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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 wortmannin on traffic of EGFRs and TRs through MVBs. 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 anti-EGFR gold (10 nm) and EGF for 1 h at 20°C in the absence of wortmannin and were then chased at 37°C for 1 h in the absence (a and b) or the presence of wortmannin (c). Cells were then permeabilized, fixed, and labeled with anti-TR antibody (5 nm gold). Note that in both the absence and the presence of wortmannin there are very few TRs on MVBs, but small vesicles labeling strongly for TRs (arrows) are often in close proximity. Bars, 0.1 μm.
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fig5: The effects of wortmannin on traffic of EGFRs and TRs through MVBs. 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 anti-EGFR gold (10 nm) and EGF for 1 h at 20°C in the absence of wortmannin and were then chased at 37°C for 1 h in the absence (a and b) or the presence of wortmannin (c). Cells were then permeabilized, fixed, and labeled with anti-TR antibody (5 nm gold). Note that in both the absence and the presence of wortmannin there are very few TRs on MVBs, but small vesicles labeling strongly for TRs (arrows) are often in close proximity. Bars, 0.1 μm.

Mentions: To determine whether there is a general inhibition of exit from MVBs in wortmannin-treated cells or whether EGFRs are specifically retained on the perimeter membrane of MVBs, we determined the effect of wortmannin treatment on removal of TRs from MVBs. Lysosomes were cross-linked in the living cell as described above. Cells were then incubated with anti-EGFR gold and EGF at 20°C in the absence of wortmannin to allow EGFR internalization but retain EGFRs in the TR-containing endosome. Cells were then chased for 1 h at 37°C in the presence or absence of wortmannin. In the absence of wortmannin, TRs would be expected to be gradually removed from the maturing MVBs. Cells were then permeabilized and immunogold labeled using an antibody to the cytoplasmic domain of the TR as described in Materials and methods. As expected, mature MVBs accumulated in the absence of wortmannin for 1 h at 37°C have very few TRs compared with surrounding vesicles and tubules (Fig. 5, a and b). In wortmannin-treated cells, although the MVBs are enlarged, there are also very few TRs on the perimeter membrane compared with surrounding vesicles and tubules (Fig. 5, b and c). Examining a single time point does not allow the effect of wortmannin on the rate of removal of TRs from the MVBs to be determined. However, counting the number of EGFR- and TR-gold particles in random thin sections shows that the ratio of EGFRs to TRs in MVBs accumulated in the presence of wortmannin compared with untreated cells is almost identical: 57:2 EGFRs to TRs in the absence of wortmannin and 51:2 in the presence of wortmannin (n = 100). Thus, although inward vesiculation is inhibited sorting of EGFRs from TRs continues.


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 traffic of EGFRs and TRs through MVBs. 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 anti-EGFR gold (10 nm) and EGF for 1 h at 20°C in the absence of wortmannin and were then chased at 37°C for 1 h in the absence (a and b) or the presence of wortmannin (c). Cells were then permeabilized, fixed, and labeled with anti-TR antibody (5 nm gold). Note that in both the absence and the presence of wortmannin there are very few TRs on MVBs, but small vesicles labeling strongly for TRs (arrows) are often in close proximity. Bars, 0.1 μm.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: The effects of wortmannin on traffic of EGFRs and TRs through MVBs. 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 anti-EGFR gold (10 nm) and EGF for 1 h at 20°C in the absence of wortmannin and were then chased at 37°C for 1 h in the absence (a and b) or the presence of wortmannin (c). Cells were then permeabilized, fixed, and labeled with anti-TR antibody (5 nm gold). Note that in both the absence and the presence of wortmannin there are very few TRs on MVBs, but small vesicles labeling strongly for TRs (arrows) are often in close proximity. Bars, 0.1 μm.
Mentions: To determine whether there is a general inhibition of exit from MVBs in wortmannin-treated cells or whether EGFRs are specifically retained on the perimeter membrane of MVBs, we determined the effect of wortmannin treatment on removal of TRs from MVBs. Lysosomes were cross-linked in the living cell as described above. Cells were then incubated with anti-EGFR gold and EGF at 20°C in the absence of wortmannin to allow EGFR internalization but retain EGFRs in the TR-containing endosome. Cells were then chased for 1 h at 37°C in the presence or absence of wortmannin. In the absence of wortmannin, TRs would be expected to be gradually removed from the maturing MVBs. Cells were then permeabilized and immunogold labeled using an antibody to the cytoplasmic domain of the TR as described in Materials and methods. As expected, mature MVBs accumulated in the absence of wortmannin for 1 h at 37°C have very few TRs compared with surrounding vesicles and tubules (Fig. 5, a and b). In wortmannin-treated cells, although the MVBs are enlarged, there are also very few TRs on the perimeter membrane compared with surrounding vesicles and tubules (Fig. 5, b and c). Examining a single time point does not allow the effect of wortmannin on the rate of removal of TRs from the MVBs to be determined. However, counting the number of EGFR- and TR-gold particles in random thin sections shows that the ratio of EGFRs to TRs in MVBs accumulated in the presence of wortmannin compared with untreated cells is almost identical: 57:2 EGFRs to TRs in the absence of wortmannin and 51:2 in the presence of wortmannin (n = 100). Thus, although inward vesiculation is inhibited sorting of EGFRs from TRs continues.

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