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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.


Compartmentalization of peroxisomal membrane proteins.A, anticorrelation between compartmentalization and colocalization of PEX5 versus PEX14 (left) and PEX11 versus PEX14 (right). Normalized frequency plots of value pairs of average nearest distance between maxima within the intensity distribution (as a measure of compartmentalization) and Pearson's colocalization test from single peroxisomes (5439 for PEX5-PEX14 and 6178 for PEX11-PEX14) are shown. B, representative patterns of the intensity distribution in the circular regions around single peroxisomes from PEX5 and PEX14 (upper two panels) and PEX11 and PEX14 (lower two panels) derived from dual color STED images and ordered from the highest to the lowest Pearson's test colocalization value (left to right as labeled). Only the extreme cases of high and low colocalization are shown; medium cases are left out (white boxes; for full sequences see supplemental Fig. 2). C, representative dual color STED images of PEX5 (green) and PEX14 (red) (upper panels) as well as PEX11 (green) and PEX14 (red) (lower panels) for strong colocalization (Pearson's test values >0.6) and low compartmentalization of both proteins (left panels) and for low colocalization (Pearson's test values <0.4) and high compartmentalization of both proteins (right panels). Scale bars, 200 nm.
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Figure 7: Compartmentalization of peroxisomal membrane proteins.A, anticorrelation between compartmentalization and colocalization of PEX5 versus PEX14 (left) and PEX11 versus PEX14 (right). Normalized frequency plots of value pairs of average nearest distance between maxima within the intensity distribution (as a measure of compartmentalization) and Pearson's colocalization test from single peroxisomes (5439 for PEX5-PEX14 and 6178 for PEX11-PEX14) are shown. B, representative patterns of the intensity distribution in the circular regions around single peroxisomes from PEX5 and PEX14 (upper two panels) and PEX11 and PEX14 (lower two panels) derived from dual color STED images and ordered from the highest to the lowest Pearson's test colocalization value (left to right as labeled). Only the extreme cases of high and low colocalization are shown; medium cases are left out (white boxes; for full sequences see supplemental Fig. 2). C, representative dual color STED images of PEX5 (green) and PEX14 (red) (upper panels) as well as PEX11 (green) and PEX14 (red) (lower panels) for strong colocalization (Pearson's test values >0.6) and low compartmentalization of both proteins (left panels) and for low colocalization (Pearson's test values <0.4) and high compartmentalization of both proteins (right panels). Scale bars, 200 nm.

Mentions: To further highlight the compartmentalized spatial distribution of the proteins at the peroxisomal membrane, we developed a more rigorous image analysis regime to examine the trends in the data. This resulted from close inspection of the two-color STED images where although there was a tendency toward a compartmentalized distribution of the proteins at the same time there was also an indication that there was a significant number of peroxisomes with a low level of colocalization, for example PEX5 and PEX14 or PEX11 and PEX14 (Fig. 5A). This is quantitatively shown by a fraction of peroxisomes exhibiting a Pearson's coefficient that tended toward zero (Fig. 5B). To confirm this tendency, we expanded our colocalization analysis. As mentioned, the spatial intensity distribution of each protein staining at a single peroxisome is usually strongly compartmentalized and characterized by multiple maxima. We determined the locations of the maxima within the intensity distribution of each protein staining. We plotted the average interchannel distance between those maxima for a region against the corresponding Pearson's correlation value both for PEX5/PEX14 and PEX11/PEX14, resulting in a reciprocal relationship between both parameters. These data show that large distances between maxima (or strongly compartmentalized protein distributions) are characterized by a low colocalization with other proteins (Fig. 7 and supplemental Fig. 2). Representative protein distribution patterns with high and low Pearson's correlation values are depicted in Fig. 7C. Peroxisomes characterized by high colocalization between PEX5 and PEX14 or PEX11 and PEX14 appear more circular and less compartmentalized compared with the low colocalization cases where multiple maxima are present. Furthermore, note the slightly stronger colocalization between PEX5 and PEX14 compared with PEX11 and PEX14; however, both comparisons still show the same tendency between colocalization and compartmentalization.


Super-resolution Microscopy Reveals Compartmentalization of Peroxisomal Membrane Proteins *
Compartmentalization of peroxisomal membrane proteins.A, anticorrelation between compartmentalization and colocalization of PEX5 versus PEX14 (left) and PEX11 versus PEX14 (right). Normalized frequency plots of value pairs of average nearest distance between maxima within the intensity distribution (as a measure of compartmentalization) and Pearson's colocalization test from single peroxisomes (5439 for PEX5-PEX14 and 6178 for PEX11-PEX14) are shown. B, representative patterns of the intensity distribution in the circular regions around single peroxisomes from PEX5 and PEX14 (upper two panels) and PEX11 and PEX14 (lower two panels) derived from dual color STED images and ordered from the highest to the lowest Pearson's test colocalization value (left to right as labeled). Only the extreme cases of high and low colocalization are shown; medium cases are left out (white boxes; for full sequences see supplemental Fig. 2). C, representative dual color STED images of PEX5 (green) and PEX14 (red) (upper panels) as well as PEX11 (green) and PEX14 (red) (lower panels) for strong colocalization (Pearson's test values >0.6) and low compartmentalization of both proteins (left panels) and for low colocalization (Pearson's test values <0.4) and high compartmentalization of both proteins (right panels). Scale bars, 200 nm.
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Figure 7: Compartmentalization of peroxisomal membrane proteins.A, anticorrelation between compartmentalization and colocalization of PEX5 versus PEX14 (left) and PEX11 versus PEX14 (right). Normalized frequency plots of value pairs of average nearest distance between maxima within the intensity distribution (as a measure of compartmentalization) and Pearson's colocalization test from single peroxisomes (5439 for PEX5-PEX14 and 6178 for PEX11-PEX14) are shown. B, representative patterns of the intensity distribution in the circular regions around single peroxisomes from PEX5 and PEX14 (upper two panels) and PEX11 and PEX14 (lower two panels) derived from dual color STED images and ordered from the highest to the lowest Pearson's test colocalization value (left to right as labeled). Only the extreme cases of high and low colocalization are shown; medium cases are left out (white boxes; for full sequences see supplemental Fig. 2). C, representative dual color STED images of PEX5 (green) and PEX14 (red) (upper panels) as well as PEX11 (green) and PEX14 (red) (lower panels) for strong colocalization (Pearson's test values >0.6) and low compartmentalization of both proteins (left panels) and for low colocalization (Pearson's test values <0.4) and high compartmentalization of both proteins (right panels). Scale bars, 200 nm.
Mentions: To further highlight the compartmentalized spatial distribution of the proteins at the peroxisomal membrane, we developed a more rigorous image analysis regime to examine the trends in the data. This resulted from close inspection of the two-color STED images where although there was a tendency toward a compartmentalized distribution of the proteins at the same time there was also an indication that there was a significant number of peroxisomes with a low level of colocalization, for example PEX5 and PEX14 or PEX11 and PEX14 (Fig. 5A). This is quantitatively shown by a fraction of peroxisomes exhibiting a Pearson's coefficient that tended toward zero (Fig. 5B). To confirm this tendency, we expanded our colocalization analysis. As mentioned, the spatial intensity distribution of each protein staining at a single peroxisome is usually strongly compartmentalized and characterized by multiple maxima. We determined the locations of the maxima within the intensity distribution of each protein staining. We plotted the average interchannel distance between those maxima for a region against the corresponding Pearson's correlation value both for PEX5/PEX14 and PEX11/PEX14, resulting in a reciprocal relationship between both parameters. These data show that large distances between maxima (or strongly compartmentalized protein distributions) are characterized by a low colocalization with other proteins (Fig. 7 and supplemental Fig. 2). Representative protein distribution patterns with high and low Pearson's correlation values are depicted in Fig. 7C. Peroxisomes characterized by high colocalization between PEX5 and PEX14 or PEX11 and PEX14 appear more circular and less compartmentalized compared with the low colocalization cases where multiple maxima are present. Furthermore, note the slightly stronger colocalization between PEX5 and PEX14 compared with PEX11 and PEX14; however, both comparisons still show the same tendency between colocalization and compartmentalization.

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.