Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins.
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 stimulated emission depletion 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 sterol carrier protein 2 (SCP2) occupies only a subregion of larger peroxisomes, highlighting the heterogeneous distribution of proteins even within the peroxisome. Finally, our data reveal 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 reorganization linked to changes occurring in the peroxisomes. Through the use of multicolor stimulated emission depletion microscopy, we have been able to characterize peroxisomes and their constituents to a yet unseen level of detail while maintaining a highly statistical approach, paving the way for equally complex biological studies in the future.
From the Medical Research Council Human Immunology Unit and.
Silvia
S
Galiani
Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, United Kingdom.
Dominic
D
Waithe
From the Medical Research Council Human Immunology Unit and.
Katharina
K
Reglinski
Institute of Physiological Chemistry, Systemic Biochemistry, Ruhr University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany, and.
Luis Daniel
LD
Cruz-Zaragoza
Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, United Kingdom.
Esther
E
Garcia
From the Medical Research Council Human Immunology Unit and MEMPHYS-Center for Biomembrane Physics, Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense, Denmark.
Mathias P
MP
Clausen
Institute of Physiological Chemistry, Systemic Biochemistry, Ruhr University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany, and.
Wolfgang
W
Schliebs
Institute of Physiological Chemistry, Systemic Biochemistry, Ruhr University Bochum, Universitätsstrasse 150, 44801 Bochum, Germany, and.
Ralf
R
Erdmann
From the Medical Research Council Human Immunology Unit and Wolfson Imaging Centre, Weatherall Institute of Molecular Medicine, University of Oxford, Headley Way, Oxford OX3 9DS, United Kingdom, christian.eggeling@rdm.ox.ac.uk.
Christian
C
Eggeling
D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008853:Microscopy; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*
D002352:Carrier Proteins; D008565:Membrane Proteins; C455539:PEX14 protein, human; C000614264:PEX5 protein, human; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; C072087:sterol carrier proteins
STED microscopy; membrane protein; membrane trafficking; microscopy; peroxisome; protein import; super-resolution optical microscopy