Limits...
Human VPS34 is required for internal vesicle formation within multivesicular endosomes.

Futter CE, Collinson LM, Backer JM, Hopkins CR - J. Cell Biol. (2001)

Bottom Line: In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation.Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs.Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom.

ABSTRACT
After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

Show MeSH

Related in: MedlinePlus

The effects of wortmannin on delivery of the EGFR to the lysosome. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with anti-EGFR gold and EGF for 1 h at 20°C, all in the absence of wortmannin. Cells were then chased for 1 h at 37°C in the absence (a) or presence of wortmannin (b and c). Note that in both the absence and the presence of wortmannin, the EGFR have, in many cases, reached the HRP-loaded lysosomes (arrows). In the presence of wortmannin, some MVBs (without HRP) are enlarged with comparatively few internal vesicles, and EGFRs on the perimeter membrane (asterisks) and some lysosomes (with HRP) are also enlarged (crosses). Bar, 0.2 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199316&req=5

fig1: The effects of wortmannin on delivery of the EGFR to the lysosome. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with anti-EGFR gold and EGF for 1 h at 20°C, all in the absence of wortmannin. Cells were then chased for 1 h at 37°C in the absence (a) or presence of wortmannin (b and c). Note that in both the absence and the presence of wortmannin, the EGFR have, in many cases, reached the HRP-loaded lysosomes (arrows). In the presence of wortmannin, some MVBs (without HRP) are enlarged with comparatively few internal vesicles, and EGFRs on the perimeter membrane (asterisks) and some lysosomes (with HRP) are also enlarged (crosses). Bar, 0.2 μm.

Mentions: We identified lysosomes by incubating cells with a pulse of HRP followed by a 2–4 h chase. As previously described (Futter et al., 1996), in HEp-2 cells this protocol labels only lysosomes. After loading lysosomes with HRP, cells were incubated at 20°C with anti-EGFR antibody conjugated to colloidal gold, which in the presence of EGF follows the normal trafficking route of the EGFR. At 20°C, EGFRs are retained in the transferrin-containing endosome, but upon transfer to 37°C activated EGFRs move into the HRP-loaded lysosome, and after 1 h at 37°C the majority of anti-EGFR gold particles are in the lysosome (Fig. 1 a). When wortmannin is included in the 37°C incubation, most MVBs (defined by the presence of anti-EGFR gold but the absence of HRP) and some lysosomes (defined by the presence of HRP) become enlarged (Fig. 1, b and c). Both MVBs and lysosomes have fewer internal vesicles and internal membranes, but it is clear that many of the anti-EGFR gold particles reach HRP-loaded lysosomes in the presence of wortmannin. Counting the number of anti-EGFR gold particles that reached HRP-filled lysosomes after 1 h at 37°C showed that delivery of EGFRs to the lysosome was only slightly inhibited (6%) by wortmannin treatment and that the majority of EGFRs (81.9%) reached the lysosome in the presence of wortmannin (Table I). In these experiments, cells were allowed to take up EGFRs at 20°C in the absence of wortmannin (to eliminate possible effects of wortmannin on internalization of EGFRs), and wortmannin was only included in the 37°C incubation. PI(3)P is likely to be depleted rapidly from cells after wortmannin addition, since the PI(3)P binding protein, EEA1, was released from endosomal membranes within 2 min of wortmannin addition at 37°C (unpublished data). However, to eliminate the possibility that some EGFRs reached the HRP-filled lysosomes before depletion of PI(3)P cells were preincubated with wortmannin at 37°C for 30 min before loading with anti-EGFR–gold/EGF at 20°C and chase at 37°C, all in the continued presence of wortmannin. Under these conditions, there was a 12% reduction in the number of EGFRs reaching the lysosome, but the majority (76.7%) of EGFRs still reached the HRP-filled lysosome.


Human VPS34 is required for internal vesicle formation within multivesicular endosomes.

Futter CE, Collinson LM, Backer JM, Hopkins CR - J. Cell Biol. (2001)

The effects of wortmannin on delivery of the EGFR to the lysosome. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with anti-EGFR gold and EGF for 1 h at 20°C, all in the absence of wortmannin. Cells were then chased for 1 h at 37°C in the absence (a) or presence of wortmannin (b and c). Note that in both the absence and the presence of wortmannin, the EGFR have, in many cases, reached the HRP-loaded lysosomes (arrows). In the presence of wortmannin, some MVBs (without HRP) are enlarged with comparatively few internal vesicles, and EGFRs on the perimeter membrane (asterisks) and some lysosomes (with HRP) are also enlarged (crosses). Bar, 0.2 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199316&req=5

fig1: The effects of wortmannin on delivery of the EGFR to the lysosome. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with anti-EGFR gold and EGF for 1 h at 20°C, all in the absence of wortmannin. Cells were then chased for 1 h at 37°C in the absence (a) or presence of wortmannin (b and c). Note that in both the absence and the presence of wortmannin, the EGFR have, in many cases, reached the HRP-loaded lysosomes (arrows). In the presence of wortmannin, some MVBs (without HRP) are enlarged with comparatively few internal vesicles, and EGFRs on the perimeter membrane (asterisks) and some lysosomes (with HRP) are also enlarged (crosses). Bar, 0.2 μm.
Mentions: We identified lysosomes by incubating cells with a pulse of HRP followed by a 2–4 h chase. As previously described (Futter et al., 1996), in HEp-2 cells this protocol labels only lysosomes. After loading lysosomes with HRP, cells were incubated at 20°C with anti-EGFR antibody conjugated to colloidal gold, which in the presence of EGF follows the normal trafficking route of the EGFR. At 20°C, EGFRs are retained in the transferrin-containing endosome, but upon transfer to 37°C activated EGFRs move into the HRP-loaded lysosome, and after 1 h at 37°C the majority of anti-EGFR gold particles are in the lysosome (Fig. 1 a). When wortmannin is included in the 37°C incubation, most MVBs (defined by the presence of anti-EGFR gold but the absence of HRP) and some lysosomes (defined by the presence of HRP) become enlarged (Fig. 1, b and c). Both MVBs and lysosomes have fewer internal vesicles and internal membranes, but it is clear that many of the anti-EGFR gold particles reach HRP-loaded lysosomes in the presence of wortmannin. Counting the number of anti-EGFR gold particles that reached HRP-filled lysosomes after 1 h at 37°C showed that delivery of EGFRs to the lysosome was only slightly inhibited (6%) by wortmannin treatment and that the majority of EGFRs (81.9%) reached the lysosome in the presence of wortmannin (Table I). In these experiments, cells were allowed to take up EGFRs at 20°C in the absence of wortmannin (to eliminate possible effects of wortmannin on internalization of EGFRs), and wortmannin was only included in the 37°C incubation. PI(3)P is likely to be depleted rapidly from cells after wortmannin addition, since the PI(3)P binding protein, EEA1, was released from endosomal membranes within 2 min of wortmannin addition at 37°C (unpublished data). However, to eliminate the possibility that some EGFRs reached the HRP-filled lysosomes before depletion of PI(3)P cells were preincubated with wortmannin at 37°C for 30 min before loading with anti-EGFR–gold/EGF at 20°C and chase at 37°C, all in the continued presence of wortmannin. Under these conditions, there was a 12% reduction in the number of EGFRs reaching the lysosome, but the majority (76.7%) of EGFRs still reached the HRP-filled lysosome.

Bottom Line: In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation.Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs.Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

View Article: PubMed Central - PubMed

Affiliation: Institute of Ophthalmology, University College London, London EC1V 9EL, United Kingdom.

ABSTRACT
After internalization from the plasma membrane, activated EGF receptors (EGFRs) are delivered to multivesicular bodies (MVBs). Within MVBs, EGFRs are removed from the perimeter membrane to internal vesicles, thereby being sorted from transferrin receptors, which recycle back to the plasma membrane. The phosphatidylinositol (PI) 3'-kinase inhibitor, wortmannin, inhibits internal vesicle formation within MVBs and causes EGFRs to remain in clusters on the perimeter membrane. Microinjection of isotype-specific inhibitory antibodies demonstrates that the PI 3'-kinase required for internal vesicle formation is hVPS34. In the presence of wortmannin, EGFRs continue to be delivered to lysosomes, showing that their removal from the recycling pathway and their delivery to lysosomes does not depend on inward vesiculation. We showed previously that tyrosine kinase-negative EGFRs fail to accumulate on internal vesicles of MVBs but are recycled rather than delivered to lysosomes. Therefore, we conclude that selection of EGFRs for inclusion on internal vesicles requires tyrosine kinase but not PI 3'-kinase activity, whereas vesicle formation requires PI 3'-kinase activity. Finally, in wortmannin-treated cells there is increased EGF-stimulated tyrosine phosphorylation when EGFRs are retained on the perimeter membrane of MVBs. Therefore, we suggest that inward vesiculation is involved directly with attenuating signal transduction.

Show MeSH
Related in: MedlinePlus