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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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Exocytosis of VSVG-ts045 occurs predominantly at the cER perimeter. (A) HEK cell transfected with YFP-VSVG-ts045 and DsRed2-ER and imaged via TRIF microscopy at the permissive temperature (32°C). Blue dots mark exocytic events that occurred ≤0.3 μm from the cER perimeter, and yellow dots mark events that occurred >0.3 μm from the cER perimeter. Of 52 exocytic events recorded in this cell, 46 of them occurred within 0.3 μm of the cER perimeter; only the first 33 events are illustrated in (A) and (B). (B) Summary of the location of YFP-VSVG-ts045 vesicle fusion over time. Same cell as in (A). Overall 84 ± 12% of exocytic events (n = 213, from 7 cells) occurred within 0.3 μm of the cER perimeter. The cER perimeter (0.3 μm) in these cells was 28 ± 8% of the area of the TIRF footprint. (C) Magnification of DsRed2-ER fluorescence from (A) overlaid with a track of vesicular movement (yellow line). Appearance of the vesicle at the PM is marked with a blue dot, and exocytosis within 0.3 μm of the cER perimeter is marked with a red +.
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Figure 6: Exocytosis of VSVG-ts045 occurs predominantly at the cER perimeter. (A) HEK cell transfected with YFP-VSVG-ts045 and DsRed2-ER and imaged via TRIF microscopy at the permissive temperature (32°C). Blue dots mark exocytic events that occurred ≤0.3 μm from the cER perimeter, and yellow dots mark events that occurred >0.3 μm from the cER perimeter. Of 52 exocytic events recorded in this cell, 46 of them occurred within 0.3 μm of the cER perimeter; only the first 33 events are illustrated in (A) and (B). (B) Summary of the location of YFP-VSVG-ts045 vesicle fusion over time. Same cell as in (A). Overall 84 ± 12% of exocytic events (n = 213, from 7 cells) occurred within 0.3 μm of the cER perimeter. The cER perimeter (0.3 μm) in these cells was 28 ± 8% of the area of the TIRF footprint. (C) Magnification of DsRed2-ER fluorescence from (A) overlaid with a track of vesicular movement (yellow line). Appearance of the vesicle at the PM is marked with a blue dot, and exocytosis within 0.3 μm of the cER perimeter is marked with a red +.

Mentions: In the case of the TfR, the measured exocytosis reflects the delivery of a recycling membrane protein, as opposed to a nascent protein trafficking directly from the Golgi to the cell surface. Thus the vesicles harboring these two different cargoes may be delivered to different microdomains on the cell surface. To examine the exocytic location of newly synthesized membrane proteins, we expressed a YFP-tagged, temperature-sensitive mutant of VSVG (YFP-VSVG-ts045), which remains unfolded in the ER at 40°C. After a switch to the permissive temperature (32°C), VSVG-ts045 can traffic through the Golgi network, where it buds off into vesicles that ultimately fuse with the PM (Presley et al., 1997; Toomre et al., 2000; Keller et al., 2001). This creates a large number of exocytotic events against a relatively nonfluorescent background, enabling the identification of the site of exocytosis even without the aid of a pH-sensitive fluorescent protein. Exocytosis was examined in HEK cells expressing both DsRed2-ER and YFP-VSVG-ts045 following the shift to the permissive temperature. Figure 6A shows a HEK cell with blue dots marking the exocytic events that occurred within 0.3 μm of the cER perimeter and yellow dots marking exocytic delivery >0.3 μm from the cER perimeter. Figure 6B summarizes the location of exocytosis over time. Out of 52 exocytic events recorded in this cell, 46 of them occurred within 0.3 μm of the cER perimeter (only the first 33 events are illustrated in Figure 6, A and B). The majority of vesicles in this cell (36 of 52) appeared first at the cER perimeter; vesicle arrival was immediately followed by fusion at the cER or transient docking prior to fusion. However, in nine of 52 of these exocytic events, the vesicle appeared first at one cER perimeter before moving to a distant cER perimeter to fuse. This behavior is illustrated in Figure 6C, which shows a vesicle track (yellow line) following arrival at the cER perimeter (blue dot). The fusion or delivery site is indicated by a red “+.” Vesicles with this behavior are also illustrated in Video S5. Together, these behaviors indicate that vesicles destined for fusion first appear in the TIRF field adjacent to cER-enriched domains. Generally, vesicles undergo fusion near the same cER domain where they first appeared. Vesicles that do not undergo fusion at the cER domain where they first appeared undergo fusion at a distant cER domain. Overall 84 ± 12% of exocytic events (n = 213, from seven cells) occurred within 0.3 μm of the cER, indicating a high degree of association between exocytosis and cER-enriched domains. The cER perimeter (0.3 μm) in these cells was 28 ± 8% of the area of the TIRF footprint, again indicating that the cER-localized delivery is not simply random.


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)

Exocytosis of VSVG-ts045 occurs predominantly at the cER perimeter. (A) HEK cell transfected with YFP-VSVG-ts045 and DsRed2-ER and imaged via TRIF microscopy at the permissive temperature (32°C). Blue dots mark exocytic events that occurred ≤0.3 μm from the cER perimeter, and yellow dots mark events that occurred >0.3 μm from the cER perimeter. Of 52 exocytic events recorded in this cell, 46 of them occurred within 0.3 μm of the cER perimeter; only the first 33 events are illustrated in (A) and (B). (B) Summary of the location of YFP-VSVG-ts045 vesicle fusion over time. Same cell as in (A). Overall 84 ± 12% of exocytic events (n = 213, from 7 cells) occurred within 0.3 μm of the cER perimeter. The cER perimeter (0.3 μm) in these cells was 28 ± 8% of the area of the TIRF footprint. (C) Magnification of DsRed2-ER fluorescence from (A) overlaid with a track of vesicular movement (yellow line). Appearance of the vesicle at the PM is marked with a blue dot, and exocytosis within 0.3 μm of the cER perimeter is marked with a red +.
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Figure 6: Exocytosis of VSVG-ts045 occurs predominantly at the cER perimeter. (A) HEK cell transfected with YFP-VSVG-ts045 and DsRed2-ER and imaged via TRIF microscopy at the permissive temperature (32°C). Blue dots mark exocytic events that occurred ≤0.3 μm from the cER perimeter, and yellow dots mark events that occurred >0.3 μm from the cER perimeter. Of 52 exocytic events recorded in this cell, 46 of them occurred within 0.3 μm of the cER perimeter; only the first 33 events are illustrated in (A) and (B). (B) Summary of the location of YFP-VSVG-ts045 vesicle fusion over time. Same cell as in (A). Overall 84 ± 12% of exocytic events (n = 213, from 7 cells) occurred within 0.3 μm of the cER perimeter. The cER perimeter (0.3 μm) in these cells was 28 ± 8% of the area of the TIRF footprint. (C) Magnification of DsRed2-ER fluorescence from (A) overlaid with a track of vesicular movement (yellow line). Appearance of the vesicle at the PM is marked with a blue dot, and exocytosis within 0.3 μm of the cER perimeter is marked with a red +.
Mentions: In the case of the TfR, the measured exocytosis reflects the delivery of a recycling membrane protein, as opposed to a nascent protein trafficking directly from the Golgi to the cell surface. Thus the vesicles harboring these two different cargoes may be delivered to different microdomains on the cell surface. To examine the exocytic location of newly synthesized membrane proteins, we expressed a YFP-tagged, temperature-sensitive mutant of VSVG (YFP-VSVG-ts045), which remains unfolded in the ER at 40°C. After a switch to the permissive temperature (32°C), VSVG-ts045 can traffic through the Golgi network, where it buds off into vesicles that ultimately fuse with the PM (Presley et al., 1997; Toomre et al., 2000; Keller et al., 2001). This creates a large number of exocytotic events against a relatively nonfluorescent background, enabling the identification of the site of exocytosis even without the aid of a pH-sensitive fluorescent protein. Exocytosis was examined in HEK cells expressing both DsRed2-ER and YFP-VSVG-ts045 following the shift to the permissive temperature. Figure 6A shows a HEK cell with blue dots marking the exocytic events that occurred within 0.3 μm of the cER perimeter and yellow dots marking exocytic delivery >0.3 μm from the cER perimeter. Figure 6B summarizes the location of exocytosis over time. Out of 52 exocytic events recorded in this cell, 46 of them occurred within 0.3 μm of the cER perimeter (only the first 33 events are illustrated in Figure 6, A and B). The majority of vesicles in this cell (36 of 52) appeared first at the cER perimeter; vesicle arrival was immediately followed by fusion at the cER or transient docking prior to fusion. However, in nine of 52 of these exocytic events, the vesicle appeared first at one cER perimeter before moving to a distant cER perimeter to fuse. This behavior is illustrated in Figure 6C, which shows a vesicle track (yellow line) following arrival at the cER perimeter (blue dot). The fusion or delivery site is indicated by a red “+.” Vesicles with this behavior are also illustrated in Video S5. Together, these behaviors indicate that vesicles destined for fusion first appear in the TIRF field adjacent to cER-enriched domains. Generally, vesicles undergo fusion near the same cER domain where they first appeared. Vesicles that do not undergo fusion at the cER domain where they first appeared undergo fusion at a distant cER domain. Overall 84 ± 12% of exocytic events (n = 213, from seven cells) occurred within 0.3 μm of the cER, indicating a high degree of association between exocytosis and cER-enriched domains. The cER perimeter (0.3 μm) in these cells was 28 ± 8% of the area of the TIRF footprint, again indicating that the cER-localized delivery is not simply random.

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