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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.

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The effects of microinjection with anti–PI 3′-kinase antibodies after incubation with EGF at 20°C. 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 incubated with 10 nm anti-EGFR gold (arrowheads) and EGF for 1 h at 20°C and were then microinjected with 20 nm BSA gold (arrows) and anti-hVPS34 (a) or anti-p110β (b) at 20°C before incubation at 20°C for a further 30 min and then chase at 37°C for 1 h. Microinjection of anti-hVPS34 caused the generation of enlarged MVBs with very few internal vesicles and EGFRs on the perimeter membrane. Microinjection of anti-p110β antibody caused the formation of small MVBs with few internal vesicles and few EGFRs. Bars, 0.2 μm.
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fig7: The effects of microinjection with anti–PI 3′-kinase antibodies after incubation with EGF at 20°C. 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 incubated with 10 nm anti-EGFR gold (arrowheads) and EGF for 1 h at 20°C and were then microinjected with 20 nm BSA gold (arrows) and anti-hVPS34 (a) or anti-p110β (b) at 20°C before incubation at 20°C for a further 30 min and then chase at 37°C for 1 h. Microinjection of anti-hVPS34 caused the generation of enlarged MVBs with very few internal vesicles and EGFRs on the perimeter membrane. Microinjection of anti-p110β antibody caused the formation of small MVBs with few internal vesicles and few EGFRs. Bars, 0.2 μm.

Mentions: Therefore, we performed further microinjection experiments to mimic the experiments using wortmannin as closely as possible. HRP-loaded lysosomes were cross-linked in the living cell, and then cells were loaded with anti-EGFR gold and EGF at 20°C. Cells were then microinjected with antibody and with 20 nm gold particles and were then incubated for a further 30 min at 20°C to allow time for the microinjected antibody to bind its antigen within the cell. Cells were then chased at 37°C for 1 h. Microinjection of the anti-hVPS34 antibody caused the formation of enlarged MVBs with very few internal vesicles and with EGFRs on the perimeter membrane, closely mimicking the effects seen with wortmannin treatment (Fig. 7 a). This effect appeared to be dose dependent. In cells with large amounts of injected 20 nm gold (indicating a larger volume of microinjected anti–PI 3′-kinase antibody), MVBs were large and had very few internal vesicles. Cells containing fewer injected gold particles seemed to have smaller MVBs with a few internal vesicles but still contained fewer internal vesicles than MVBs of control cells (unpublished data). Therefore, we conclude that the inhibition of inward vesiculation within the MVB by wortmannin is due to inhibition of the PI 3′-kinase hVPS34. Large doses of anti-p110β antibody sometimes led to a decrease in the size of MVBs and some accumulation of EGFRs in small vesicles and tubules (Fig. 7 b). This is an effect not observed with wortmannin treatment. p110β may be involved in an early step in the maturation of MVBs and is presumably more effectively inhibited by microinjection than by wortmannin treatment.


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 after incubation with EGF at 20°C. 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 incubated with 10 nm anti-EGFR gold (arrowheads) and EGF for 1 h at 20°C and were then microinjected with 20 nm BSA gold (arrows) and anti-hVPS34 (a) or anti-p110β (b) at 20°C before incubation at 20°C for a further 30 min and then chase at 37°C for 1 h. Microinjection of anti-hVPS34 caused the generation of enlarged MVBs with very few internal vesicles and EGFRs on the perimeter membrane. Microinjection of anti-p110β antibody caused the formation of small MVBs with few internal vesicles and few EGFRs. Bars, 0.2 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: The effects of microinjection with anti–PI 3′-kinase antibodies after incubation with EGF at 20°C. 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 incubated with 10 nm anti-EGFR gold (arrowheads) and EGF for 1 h at 20°C and were then microinjected with 20 nm BSA gold (arrows) and anti-hVPS34 (a) or anti-p110β (b) at 20°C before incubation at 20°C for a further 30 min and then chase at 37°C for 1 h. Microinjection of anti-hVPS34 caused the generation of enlarged MVBs with very few internal vesicles and EGFRs on the perimeter membrane. Microinjection of anti-p110β antibody caused the formation of small MVBs with few internal vesicles and few EGFRs. Bars, 0.2 μm.
Mentions: Therefore, we performed further microinjection experiments to mimic the experiments using wortmannin as closely as possible. HRP-loaded lysosomes were cross-linked in the living cell, and then cells were loaded with anti-EGFR gold and EGF at 20°C. Cells were then microinjected with antibody and with 20 nm gold particles and were then incubated for a further 30 min at 20°C to allow time for the microinjected antibody to bind its antigen within the cell. Cells were then chased at 37°C for 1 h. Microinjection of the anti-hVPS34 antibody caused the formation of enlarged MVBs with very few internal vesicles and with EGFRs on the perimeter membrane, closely mimicking the effects seen with wortmannin treatment (Fig. 7 a). This effect appeared to be dose dependent. In cells with large amounts of injected 20 nm gold (indicating a larger volume of microinjected anti–PI 3′-kinase antibody), MVBs were large and had very few internal vesicles. Cells containing fewer injected gold particles seemed to have smaller MVBs with a few internal vesicles but still contained fewer internal vesicles than MVBs of control cells (unpublished data). Therefore, we conclude that the inhibition of inward vesiculation within the MVB by wortmannin is due to inhibition of the PI 3′-kinase hVPS34. Large doses of anti-p110β antibody sometimes led to a decrease in the size of MVBs and some accumulation of EGFRs in small vesicles and tubules (Fig. 7 b). This is an effect not observed with wortmannin treatment. p110β may be involved in an early step in the maturation of MVBs and is presumably more effectively inhibited by microinjection than by wortmannin treatment.

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