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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 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 incubated with anti-EGFR gold and EGF at 20°C in the absence of wortmannin and then chased at 37°C in the absence (a) or the presence of wortmannin (b and c). Note that in the absence of wortmannin, MVBs with many internal vesicles and anti-EGFR gold predominantly on the internal vesicles accumulate (asterisks). In the presence of wortmannin, enlarged MVBs with very few internal vesicles accumulate (crosses), and the EGFR are clustered (arrowheads) on the perimeter membrane of the enlarged MVBs. L, lysosome. Bars: (a and b) 0.1 μm; (c) 0.5 μm.
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fig3: The effects of wortmannin 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 incubated with anti-EGFR gold and EGF at 20°C in the absence of wortmannin and then chased at 37°C in the absence (a) or the presence of wortmannin (b and c). Note that in the absence of wortmannin, MVBs with many internal vesicles and anti-EGFR gold predominantly on the internal vesicles accumulate (asterisks). In the presence of wortmannin, enlarged MVBs with very few internal vesicles accumulate (crosses), and the EGFR are clustered (arrowheads) on the perimeter membrane of the enlarged MVBs. L, lysosome. Bars: (a and b) 0.1 μm; (c) 0.5 μm.

Mentions: Incubation of living cells with DAB/H2O2 crosslinks HRP-loaded lysosomes, preventing fusion between MVBs and lysosomes, and causes MVBs to accumulate (Futter et al., 1996). This technique allows the development of the MVB to be followed in the absence of endosome–lysosome fusion and is particularly useful for studying inward vesiculation. To quantify the effects of wortmannin on inward vesiculation, HRP-loaded lysosomes were cross-linked and cells were then incubated with anti-EGFR gold and EGF at 20°C followed by a 1 h chase at 37°C in the presence or absence of wortmannin. As shown in Fig. 3 a, in the absence of wortmannin MVBs with many internal vesicles accumulate, and the EGFR gold is primarily on the internal vesicles. In the presence of wortmannin enlarged anti-EGFR, gold-containing vacuoles accumulate (Fig. 3, b and c). These vacuoles contain very few internal vesicles, and the majority of the gold-labeled EGFRs is on the perimeter membrane. These EGFRs are not evenly distributed on the perimeter membrane but are clustered, and where an internal vesicle does form it is usually labeled with anti-EGFR gold (Fig. 3 b).


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 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 incubated with anti-EGFR gold and EGF at 20°C in the absence of wortmannin and then chased at 37°C in the absence (a) or the presence of wortmannin (b and c). Note that in the absence of wortmannin, MVBs with many internal vesicles and anti-EGFR gold predominantly on the internal vesicles accumulate (asterisks). In the presence of wortmannin, enlarged MVBs with very few internal vesicles accumulate (crosses), and the EGFR are clustered (arrowheads) on the perimeter membrane of the enlarged MVBs. L, lysosome. Bars: (a and b) 0.1 μm; (c) 0.5 μm.
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Related In: Results  -  Collection

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

fig3: The effects of wortmannin 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 incubated with anti-EGFR gold and EGF at 20°C in the absence of wortmannin and then chased at 37°C in the absence (a) or the presence of wortmannin (b and c). Note that in the absence of wortmannin, MVBs with many internal vesicles and anti-EGFR gold predominantly on the internal vesicles accumulate (asterisks). In the presence of wortmannin, enlarged MVBs with very few internal vesicles accumulate (crosses), and the EGFR are clustered (arrowheads) on the perimeter membrane of the enlarged MVBs. L, lysosome. Bars: (a and b) 0.1 μm; (c) 0.5 μm.
Mentions: Incubation of living cells with DAB/H2O2 crosslinks HRP-loaded lysosomes, preventing fusion between MVBs and lysosomes, and causes MVBs to accumulate (Futter et al., 1996). This technique allows the development of the MVB to be followed in the absence of endosome–lysosome fusion and is particularly useful for studying inward vesiculation. To quantify the effects of wortmannin on inward vesiculation, HRP-loaded lysosomes were cross-linked and cells were then incubated with anti-EGFR gold and EGF at 20°C followed by a 1 h chase at 37°C in the presence or absence of wortmannin. As shown in Fig. 3 a, in the absence of wortmannin MVBs with many internal vesicles accumulate, and the EGFR gold is primarily on the internal vesicles. In the presence of wortmannin enlarged anti-EGFR, gold-containing vacuoles accumulate (Fig. 3, b and c). These vacuoles contain very few internal vesicles, and the majority of the gold-labeled EGFRs is on the perimeter membrane. These EGFRs are not evenly distributed on the perimeter membrane but are clustered, and where an internal vesicle does form it is usually labeled with anti-EGFR gold (Fig. 3 b).

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