Development of a genetically encoded fluorescent probe for sensing molecular crowding in mitochondria

Molecular crowding (MC) is a phenomenon naturally occurring in all intracellular compartments due to the high concentrations of ions and biological molecules (primarily proteins) densely populating the cellular milieu. Multiple factors, including the size and broad chemical diversity of different crowders (e.g. ions, small organic compounds, polymers), as well as their complex interactions with the solvent, contribute to establish an environment with extremely variable context-depending crowding states. Furthermore, MC continuously undergoes to rapid changes, in response to both physiological processes and environmental stress, along with modifications in cellular architecture and metabolism. Given its key role in controlling protein stability, interactions and functionality, it represents a cellular parameter that needs to be reliably measured. Although FRET-based probes had already been developed and proposed as suitable and promising tools in this field, their application within the mitochondrial compartment is poorly explored. To this aim, we developed a fluorescent genetically encoded probe specifically targeted to organelle matrix in order to monitor local MC dynamics occurring in cellular models during physiological and pathological conditions.