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 microinjection with anti–PI 3′-kinase antibodies on inward vesiculation in cells where the lysosomes have been cross-linked. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with DAB/H2O2 at 4°C to crosslink the lysosomes. Cells were then microinjected at 37°C with 20 nm BSA gold (arrows) alone (a) or coinjected with anti-hVPS34 antibody (b), anti-p110α antibody (c), or anti-p110β antibody (d). Cells were then incubated with 10 nm anti-EGFR gold (arrowheads) and EGF for 1 h at 20°C and were then chased at 37°C for 1 h. Microinjection with the anti-hVPS34 antibody caused the generation of enlarged MVBs containing few internal vesicles (asterisks). Microinjection with anti-p110α had no effect on MVB formation, whereas microinjection with p110β in some cases caused the generation of MVBs with comparatively few EGFRs, but they were not enlarged. Bars, 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199316&req=5

fig6: The effects of microinjection with anti–PI 3′-kinase antibodies on inward vesiculation in cells where the lysosomes have been cross-linked. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with DAB/H2O2 at 4°C to crosslink the lysosomes. Cells were then microinjected at 37°C with 20 nm BSA gold (arrows) alone (a) or coinjected with anti-hVPS34 antibody (b), anti-p110α antibody (c), or anti-p110β antibody (d). Cells were then incubated with 10 nm anti-EGFR gold (arrowheads) and EGF for 1 h at 20°C and were then chased at 37°C for 1 h. Microinjection with the anti-hVPS34 antibody caused the generation of enlarged MVBs containing few internal vesicles (asterisks). Microinjection with anti-p110α had no effect on MVB formation, whereas microinjection with p110β in some cases caused the generation of MVBs with comparatively few EGFRs, but they were not enlarged. Bars, 0.5 μm.

Mentions: To determine which PI 3′-kinase is involved in inward vesiculation, isotype-specific inhibitory antibodies to the p110α and p110β subunits of the type 1 kinases and to hVPS 34 (the type III kinase) were assessed for their effects on inward vesiculation. These antibodies have been shown to inhibit the respective PI 3′-kinase activities when microinjected into cells (Siddhanta et al., 1998). HRP-loaded lysosomes were cross-linked in the living cell, and then cells were microinjected with anti–PI 3′-kinase antibody and with 20 nm gold in order to locate the microinjected cells. Cells were then allowed to recover for a further 2 h at 37°C before incubation with anti-EGFR gold and EGF at 20°C. Cells were then chased at 37°C for 1 h before processing for EM. Control experiments were performed to confirm that the morphology of the cells, and the formation of MVBs was not affected by microinjection with 20 nm gold. The microinjected 20 nm gold was distributed frequently throughout the cytoplasm as single particles, although occasionally clusters of gold were observed in the cytoplasm or enclosed within a limiting membrane (Fig. 6 a). Microinjection of anti-p110α antibody did not affect the morphology of the MVB at any dose of antibody (Fig. 6 c). Microinjection of anti-p110β antibody did not appear to affect the morphology of the MVB at low doses. However, cells injected with larger doses of antibody had unusually small MVBs with very few internal vesicles, and EGFRs were often found in small vesicles and tubules rather than MVBs (Fig. 6 d). This suggests that p110β is involved in early events in endocytic processing and may be involved in the delivery of membrane to the MVB. In cells microinjected with anti-hVPS34 MVBs had a reduced number of internal vesicles and the EGFRs were primarily on the perimeter membrane (Fig. 6 b). Although in some cases these MVBs were enlarged, they were not as large as those induced by treatment with wortmannin. It is possible that this difference in the results of anti–PI 3′-kinase antibody injection and wortmannin treatment could be explained by differences in the timing of PI 3′-kinase inhibition. Anti–PI 3′-kinase antibodies were injected before the addition of anti-EGFR gold and EGF, whereas wortmannin was added to the cells after they had been incubated with anti-EGFR gold and EGF at 20°C.


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 microinjection with anti–PI 3′-kinase antibodies on inward vesiculation in cells where the lysosomes have been cross-linked. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with DAB/H2O2 at 4°C to crosslink the lysosomes. Cells were then microinjected at 37°C with 20 nm BSA gold (arrows) alone (a) or coinjected with anti-hVPS34 antibody (b), anti-p110α antibody (c), or anti-p110β antibody (d). Cells were then incubated with 10 nm anti-EGFR gold (arrowheads) and EGF for 1 h at 20°C and were then chased at 37°C for 1 h. Microinjection with the anti-hVPS34 antibody caused the generation of enlarged MVBs containing few internal vesicles (asterisks). Microinjection with anti-p110α had no effect on MVB formation, whereas microinjection with p110β in some cases caused the generation of MVBs with comparatively few EGFRs, but they were not enlarged. Bars, 0.5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: The effects of microinjection with anti–PI 3′-kinase antibodies on inward vesiculation in cells where the lysosomes have been cross-linked. HEp-2 cells were incubated with HRP for 30 min at 37°C, chased for 3 h at 37°C, and then incubated with DAB/H2O2 at 4°C to crosslink the lysosomes. Cells were then microinjected at 37°C with 20 nm BSA gold (arrows) alone (a) or coinjected with anti-hVPS34 antibody (b), anti-p110α antibody (c), or anti-p110β antibody (d). Cells were then incubated with 10 nm anti-EGFR gold (arrowheads) and EGF for 1 h at 20°C and were then chased at 37°C for 1 h. Microinjection with the anti-hVPS34 antibody caused the generation of enlarged MVBs containing few internal vesicles (asterisks). Microinjection with anti-p110α had no effect on MVB formation, whereas microinjection with p110β in some cases caused the generation of MVBs with comparatively few EGFRs, but they were not enlarged. Bars, 0.5 μm.
Mentions: To determine which PI 3′-kinase is involved in inward vesiculation, isotype-specific inhibitory antibodies to the p110α and p110β subunits of the type 1 kinases and to hVPS 34 (the type III kinase) were assessed for their effects on inward vesiculation. These antibodies have been shown to inhibit the respective PI 3′-kinase activities when microinjected into cells (Siddhanta et al., 1998). HRP-loaded lysosomes were cross-linked in the living cell, and then cells were microinjected with anti–PI 3′-kinase antibody and with 20 nm gold in order to locate the microinjected cells. Cells were then allowed to recover for a further 2 h at 37°C before incubation with anti-EGFR gold and EGF at 20°C. Cells were then chased at 37°C for 1 h before processing for EM. Control experiments were performed to confirm that the morphology of the cells, and the formation of MVBs was not affected by microinjection with 20 nm gold. The microinjected 20 nm gold was distributed frequently throughout the cytoplasm as single particles, although occasionally clusters of gold were observed in the cytoplasm or enclosed within a limiting membrane (Fig. 6 a). Microinjection of anti-p110α antibody did not affect the morphology of the MVB at any dose of antibody (Fig. 6 c). Microinjection of anti-p110β antibody did not appear to affect the morphology of the MVB at low doses. However, cells injected with larger doses of antibody had unusually small MVBs with very few internal vesicles, and EGFRs were often found in small vesicles and tubules rather than MVBs (Fig. 6 d). This suggests that p110β is involved in early events in endocytic processing and may be involved in the delivery of membrane to the MVB. In cells microinjected with anti-hVPS34 MVBs had a reduced number of internal vesicles and the EGFRs were primarily on the perimeter membrane (Fig. 6 b). Although in some cases these MVBs were enlarged, they were not as large as those induced by treatment with wortmannin. It is possible that this difference in the results of anti–PI 3′-kinase antibody injection and wortmannin treatment could be explained by differences in the timing of PI 3′-kinase inhibition. Anti–PI 3′-kinase antibodies were injected before the addition of anti-EGFR gold and EGF, whereas wortmannin was added to the cells after they had been incubated with anti-EGFR gold and EGF at 20°C.

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