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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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Thin-section EM of cER in HEK cells. (A) EM micrograph of an ultrathin section (<100 nm) through the basal membrane (B) of the cell. Within 200 nm of the ER (ER) on the right, there are four vesicular structures (V), which are likely docked at the PM. (B) EM micrograph illustrates ER making a small contact point (black arrow) with the PM (PM) in this section. Endocytic structures (white arrows) and possible vesicular structures are within 200 nm of the ER–PM contact point. (C) This EM micrograph illustrates a typical region of cER. Here the ER runs parallel to the PM but at a distance >100 nm from the PM. The ER turns to make perpendicular contacts with PM (black arrows). (D) This micrograph illustrates a similar situation as in (C), with the ER running parallel to and making contacts with the PM. Microtubules (MT) run underneath the cER at a distance far out of the TIRF field. All micrographs were acquired at 100,000× magnification. All scale bars are 200 nm. (B = basal membrane; V = vesicular structure; PM = cross-sectioned PM; MT = microtubules; T = transverse-sectioned PM; black arrows = ER–PM contacts; white arrows = endocytic structures).
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Figure 8: Thin-section EM of cER in HEK cells. (A) EM micrograph of an ultrathin section (<100 nm) through the basal membrane (B) of the cell. Within 200 nm of the ER (ER) on the right, there are four vesicular structures (V), which are likely docked at the PM. (B) EM micrograph illustrates ER making a small contact point (black arrow) with the PM (PM) in this section. Endocytic structures (white arrows) and possible vesicular structures are within 200 nm of the ER–PM contact point. (C) This EM micrograph illustrates a typical region of cER. Here the ER runs parallel to the PM but at a distance >100 nm from the PM. The ER turns to make perpendicular contacts with PM (black arrows). (D) This micrograph illustrates a similar situation as in (C), with the ER running parallel to and making contacts with the PM. Microtubules (MT) run underneath the cER at a distance far out of the TIRF field. All micrographs were acquired at 100,000× magnification. All scale bars are 200 nm. (B = basal membrane; V = vesicular structure; PM = cross-sectioned PM; MT = microtubules; T = transverse-sectioned PM; black arrows = ER–PM contacts; white arrows = endocytic structures).

Mentions: The cER as viewed with TIRF-based light microscopy exhibited both tubular and punctate structures. At an ultrastructural level, we expected to see a similar pattern of ER running underneath PM. Our data also suggested that we would find both vesicular and endosomal structures near the cER. We analyzed the cER in glutaraldehyde-fixed HEK cells using thin-section electron microscopy (EM). To observe EM thin sections that most closely relate to our TIRF images, we began sectioning at the basal surface of the cells. Figure 8A gives us a TIRF-like snapshot of the basal membrane of the cell. Here we see ER that is likely within 100 nm of the PM, as it occupies the same plane as the sectioned basal membrane (B). Within 200 nm of the ER perimeter, we see four vesicular structures (V) that seem to be docked at the PM. These structures appear less dense in the center, suggesting they may be continuous with the extracellular space and thus in the process of exo- or endocytosis. Figure 8B is a deeper section, in which the ER makes a very small point of contact (black arrow) with the PM. We observed here both endocytic structures (white arrows) and vesicular structures in the immediate vicinity of the ER. The EM micrograph in Figure 8C illustrates a typical region of cER. Here the ER runs parallel to the PM at a distance >100 nm away, likely not within the field of view that can be imaged by TIRF microscopy. The ER turns outward to make occasional perpendicular contacts (black arrows) with the PM. Figure 8D illustrates a similar situation, with the ER running parallel to the transversely sectioned PM (T) and occasionally making close contact with the PM. Here some of the tubular ER running parallel to the PM is close enough (<100 nm) to be observed in the TIR imaging plane. This is consistent with the often tubular appearance of the cER in TIRF. Microtubules run underneath the ER at a distance far beyond TIR imaging plane. Distinct ER–PM junctions like those found in subsurface cisterns in neurons (Rosenbluth, 1962) or induced by STIM1 in response to ER Ca2+ depletion (Orci et al., 2009) were rarely observed in our HEK cells.


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)

Thin-section EM of cER in HEK cells. (A) EM micrograph of an ultrathin section (<100 nm) through the basal membrane (B) of the cell. Within 200 nm of the ER (ER) on the right, there are four vesicular structures (V), which are likely docked at the PM. (B) EM micrograph illustrates ER making a small contact point (black arrow) with the PM (PM) in this section. Endocytic structures (white arrows) and possible vesicular structures are within 200 nm of the ER–PM contact point. (C) This EM micrograph illustrates a typical region of cER. Here the ER runs parallel to the PM but at a distance >100 nm from the PM. The ER turns to make perpendicular contacts with PM (black arrows). (D) This micrograph illustrates a similar situation as in (C), with the ER running parallel to and making contacts with the PM. Microtubules (MT) run underneath the cER at a distance far out of the TIRF field. All micrographs were acquired at 100,000× magnification. All scale bars are 200 nm. (B = basal membrane; V = vesicular structure; PM = cross-sectioned PM; MT = microtubules; T = transverse-sectioned PM; black arrows = ER–PM contacts; white arrows = endocytic structures).
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Related In: Results  -  Collection

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Figure 8: Thin-section EM of cER in HEK cells. (A) EM micrograph of an ultrathin section (<100 nm) through the basal membrane (B) of the cell. Within 200 nm of the ER (ER) on the right, there are four vesicular structures (V), which are likely docked at the PM. (B) EM micrograph illustrates ER making a small contact point (black arrow) with the PM (PM) in this section. Endocytic structures (white arrows) and possible vesicular structures are within 200 nm of the ER–PM contact point. (C) This EM micrograph illustrates a typical region of cER. Here the ER runs parallel to the PM but at a distance >100 nm from the PM. The ER turns to make perpendicular contacts with PM (black arrows). (D) This micrograph illustrates a similar situation as in (C), with the ER running parallel to and making contacts with the PM. Microtubules (MT) run underneath the cER at a distance far out of the TIRF field. All micrographs were acquired at 100,000× magnification. All scale bars are 200 nm. (B = basal membrane; V = vesicular structure; PM = cross-sectioned PM; MT = microtubules; T = transverse-sectioned PM; black arrows = ER–PM contacts; white arrows = endocytic structures).
Mentions: The cER as viewed with TIRF-based light microscopy exhibited both tubular and punctate structures. At an ultrastructural level, we expected to see a similar pattern of ER running underneath PM. Our data also suggested that we would find both vesicular and endosomal structures near the cER. We analyzed the cER in glutaraldehyde-fixed HEK cells using thin-section electron microscopy (EM). To observe EM thin sections that most closely relate to our TIRF images, we began sectioning at the basal surface of the cells. Figure 8A gives us a TIRF-like snapshot of the basal membrane of the cell. Here we see ER that is likely within 100 nm of the PM, as it occupies the same plane as the sectioned basal membrane (B). Within 200 nm of the ER perimeter, we see four vesicular structures (V) that seem to be docked at the PM. These structures appear less dense in the center, suggesting they may be continuous with the extracellular space and thus in the process of exo- or endocytosis. Figure 8B is a deeper section, in which the ER makes a very small point of contact (black arrow) with the PM. We observed here both endocytic structures (white arrows) and vesicular structures in the immediate vicinity of the ER. The EM micrograph in Figure 8C illustrates a typical region of cER. Here the ER runs parallel to the PM at a distance >100 nm away, likely not within the field of view that can be imaged by TIRF microscopy. The ER turns outward to make occasional perpendicular contacts (black arrows) with the PM. Figure 8D illustrates a similar situation, with the ER running parallel to the transversely sectioned PM (T) and occasionally making close contact with the PM. Here some of the tubular ER running parallel to the PM is close enough (<100 nm) to be observed in the TIR imaging plane. This is consistent with the often tubular appearance of the cER in TIRF. Microtubules run underneath the ER at a distance far beyond TIR imaging plane. Distinct ER–PM junctions like those found in subsurface cisterns in neurons (Rosenbluth, 1962) or induced by STIM1 in response to ER Ca2+ depletion (Orci et al., 2009) were rarely observed in our HEK cells.

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