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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 the number of internal vesicles per MVB. 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 or the presence of wortmannin. The total number of internal vesicles in 13 MVBs per treatment was estimated by analysis of 70-nm serial sections.
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fig4: The effects of wortmannin on the number of internal vesicles per MVB. 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 or the presence of wortmannin. The total number of internal vesicles in 13 MVBs per treatment was estimated by analysis of 70-nm serial sections.

Mentions: The major change in volume of MVBs induced by wortmannin treatment may alter the probability of sectioning through internal vesicles in any given thin section. To accurately quantitate the number of internal vesicles per MVB, we therefore analyzed serial sections to reconstruct individual MVBs in their entirety. As shown in Table II, MVBs formed in the presence of wortmannin have fivefold fewer internal vesicles than those formed in the absence of wortmannin and have approximately twice the diameter. As shown in Fig. 4, the number of internal vesicles per MVB is extremely variable, particularly in control cells, but the majority of MVBs have 10–40 internal vesicles per MVB. In contrast, in wortmannin-treated cells the majority of MVBs have less than five internal vesicles. Estimating the total membrane area of the MVB, assuming all internal vesicles to have a diameter of 50 nm, indicates that wortmannin-treated vacuoles have approximately twice as much membrane as control cells (Table II), suggesting that the vacuolar enlargement cannot be explained solely by a failure to inwardly vesiculate. Inhibition of exit from the MVB as has been suggested by others (Reaves et al., 1996; Kundra and Kornfeld, 1998) may, therefore, also contribute to the vacuolar enlargement. MVBs allowed to accumulate for 1 h in the presence of wortmannin have up to 50% fewer gold particles than those that accumulate in the absence of wortmannin. We can assume that the entry of EGFRs into the MVBs is not inhibited by wortmannin, since in cells where the lysosomes have not been cross-linked EGFRs are efficiently delivered to the lysosome (see above). Therefore, it is likely that some loss of EGFRs from the perimeter membrane of the MVB does occur in wortmannin-treated cells when MVB–lysosome fusion is prevented.


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 the number of internal vesicles per MVB. 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 or the presence of wortmannin. The total number of internal vesicles in 13 MVBs per treatment was estimated by analysis of 70-nm serial sections.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: The effects of wortmannin on the number of internal vesicles per MVB. 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 or the presence of wortmannin. The total number of internal vesicles in 13 MVBs per treatment was estimated by analysis of 70-nm serial sections.
Mentions: The major change in volume of MVBs induced by wortmannin treatment may alter the probability of sectioning through internal vesicles in any given thin section. To accurately quantitate the number of internal vesicles per MVB, we therefore analyzed serial sections to reconstruct individual MVBs in their entirety. As shown in Table II, MVBs formed in the presence of wortmannin have fivefold fewer internal vesicles than those formed in the absence of wortmannin and have approximately twice the diameter. As shown in Fig. 4, the number of internal vesicles per MVB is extremely variable, particularly in control cells, but the majority of MVBs have 10–40 internal vesicles per MVB. In contrast, in wortmannin-treated cells the majority of MVBs have less than five internal vesicles. Estimating the total membrane area of the MVB, assuming all internal vesicles to have a diameter of 50 nm, indicates that wortmannin-treated vacuoles have approximately twice as much membrane as control cells (Table II), suggesting that the vacuolar enlargement cannot be explained solely by a failure to inwardly vesiculate. Inhibition of exit from the MVB as has been suggested by others (Reaves et al., 1996; Kundra and Kornfeld, 1998) may, therefore, also contribute to the vacuolar enlargement. MVBs allowed to accumulate for 1 h in the presence of wortmannin have up to 50% fewer gold particles than those that accumulate in the absence of wortmannin. We can assume that the entry of EGFRs into the MVBs is not inhibited by wortmannin, since in cells where the lysosomes have not been cross-linked EGFRs are efficiently delivered to the lysosome (see above). Therefore, it is likely that some loss of EGFRs from the perimeter membrane of the MVB does occur in wortmannin-treated cells when MVB–lysosome fusion is prevented.

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