Early phases of protein aggregation probed in vivo

Cellular misfolding and aggregation of certain proteins cause neurodegenerative disorders like Huntington, Parkinson, Alzheimer and prion diseases. The diseases may be predominantly caused by “gain-of-function” proteotoxicity, with misfolded proteins and prefibrillar aggregates being probably the primary toxic agents. The formation of inclusion bodies via a number of different soluble oligomeric aggregates and unstable intermediates are common characteristics and complicate the understanding of underlying molecular principles. Folding and aggregation pathways of proteins are predominantly investigated in vitro, in environments ranging from dilute solution to cryogenic crystals. These results are extrapolated to explain protein dynamics in the living cell, generally without the ability to directly compare the cellular environment with in vitro conditions. Particularly early events of protein aggregation are highly sensitive to solvent conditions, justifying the desire to study these events directly in neurons. We developed the temperature-jump fluorescence microscope to spatio-temporally resolve fast protein folding and stability inside a single living cell. We demonstrated the method by probing a fluorescent phosphoglycerate kinase construct in a bone marrow cell. The same instrument was also used to perform the comparative in vitro measurement in dilute buffer and crowded environments. Investigating an ensemble of cells, each cell has its own unique kinetic signature that can differ substantially from the in vitro result. Variations in the cytoplasmic environment are important modulators of the protein folding and aggregation free energy landscape. We quantitate these variations with a statistical analysis of multiple cells and compare dynamics on the nm length scale with μm length scale diffusion processes.