Biophysical Imaging

Peroxisomes are cell organelles with a size of 0.3-0.6 μm in mammalian cells, fulfilling many different functions like the oxidation of fatty acids, detoxification of reactive oxygen species as well as the biosynthesis of different lipids. The import of proteins into peroxisomes is unique since peroxisomes import already folded and even oligomerized proteins from the cytosol. Any malfunction on the import process will impair the function of peroxisomes and lead to severe related diseases. Most proteins destined for the peroxisomal matrix are characterized by a PTS1 (Peroxisomal Targeting Sequence 1) that is recognized by the peroxisomal import receptor PEX5, which binds them in the cytosol and directs them to the peroxisomal membrane. We here present our extended study to characterize the molecular diffusion and interaction dynamics of PEX5 and PTS1 cargo proteins in the cytosol of live human fibroblasts using Fluorescence Correlation Spectroscopy (FCS) in combination with multi-colour (FCCS) and super-resolution (STED-FCS) detection. We observed an unexpectedly slow diffusion of PEX5 with and without cargo (2-times slower than for similarly sized cytosolic proteins). A deep analysis on different variants of PEX5 link this slowed down diffusion to the protein’s N-Terminal part, which is known to be involved in the in integration of PEX5 into the peroxisomal membrane. To further characterize the dynamics of PEX5 in the cytosol, we implemented aberration corrections using adaptive optics, which helped us to precisely determine the diffusion mode of cytosolic PEX5.