest. Don’t just follow the protocol but design your own experiments!

2. In vitro experimentation: Evolution has found surprising and efficient ways to allow cells to adapt to their environment. We will investigate redox switches in metabolic enzymes that re-shape metabolite fluxes in order to provide the cell with the resources they need to survive challenges such as excess ROS.

3. Mammalian tissue culture and differentiation: The various cells of our bodies generate energy in different ways – neurons depend on oxidative phosphorylation, heart muscle cells rely on fatty acid oxidation, whereas stem cells predominantly rely on glycolysis. Consequently, cellular metabolism and in particular, redox metabolism undergoes dramatic re-programming during differentiation.

Methodology

• baker’s yeast and mammalian tissue culture
• in vitro biochemical assays
• assessment of cellular behavior upon redox stress (proliferation, cell death)
• genetically encoded fluorescent proteins as tools to measure small redox molecules
• redox stress response pathway analysis in cells
• experiments to determine protein redox states in intact cells and in vitro
• redox processes during cellular differentiation

Phenotypic assays in yeast can probe various aspects of tolerance to redox stress. Drop dilution assays can be used to measure growth under oxidative stress applied via solid media, growth curves track changes in growth rate, Halo assays evaluate sensitivity to oxidative agents by measuring inhibition zones around applied ROS sources and  survival assays evaluate resistance to acute stresses. Genetically different yeast strains can be compared to understand how genetic variations influence oxidative stress tolerance.

Genetically encoded redox sensors like roGFP2, Peredox, and NapStar monitor redox couples (e.g., GSH:GSSG, NADPH:NADP) and hydrogen peroxide (H₂O₂) levels in living cells, providing real-time insights into cellular health, stress, and disease mechanisms. These sensors can be targeted to specific cellular compartments like mitochondria to study localized redox dynamics. We will use these sensors to track redox dynamics in yeast culture and mammalian cells.

In vitro enzyme kinetics will allow us to understand the biochemistry of redox switches. We will specifically look at the "GAPDH redox switch", which shifts GAPDH’s function from a glycolytic enzyme to a safeguard of reductive capacity.  The activity of the wild-type enzyme as well as that of mutant proteins that differ in terms of their sensitivity to oxidative modification will be assessed. Furthermore, we will explore the role of this switch in cellular systems.

Models of cellular differentiation can serve to study the maturation of mitochondria, alterations in cellular metabolism and cell morphological transitions. You will get hands-on experience with biochemical and imaging techniques that allow us to ask questions like: Which cellular processes accompany the differentiation of a myoblast precursor into a mature muscle cell? Is this straight path or are different directions possible in this process? What happens to the state of individual redox couples?