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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.


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STED imaging of peroxisomal membrane and matrix.A, peroxisomal protein import process. A sketch of a peroxisome (left) and a close-up of a part of the peroxisomal membrane (right) with peroxisomal import receptor PEX5 (blue); membrane protein PEX14 (orange), both components of the translocation pore; PTS1 cargo protein (green); and proliferation factor PEX11 (yellow). The cargo receptor PEX5 binds PTS1-containing cargo proteins in the cytosol, directs them to the peroxisomal membrane where PEX5 becomes part of the translocation pore, the PTS1-containing cargo proteins become imported, and PEX5 is released afterward. B, representative dual color confocal (upper left) and STED (lower right) images of fixed human fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 and immunostained for PEX14 (red; Abberior STAR 600 secondary antibody) and GFP-SCP2 (green; GFP nanobooster Abberior STAR 635P); overview (main panel) and zoom (inset; STED) of area marked in the overview. Arrows, examples of pointlike and ringlike SCP2 intensity patterns surrounded by PEX14, i.e. the peroxisomal membrane. Scale bars, 5 (overview) and 1 μm (inset).
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Figure 1: STED imaging of peroxisomal membrane and matrix.A, peroxisomal protein import process. A sketch of a peroxisome (left) and a close-up of a part of the peroxisomal membrane (right) with peroxisomal import receptor PEX5 (blue); membrane protein PEX14 (orange), both components of the translocation pore; PTS1 cargo protein (green); and proliferation factor PEX11 (yellow). The cargo receptor PEX5 binds PTS1-containing cargo proteins in the cytosol, directs them to the peroxisomal membrane where PEX5 becomes part of the translocation pore, the PTS1-containing cargo proteins become imported, and PEX5 is released afterward. B, representative dual color confocal (upper left) and STED (lower right) images of fixed human fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 and immunostained for PEX14 (red; Abberior STAR 600 secondary antibody) and GFP-SCP2 (green; GFP nanobooster Abberior STAR 635P); overview (main panel) and zoom (inset; STED) of area marked in the overview. Arrows, examples of pointlike and ringlike SCP2 intensity patterns surrounded by PEX14, i.e. the peroxisomal membrane. Scale bars, 5 (overview) and 1 μm (inset).

Mentions: Peroxisomes (see Fig. 1A) are small, membrane-enclosed organelles that fulfill many different functions of eukaryotic cells, including a variety of metabolic reactions (1). Peroxisomes contain a diverse collection of densely packed enzymes whose composition is largely dependent on the host organism, cell type, and tissue (2, 3). Peroxisomal function and protein composition, at a given time, are largely dependent on changes occurring in the external environment to which it responds dynamically (4). In human cells, peroxisomes are involved in the β-oxidation of fatty acids, reactive oxygen species detoxification, and biosynthesis of different lipids, including plasmalogen, the main component of the myelin sheath (5). Because of its crucial roles in human metabolism, defects in peroxisomal function can cause many severe diseases (6, 7). Only a fundamental understanding of peroxisomal biogenesis and assembly will help identify ways to control these diseases, leading to novel therapeutic strategies.


Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins *
STED imaging of peroxisomal membrane and matrix.A, peroxisomal protein import process. A sketch of a peroxisome (left) and a close-up of a part of the peroxisomal membrane (right) with peroxisomal import receptor PEX5 (blue); membrane protein PEX14 (orange), both components of the translocation pore; PTS1 cargo protein (green); and proliferation factor PEX11 (yellow). The cargo receptor PEX5 binds PTS1-containing cargo proteins in the cytosol, directs them to the peroxisomal membrane where PEX5 becomes part of the translocation pore, the PTS1-containing cargo proteins become imported, and PEX5 is released afterward. B, representative dual color confocal (upper left) and STED (lower right) images of fixed human fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 and immunostained for PEX14 (red; Abberior STAR 600 secondary antibody) and GFP-SCP2 (green; GFP nanobooster Abberior STAR 635P); overview (main panel) and zoom (inset; STED) of area marked in the overview. Arrows, examples of pointlike and ringlike SCP2 intensity patterns surrounded by PEX14, i.e. the peroxisomal membrane. Scale bars, 5 (overview) and 1 μm (inset).
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Figure 1: STED imaging of peroxisomal membrane and matrix.A, peroxisomal protein import process. A sketch of a peroxisome (left) and a close-up of a part of the peroxisomal membrane (right) with peroxisomal import receptor PEX5 (blue); membrane protein PEX14 (orange), both components of the translocation pore; PTS1 cargo protein (green); and proliferation factor PEX11 (yellow). The cargo receptor PEX5 binds PTS1-containing cargo proteins in the cytosol, directs them to the peroxisomal membrane where PEX5 becomes part of the translocation pore, the PTS1-containing cargo proteins become imported, and PEX5 is released afterward. B, representative dual color confocal (upper left) and STED (lower right) images of fixed human fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 and immunostained for PEX14 (red; Abberior STAR 600 secondary antibody) and GFP-SCP2 (green; GFP nanobooster Abberior STAR 635P); overview (main panel) and zoom (inset; STED) of area marked in the overview. Arrows, examples of pointlike and ringlike SCP2 intensity patterns surrounded by PEX14, i.e. the peroxisomal membrane. Scale bars, 5 (overview) and 1 μm (inset).
Mentions: Peroxisomes (see Fig. 1A) are small, membrane-enclosed organelles that fulfill many different functions of eukaryotic cells, including a variety of metabolic reactions (1). Peroxisomes contain a diverse collection of densely packed enzymes whose composition is largely dependent on the host organism, cell type, and tissue (2, 3). Peroxisomal function and protein composition, at a given time, are largely dependent on changes occurring in the external environment to which it responds dynamically (4). In human cells, peroxisomes are involved in the β-oxidation of fatty acids, reactive oxygen species detoxification, and biosynthesis of different lipids, including plasmalogen, the main component of the myelin sheath (5). Because of its crucial roles in human metabolism, defects in peroxisomal function can cause many severe diseases (6, 7). Only a fundamental understanding of peroxisomal biogenesis and assembly will help identify ways to control these diseases, leading to novel therapeutic strategies.

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.


Related in: MedlinePlus