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First published online 9 March 2004
doi: 10.1242/jcs.00998

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ATPase-deficient hVPS4 impairs formation of internal endosomal vesicles and stabilizes bilayered clathrin coats on endosomal vacuoles

Department of Cell Biology, University Medical Center and Institute for Biomembranes, 3584 CX Utrecht, The Netherlands

View larger version (148K): [in a new window]   Fig. 1. Overexpression of hVPS4EQ induces enlarged endosomal vacuoles. Cryosections of non-transfected HeLa cells or cells transfected with GFP-hVPS4wt or GFP-hVPSEQ were labeled for GFP (10 nm gold). (A) In non-transfected cells, EEs appear as electron-lucent vacuoles containing few internal vesicles. Only rarely can an inward budding profile (i.e. forming internal vesicle) be observed (arrow). (B) hVPS4wt is mainly cytosolic and the morphology of endosomal vacuoles (EV) is unaffected. (C,D) Overexpression of the ATPase-deficient hVPS4EQ leads to enlarged EVs. Some EVs display lipid particles without a limiting membrane (C). In others, unusually many inward budding profiles are seen (arrows in D). M, mitochondrion; PM, plasma membrane. Scale bars, 200 nm.

View larger version (101K): [in a new window]   Fig. 4. Incorporation of EGFR into internal endosomal vesicles is inhibited by hVPS4EQ. HeLa cells transfected with GFP-hVPS4wt or GFP-hVPS4EQ and incubated for 20 minutes with EGF were double labeled for GFP (15 nm gold) and EGFR (10 nm gold). (A) EGFR is found on internal vesicles of LEs in cells expressing hVPS4wt. (B) In cells expressing hVPS4EQ, EGFR (arrowheads) is retained at the limiting membrane of endosomal vacuoles (EV). L, lysosome; M, mitochondrion. Scale bars, 200 nm.

View larger version (177K): [in a new window]   Fig. 3. hVPS4 function is required for the disassembly of bilayered clathrin coats on endosomal vacuoles. HeLa cells transfected with GFP, GFP-hVPS4wt or GFP-hVPS4EQ were double labeled for GFP (15 nm gold) and (A-C) Hrs (10 nm gold) or (D) clathrin (10 nm gold). (A) Cells expressing GFP (15 nm gold) show Hrs (10 nm gold) in the characteristic flat bilayered coats on endosomal vacuoles (EV). (B) Also, in hVPS4wt cells, Hrs (10 nm gold) is present in flat bilayered coats. Inward budding profiles are formed next to the coat (arrow). (C,D) Cells overexpressing hVPS4EQ show endosomal vacuoles positive for Hrs (10 nm gold in C) and clathrin (10 nm gold in D). The coat is assembled in an orderly fashion, displaying the characteristic two layers and label for hVPS4EQ is present not within the coated areas but at the rims (arrowheads). Scale bars, 200 nm.

View larger version (178K): [in a new window]   Fig. 2. hVPS4EQ is present on early and late endosomes. (A) In hVPS4wt (15 nm gold) cells, TfR (10 nm gold) is present on EE vacuoles and emerging recycling tubules (arrows). (B) In hVPS4EQ cells, TfR labeling (10 nm gold) is present on normally shaped EEs that also contain hVPS4EQ (15 nm gold). The arrows point to recycling tubules that contain most TfR. (C-E) HeLa cells incubated for 5 minutes with BSA conjugated to 5 nm gold and labeled for CD63 (10 nm gold). BSA-gold is found in EEs but not in LEs or lysosomes (L). CD63 is enriched on internal membranes of LEs and lysosomes, which differ by the presence of internal vesicles or membrane sheets, respectively. (F) hVPS4EQ can also be found on swollen vacuoles that contain internal membranes positive for CD63 (10 nm gold), defining them as LEs. Label for CD63 is present on an inward budding profile (arrow) showing a distinct electron-dense cytoplasmic layer that is also seen at other parts of the limiting membrane. PM, plasma membrane. Scale bar, 200 nm.

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