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Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs.

Fox PD, Haberkorn CJ, Weigel AV, Higgins JL, Akin EJ, Kennedy MJ, Krapf D, Tamkun MM - Mol. Biol. Cell (2013)

Bottom Line: In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM).By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER.Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA.

ABSTRACT
In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

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Euclidean distance mapping analysis of the sites of TfR exocytosis relative to the cER perimeter. Cumulative distribution functions (CDFs) comparing the distance of TfR exocytic sites from the cER (red) with the control case (black). Distances from the cER were determined by EDMs generated from images of the cER. (A) Summary data obtained using the entire TIRF footprint as determined from the low-level TfR fluorescence. The curve for the TfR plots the cumulative individual distances between the ER and exocytic locations. The control curve summarizes the distance to the ER for all the pixels present. The mean distance from the ER for TfR delivery was 0.25 ± 0.38 μm (mean ± SD, n = 131, from 5 cells), while the distance for the control pixels was 1.5 ± 2.6 μm, p < 0.0001. (B) Results derived from the region of the cell footprints highly enriched in cER. The mean distance from the cER for TfR delivery was 0.17 ± 0.24 μm (n = 114, from 5 cells), while the distance for the control pixels averaged 0.25 ± 0.33 μm (n = 1.1 × 107 pixels), p < 0.05, assuming equal variance. The image crop forces the two curves together because now all pixels are relatively close to the ER.
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Figure 3: Euclidean distance mapping analysis of the sites of TfR exocytosis relative to the cER perimeter. Cumulative distribution functions (CDFs) comparing the distance of TfR exocytic sites from the cER (red) with the control case (black). Distances from the cER were determined by EDMs generated from images of the cER. (A) Summary data obtained using the entire TIRF footprint as determined from the low-level TfR fluorescence. The curve for the TfR plots the cumulative individual distances between the ER and exocytic locations. The control curve summarizes the distance to the ER for all the pixels present. The mean distance from the ER for TfR delivery was 0.25 ± 0.38 μm (mean ± SD, n = 131, from 5 cells), while the distance for the control pixels was 1.5 ± 2.6 μm, p < 0.0001. (B) Results derived from the region of the cell footprints highly enriched in cER. The mean distance from the cER for TfR delivery was 0.17 ± 0.24 μm (n = 114, from 5 cells), while the distance for the control pixels averaged 0.25 ± 0.33 μm (n = 1.1 × 107 pixels), p < 0.05, assuming equal variance. The image crop forces the two curves together because now all pixels are relatively close to the ER.

Mentions: If delivery to the PM occurred at random locations, independent of the ER, the distribution of distances between delivery location and cER would be, within statistical error, indistinguishable from the distribution of the EDM. This is a consequence of the EDM being the distribution of distances to the cER as computed for the pixels in the whole membrane image. Figure 3A shows the cumulative distribution function (CDF) of the distance between delivery and the cER as compared with the CDF of the distances of random pixels as obtained when the EDM of the entire TIRF footprint was used. The separation of the experimental and control curves indicates a clear preference for exocytosis at the cER, with 75% of the delivery events occurring within 0.25 μm of the cER perimeter, while the control curve generated from all the pixels in the footprint shows 75% of these pixels were within 1.75 μm of the ER. The mean distance from the ER for TfR delivery was 0.25 ± 0.38 μm (mean ± SD, n = 131, from 5 cells), while the distance for the control pixels was 1.5 ± 2.6 μm, p < 0.0001. The control curve was generated from 1.05 × 107 pixels. However, one could argue that delivery simply preferred the general region of the basal cell surface that was enriched in cER with no direct association with the cER itself. Therefore, we repeated this EDM analysis on regions of interest highly enriched in cER (Figure 3B). The results in cER-dense regions again indicate a preference for the cER. The separation between the curves is naturally reduced because the control pixels are now on average closer to the ER in the cropped images. The mean distance from the cER for TfR delivery was 0.17 ± 0.24 μm (mean ± SD, n = 114, from 5 cells), while the distance for the control pixels was 0.25 ± 0.33 μm, p < 0.01, assuming equal variance.


Plasma membrane domains enriched in cortical endoplasmic reticulum function as membrane protein trafficking hubs.

Fox PD, Haberkorn CJ, Weigel AV, Higgins JL, Akin EJ, Kennedy MJ, Krapf D, Tamkun MM - Mol. Biol. Cell (2013)

Euclidean distance mapping analysis of the sites of TfR exocytosis relative to the cER perimeter. Cumulative distribution functions (CDFs) comparing the distance of TfR exocytic sites from the cER (red) with the control case (black). Distances from the cER were determined by EDMs generated from images of the cER. (A) Summary data obtained using the entire TIRF footprint as determined from the low-level TfR fluorescence. The curve for the TfR plots the cumulative individual distances between the ER and exocytic locations. The control curve summarizes the distance to the ER for all the pixels present. The mean distance from the ER for TfR delivery was 0.25 ± 0.38 μm (mean ± SD, n = 131, from 5 cells), while the distance for the control pixels was 1.5 ± 2.6 μm, p < 0.0001. (B) Results derived from the region of the cell footprints highly enriched in cER. The mean distance from the cER for TfR delivery was 0.17 ± 0.24 μm (n = 114, from 5 cells), while the distance for the control pixels averaged 0.25 ± 0.33 μm (n = 1.1 × 107 pixels), p < 0.05, assuming equal variance. The image crop forces the two curves together because now all pixels are relatively close to the ER.
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Related In: Results  -  Collection

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Figure 3: Euclidean distance mapping analysis of the sites of TfR exocytosis relative to the cER perimeter. Cumulative distribution functions (CDFs) comparing the distance of TfR exocytic sites from the cER (red) with the control case (black). Distances from the cER were determined by EDMs generated from images of the cER. (A) Summary data obtained using the entire TIRF footprint as determined from the low-level TfR fluorescence. The curve for the TfR plots the cumulative individual distances between the ER and exocytic locations. The control curve summarizes the distance to the ER for all the pixels present. The mean distance from the ER for TfR delivery was 0.25 ± 0.38 μm (mean ± SD, n = 131, from 5 cells), while the distance for the control pixels was 1.5 ± 2.6 μm, p < 0.0001. (B) Results derived from the region of the cell footprints highly enriched in cER. The mean distance from the cER for TfR delivery was 0.17 ± 0.24 μm (n = 114, from 5 cells), while the distance for the control pixels averaged 0.25 ± 0.33 μm (n = 1.1 × 107 pixels), p < 0.05, assuming equal variance. The image crop forces the two curves together because now all pixels are relatively close to the ER.
Mentions: If delivery to the PM occurred at random locations, independent of the ER, the distribution of distances between delivery location and cER would be, within statistical error, indistinguishable from the distribution of the EDM. This is a consequence of the EDM being the distribution of distances to the cER as computed for the pixels in the whole membrane image. Figure 3A shows the cumulative distribution function (CDF) of the distance between delivery and the cER as compared with the CDF of the distances of random pixels as obtained when the EDM of the entire TIRF footprint was used. The separation of the experimental and control curves indicates a clear preference for exocytosis at the cER, with 75% of the delivery events occurring within 0.25 μm of the cER perimeter, while the control curve generated from all the pixels in the footprint shows 75% of these pixels were within 1.75 μm of the ER. The mean distance from the ER for TfR delivery was 0.25 ± 0.38 μm (mean ± SD, n = 131, from 5 cells), while the distance for the control pixels was 1.5 ± 2.6 μm, p < 0.0001. The control curve was generated from 1.05 × 107 pixels. However, one could argue that delivery simply preferred the general region of the basal cell surface that was enriched in cER with no direct association with the cER itself. Therefore, we repeated this EDM analysis on regions of interest highly enriched in cER (Figure 3B). The results in cER-dense regions again indicate a preference for the cER. The separation between the curves is naturally reduced because the control pixels are now on average closer to the ER in the cropped images. The mean distance from the cER for TfR delivery was 0.17 ± 0.24 μm (mean ± SD, n = 114, from 5 cells), while the distance for the control pixels was 0.25 ± 0.33 μm, p < 0.01, assuming equal variance.

Bottom Line: In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM).By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER.Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA.

ABSTRACT
In mammalian cells, the cortical endoplasmic reticulum (cER) is a network of tubules and cisterns that lie in close apposition to the plasma membrane (PM). We provide evidence that PM domains enriched in underlying cER function as trafficking hubs for insertion and removal of PM proteins in HEK 293 cells. By simultaneously visualizing cER and various transmembrane protein cargoes with total internal reflectance fluorescence microscopy, we demonstrate that the majority of exocytotic delivery events for a recycled membrane protein or for a membrane protein being delivered to the PM for the first time occur at regions enriched in cER. Likewise, we observed recurring clathrin clusters and functional endocytosis of PM proteins preferentially at the cER-enriched regions. Thus the cER network serves to organize the molecular machinery for both insertion and removal of cell surface proteins, highlighting a novel role for these unique cellular microdomains in membrane trafficking.

Show MeSH
Related in: MedlinePlus