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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.


Colocalization study of proteins at peroxisomes.A, representative confocal (upper left) and STED (lower right) images of fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 (blue; only confocal and only in top panels), fixed, and immunolabeled for PEX14 (red) and PEX5 (green) (left), PEX14 (red) and PEX11 (green) (middle), and TOM20 (green) and PEX5 (red) (right). Overviews (upper panels) and zooms (insets) of regions marked in overviews are shown. Scale bars, 5 (overviews) and 1 μm (insets). B, Pearson's test colocalization analysis of different peroxisomal proteins. Upper left panel, scheme of the analysis procedure. Circular patches surrounding GFP-SCP2 signal (peroxisomal regions of interest (ROI)) and non-GFP-SCP2 signal (random regions of interest) were selected, and colocalization values were calculated using a pixel-wise Pearson's test. Upper right and lower panels, frequency histogram of Pearson's test values (−1, opposing colocalization; 0, no colocalization; 1, maximum colocalization) for PEX5 versus PEX14 (upper right), PEX11 versus PEX14 (lower left), and TOM20 versus PEX5 (lower right) and for random regions of interest (dashed lines), peroxisomal (Perox) regions of interest (solid lines), and flipped (dotted lines) (number of data points: PEX5-PEX14, 5439; PEX11-PEX14, 6178; TOM20-PEX5, 4305).
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Figure 5: Colocalization study of proteins at peroxisomes.A, representative confocal (upper left) and STED (lower right) images of fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 (blue; only confocal and only in top panels), fixed, and immunolabeled for PEX14 (red) and PEX5 (green) (left), PEX14 (red) and PEX11 (green) (middle), and TOM20 (green) and PEX5 (red) (right). Overviews (upper panels) and zooms (insets) of regions marked in overviews are shown. Scale bars, 5 (overviews) and 1 μm (insets). B, Pearson's test colocalization analysis of different peroxisomal proteins. Upper left panel, scheme of the analysis procedure. Circular patches surrounding GFP-SCP2 signal (peroxisomal regions of interest (ROI)) and non-GFP-SCP2 signal (random regions of interest) were selected, and colocalization values were calculated using a pixel-wise Pearson's test. Upper right and lower panels, frequency histogram of Pearson's test values (−1, opposing colocalization; 0, no colocalization; 1, maximum colocalization) for PEX5 versus PEX14 (upper right), PEX11 versus PEX14 (lower left), and TOM20 versus PEX5 (lower right) and for random regions of interest (dashed lines), peroxisomal (Perox) regions of interest (solid lines), and flipped (dotted lines) (number of data points: PEX5-PEX14, 5439; PEX11-PEX14, 6178; TOM20-PEX5, 4305).

Mentions: Through using dual color STED imaging, we were able to relate the spatial staining of more than one protein simultaneously with a 60-nm spatial resolution. Fig. 5A shows representative dual color STED images from our study on peroxisomal proteins. We analyzed three different conditions, each a different pairing of immunolabeled protein, in fixed fibroblasts: PEX5 versus PEX14, PEX11 versus PEX14, and TOM20 versus PEX5. As before, the signal of the GFP-SCP2 was additionally recorded in confocal mode as a means of identifying actively importing peroxisomes. We analyzed more than 30 images for each condition acquired on at least 10 different cells from at least three separate samples. Our analysis compares the spatial distribution of the proteins within each peroxisomal region first by performing a pixel-wise Pearson's colocalization test to quantify the colocalization of PEX5/PEX14, PEX11/PEX14, and TOM20/PEX5 (values of 1 indicate complete colocalization, 0 indicates no colocalization, and −1 indicates opposing colocalization). Fig. 5B shows the histogram of colocalization values over the entire peroxisome population (solid line). PEX5/PEX14 show the highest colocalization with a median Pearson's value of 0.55 compared with 0.45 for PEX11/PEX14 and 0.22 for TOM20/PEX5.


Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins *
Colocalization study of proteins at peroxisomes.A, representative confocal (upper left) and STED (lower right) images of fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 (blue; only confocal and only in top panels), fixed, and immunolabeled for PEX14 (red) and PEX5 (green) (left), PEX14 (red) and PEX11 (green) (middle), and TOM20 (green) and PEX5 (red) (right). Overviews (upper panels) and zooms (insets) of regions marked in overviews are shown. Scale bars, 5 (overviews) and 1 μm (insets). B, Pearson's test colocalization analysis of different peroxisomal proteins. Upper left panel, scheme of the analysis procedure. Circular patches surrounding GFP-SCP2 signal (peroxisomal regions of interest (ROI)) and non-GFP-SCP2 signal (random regions of interest) were selected, and colocalization values were calculated using a pixel-wise Pearson's test. Upper right and lower panels, frequency histogram of Pearson's test values (−1, opposing colocalization; 0, no colocalization; 1, maximum colocalization) for PEX5 versus PEX14 (upper right), PEX11 versus PEX14 (lower left), and TOM20 versus PEX5 (lower right) and for random regions of interest (dashed lines), peroxisomal (Perox) regions of interest (solid lines), and flipped (dotted lines) (number of data points: PEX5-PEX14, 5439; PEX11-PEX14, 6178; TOM20-PEX5, 4305).
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Figure 5: Colocalization study of proteins at peroxisomes.A, representative confocal (upper left) and STED (lower right) images of fibroblast cells transfected with the peroxisomal matrix marker GFP-SCP2 (blue; only confocal and only in top panels), fixed, and immunolabeled for PEX14 (red) and PEX5 (green) (left), PEX14 (red) and PEX11 (green) (middle), and TOM20 (green) and PEX5 (red) (right). Overviews (upper panels) and zooms (insets) of regions marked in overviews are shown. Scale bars, 5 (overviews) and 1 μm (insets). B, Pearson's test colocalization analysis of different peroxisomal proteins. Upper left panel, scheme of the analysis procedure. Circular patches surrounding GFP-SCP2 signal (peroxisomal regions of interest (ROI)) and non-GFP-SCP2 signal (random regions of interest) were selected, and colocalization values were calculated using a pixel-wise Pearson's test. Upper right and lower panels, frequency histogram of Pearson's test values (−1, opposing colocalization; 0, no colocalization; 1, maximum colocalization) for PEX5 versus PEX14 (upper right), PEX11 versus PEX14 (lower left), and TOM20 versus PEX5 (lower right) and for random regions of interest (dashed lines), peroxisomal (Perox) regions of interest (solid lines), and flipped (dotted lines) (number of data points: PEX5-PEX14, 5439; PEX11-PEX14, 6178; TOM20-PEX5, 4305).
Mentions: Through using dual color STED imaging, we were able to relate the spatial staining of more than one protein simultaneously with a 60-nm spatial resolution. Fig. 5A shows representative dual color STED images from our study on peroxisomal proteins. We analyzed three different conditions, each a different pairing of immunolabeled protein, in fixed fibroblasts: PEX5 versus PEX14, PEX11 versus PEX14, and TOM20 versus PEX5. As before, the signal of the GFP-SCP2 was additionally recorded in confocal mode as a means of identifying actively importing peroxisomes. We analyzed more than 30 images for each condition acquired on at least 10 different cells from at least three separate samples. Our analysis compares the spatial distribution of the proteins within each peroxisomal region first by performing a pixel-wise Pearson's colocalization test to quantify the colocalization of PEX5/PEX14, PEX11/PEX14, and TOM20/PEX5 (values of 1 indicate complete colocalization, 0 indicates no colocalization, and −1 indicates opposing colocalization). Fig. 5B shows the histogram of colocalization values over the entire peroxisome population (solid line). PEX5/PEX14 show the highest colocalization with a median Pearson's value of 0.55 compared with 0.45 for PEX11/PEX14 and 0.22 for TOM20/PEX5.

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.