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Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins *

View Article: PubMed Central - PubMed

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 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.

No MeSH data available.


STED imaging of selected peroxisomal and mitochondrial membrane proteins. Representative dual color confocal (upper left) and STED (lower right) images of fixed human fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 (green; always confocal) and immunostained (red) for PEX5 (upper left panel), PEX14 (lower left panel), PEX11 (upper right panel), and TOM20 (lower right panel), overviews (main panels), and zooms (insets; STED) of areas marked in the overviews are shown. Scale bars, 5 (overviews) and 1 μm (insets).
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Figure 2: STED imaging of selected peroxisomal and mitochondrial membrane proteins. Representative dual color confocal (upper left) and STED (lower right) images of fixed human fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 (green; always confocal) and immunostained (red) for PEX5 (upper left panel), PEX14 (lower left panel), PEX11 (upper right panel), and TOM20 (lower right panel), overviews (main panels), and zooms (insets; STED) of areas marked in the overviews are shown. Scale bars, 5 (overviews) and 1 μm (insets).

Mentions: For further investigations on the heterogeneity in protein organization at peroxisomes, the GFP-SCP2 was used as a reference marker to identify peroxisomes processing an active import of PTS1 proteins (note that SCP2 is a PTS1 protein). Only cells with a bright expression and peroxisomal localization of the GFP-SCP2, indicated by the characteristic punctate pattern of peroxisomal staining, were used for further analysis (Fig. 2). The GFP-SCP2 signal was acquired in the confocal modality, and the spatial distribution of the peroxisomal proteins (peroxins) PEX14, PEX5, and PEX11,and the mitochondrial outer membrane protein TOM20 in the area of the identified peroxisomes was studied with the STED microscope. PEX14 and PEX11 reside in the membrane of peroxisomes, whereas the receptor PEX5 does so only transiently, switching between cytoplasmic and membrane-bound states as it shuttles PTS1-containing proteins into the peroxisome (Fig. 1A). For studying membrane-bound PEX5, only an antibody specifically recognizing the membrane-bound conformation (17) was used.


Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins *
STED imaging of selected peroxisomal and mitochondrial membrane proteins. Representative dual color confocal (upper left) and STED (lower right) images of fixed human fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 (green; always confocal) and immunostained (red) for PEX5 (upper left panel), PEX14 (lower left panel), PEX11 (upper right panel), and TOM20 (lower right panel), overviews (main panels), and zooms (insets; STED) of areas marked in the overviews are shown. Scale bars, 5 (overviews) and 1 μm (insets).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016101&req=5

Figure 2: STED imaging of selected peroxisomal and mitochondrial membrane proteins. Representative dual color confocal (upper left) and STED (lower right) images of fixed human fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 (green; always confocal) and immunostained (red) for PEX5 (upper left panel), PEX14 (lower left panel), PEX11 (upper right panel), and TOM20 (lower right panel), overviews (main panels), and zooms (insets; STED) of areas marked in the overviews are shown. Scale bars, 5 (overviews) and 1 μm (insets).
Mentions: For further investigations on the heterogeneity in protein organization at peroxisomes, the GFP-SCP2 was used as a reference marker to identify peroxisomes processing an active import of PTS1 proteins (note that SCP2 is a PTS1 protein). Only cells with a bright expression and peroxisomal localization of the GFP-SCP2, indicated by the characteristic punctate pattern of peroxisomal staining, were used for further analysis (Fig. 2). The GFP-SCP2 signal was acquired in the confocal modality, and the spatial distribution of the peroxisomal proteins (peroxins) PEX14, PEX5, and PEX11,and the mitochondrial outer membrane protein TOM20 in the area of the identified peroxisomes was studied with the STED microscope. PEX14 and PEX11 reside in the membrane of peroxisomes, whereas the receptor PEX5 does so only transiently, switching between cytoplasmic and membrane-bound states as it shuttles PTS1-containing proteins into the peroxisome (Fig. 1A). For studying membrane-bound PEX5, only an antibody specifically recognizing the membrane-bound conformation (17) was used.

View Article: PubMed Central - PubMed

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 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.

No MeSH data available.