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Rapid transport of internalized P-selectin to late endosomes and the TGN: roles in regulating cell surface expression and recycling to secretory granules.

Straley KS, Green SA - J. Cell Biol. (2000)

Bottom Line: Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles.We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins.This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, Virginia 22908-0732, USA.

ABSTRACT
Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles. We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins. P-selectin, a transmembrane cell adhesion protein involved in inflammation, is sorted from recycling cell surface receptors (e.g., low density lipoprotein [LDL] receptor) in endosomes, and is transported from the cell surface to the TGN with a half-time of 20-25 min, six to seven times faster than LDL receptor. Native P-selectin colocalizes with LDL, which is efficiently transported to lysosomes, for 20 min after internalization, but a deletion mutant deficient in endosomal sorting activity rapidly separates from the LDL pathway. Thus, P-selectin is sorted from LDL receptor in early endosomes, driving P-selectin rapidly into late endosomes. P-selectin then recycles to the TGN as efficiently as other receptors. Thus, the primary effect of early endosomal sorting of P-selectin is its rapid delivery to the TGN, with rapid turnover in lysosomes a secondary effect of frequent passage through late endosomes. This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors.

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Quantitation of S12 antibody and LDL colocalization. Images such as those shown in Fig. 5 were used to determine the number of LDL-containing structures that also contained S12 antibody. Images containing a total of 15–21 cells, with an average of 926 LDL-positive vesicles, were analyzed for each data point. Numbers represent the percentage of all LDL-positive vesicles that also contained S12 antibody for the entire sample. Similar results were obtained in two independent experiments.
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Figure 6: Quantitation of S12 antibody and LDL colocalization. Images such as those shown in Fig. 5 were used to determine the number of LDL-containing structures that also contained S12 antibody. Images containing a total of 15–21 cells, with an average of 926 LDL-positive vesicles, were analyzed for each data point. Numbers represent the percentage of all LDL-positive vesicles that also contained S12 antibody for the entire sample. Similar results were obtained in two independent experiments.

Mentions: To determine whether P-selectin was delivered to the TGN via late endosomes or directly from early endosomes, the transport pathway of internalized P-selectin was compared with that of internalized LDL, using mAb S12 to track internalized P-selectin. This antibody dissociated from P-selectin with a half-time of 156 min at 37°C and pH 5.5, conditions typical of late endosomes (Mellman 1996; and see online supplemental Figure S4, http//:www.jcb.org/cgi/content/full/151/1/107/DC1). CHO cells expressing P-selectin, or P-selectin-ΔC1, which is deficient in endosomal sorting (Green et al. 1994), were incubated for 5 min with DiI-LDL and Alexa 488-labeled mAb S12. Cells were recultured for up to 60 min before fixation. Epifluorescence images were collected and analyzed after digital deconvolution (see Materials and Methods). In both cell lines, S12 antibody was observed in numerous punctate structures throughout the cytoplasm, and in a concentration of structures at one pole of the nucleus, visible in epifluorescence, but not in the deconvolved images (our unpublished observation). The juxtanuclear staining was not prominent after 5 min of chase, but the pattern of S12 labeling did not change or diminish significantly between 10 and 60 min of chase. At early chase times, LDL was observed in a smaller number of structures, many of which contained S12 antibody, in both native P-selectin (Fig. 5 A) and P-selectin-ΔC1 cells (Fig. 5 B). After 20 min of reculture, few LDL-labeled structures in P-selectin-ΔC1 cells contained detectable levels of S12 antibody (Fig. 5 D), whereas in cells expressing native P-selectin, double-labeled structures persisted (Fig. 5 C). After 40 min of reculture, few double-labeled structures were seen in either cell line (Fig. 5E and Fig. F). Quantitation of double labeling on deconvolved images showed that LDL separated rapidly from S12 antibody in P-selectin-ΔC1 cells, whereas little separation occurred in cells expressing native P-selectin for 20 min (Fig. 6). The clear difference between the two cell lines indicates that binding of S12 antibody does not divert P-selectin-ΔC1 to the lysosomal pathway, which can occur when using cross-linking antibodies (Mellman and Plutner 1984; Gartung et al. 1985). This result indicates that native P-selectin, unlike the efficiently recycled P-selectin-ΔC1 mutant, becomes concentrated in LDL-containing carrier vesicles (maturing endosomes) with relatively high efficiency soon after internalization, indicating selective sorting in early endosomes for efficient delivery to late endosomes en route to the TGN.


Rapid transport of internalized P-selectin to late endosomes and the TGN: roles in regulating cell surface expression and recycling to secretory granules.

Straley KS, Green SA - J. Cell Biol. (2000)

Quantitation of S12 antibody and LDL colocalization. Images such as those shown in Fig. 5 were used to determine the number of LDL-containing structures that also contained S12 antibody. Images containing a total of 15–21 cells, with an average of 926 LDL-positive vesicles, were analyzed for each data point. Numbers represent the percentage of all LDL-positive vesicles that also contained S12 antibody for the entire sample. Similar results were obtained in two independent experiments.
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Related In: Results  -  Collection

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Figure 6: Quantitation of S12 antibody and LDL colocalization. Images such as those shown in Fig. 5 were used to determine the number of LDL-containing structures that also contained S12 antibody. Images containing a total of 15–21 cells, with an average of 926 LDL-positive vesicles, were analyzed for each data point. Numbers represent the percentage of all LDL-positive vesicles that also contained S12 antibody for the entire sample. Similar results were obtained in two independent experiments.
Mentions: To determine whether P-selectin was delivered to the TGN via late endosomes or directly from early endosomes, the transport pathway of internalized P-selectin was compared with that of internalized LDL, using mAb S12 to track internalized P-selectin. This antibody dissociated from P-selectin with a half-time of 156 min at 37°C and pH 5.5, conditions typical of late endosomes (Mellman 1996; and see online supplemental Figure S4, http//:www.jcb.org/cgi/content/full/151/1/107/DC1). CHO cells expressing P-selectin, or P-selectin-ΔC1, which is deficient in endosomal sorting (Green et al. 1994), were incubated for 5 min with DiI-LDL and Alexa 488-labeled mAb S12. Cells were recultured for up to 60 min before fixation. Epifluorescence images were collected and analyzed after digital deconvolution (see Materials and Methods). In both cell lines, S12 antibody was observed in numerous punctate structures throughout the cytoplasm, and in a concentration of structures at one pole of the nucleus, visible in epifluorescence, but not in the deconvolved images (our unpublished observation). The juxtanuclear staining was not prominent after 5 min of chase, but the pattern of S12 labeling did not change or diminish significantly between 10 and 60 min of chase. At early chase times, LDL was observed in a smaller number of structures, many of which contained S12 antibody, in both native P-selectin (Fig. 5 A) and P-selectin-ΔC1 cells (Fig. 5 B). After 20 min of reculture, few LDL-labeled structures in P-selectin-ΔC1 cells contained detectable levels of S12 antibody (Fig. 5 D), whereas in cells expressing native P-selectin, double-labeled structures persisted (Fig. 5 C). After 40 min of reculture, few double-labeled structures were seen in either cell line (Fig. 5E and Fig. F). Quantitation of double labeling on deconvolved images showed that LDL separated rapidly from S12 antibody in P-selectin-ΔC1 cells, whereas little separation occurred in cells expressing native P-selectin for 20 min (Fig. 6). The clear difference between the two cell lines indicates that binding of S12 antibody does not divert P-selectin-ΔC1 to the lysosomal pathway, which can occur when using cross-linking antibodies (Mellman and Plutner 1984; Gartung et al. 1985). This result indicates that native P-selectin, unlike the efficiently recycled P-selectin-ΔC1 mutant, becomes concentrated in LDL-containing carrier vesicles (maturing endosomes) with relatively high efficiency soon after internalization, indicating selective sorting in early endosomes for efficient delivery to late endosomes en route to the TGN.

Bottom Line: Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles.We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins.This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia Health System, School of Medicine, Charlottesville, Virginia 22908-0732, USA.

ABSTRACT
Prior studies on receptor recycling through late endosomes and the TGN have suggested that such traffic may be largely limited to specialized proteins that reside in these organelles. We present evidence that efficient recycling along this pathway is functionally important for nonresident proteins. P-selectin, a transmembrane cell adhesion protein involved in inflammation, is sorted from recycling cell surface receptors (e.g., low density lipoprotein [LDL] receptor) in endosomes, and is transported from the cell surface to the TGN with a half-time of 20-25 min, six to seven times faster than LDL receptor. Native P-selectin colocalizes with LDL, which is efficiently transported to lysosomes, for 20 min after internalization, but a deletion mutant deficient in endosomal sorting activity rapidly separates from the LDL pathway. Thus, P-selectin is sorted from LDL receptor in early endosomes, driving P-selectin rapidly into late endosomes. P-selectin then recycles to the TGN as efficiently as other receptors. Thus, the primary effect of early endosomal sorting of P-selectin is its rapid delivery to the TGN, with rapid turnover in lysosomes a secondary effect of frequent passage through late endosomes. This endosomal sorting event provides a mechanism for efficiently recycling secretory granule membrane proteins and, more generally, for downregulating cell surface receptors.

Show MeSH
Related in: MedlinePlus