Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins.

Abstract Membrane associated events during peroxisomal protein import processes play an essential role in peroxisome functionality. Many details of these processes are not known due to missing spatial resolution of technologies capable of investigating peroxisomes directly in the cell. Here, we present the use of super resolution optical STED microscopy to investigate with sub-60-nm resolution the heterogeneous spatial organization of the peroxisomal proteins PEX5, PEX14 and PEX11 around actively importing peroxisomes, showing distinct differences between these peroxins. Moreover, imported protein SCP2 occupies only a sub-region of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveals subpopulations of peroxisomes showing only weak colocalization between PEX14 and PEX5 or PEX11, but at the same time a clear compartmentalized organization. This compartmentalization, which was less evident in cases of strong colocalization, indicates dynamic protein re-organization linked to changes occuring in the peroxisomes. Through employing multicolour STED microscopy, we have been able to characterise peroxisomes and their constituents to a yet unseen level of detail, whilst maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.