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First published online January 27, 2006
doi: 10.1242/10.1242/jcs.02752

Journal of Cell Science 119, 571-581 (2006)
Published by The Company of Biologists 2006
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Phosphatidylinositol 4-kinase is required for endosomal trafficking and degradation of the EGF receptor

Shane Minogue1,*, Mark G. Waugh1, Maria Antonietta De Matteis2, David J. Stephens3, Fedor Berditchevski4 and J. Justin Hsuan1

1 Centre for Molecular Cell Biology, Department of Medicine, Royal Free and University College Medical School, University College London, Rowland Hill Street, London, NW3 2PF, UK
2 Department of Cell Biology and Oncology, Consorzio Mario Negri Sud, Via Nazionale, 66030 Santa Maria Imbaro (Chieti), Italy
3 Department of Biochemistry, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK
4 CRUK Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham, B15 2TA, UK


Figure 1
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  		Fig. 1. The subcellular distribution of PtdIns4KII{alpha} in HT1080 cells. (A) Live cells expressing GFP-PtdIns4KII{alpha} were imaged by confocal laser-scanning microscopy. (B) Detail of plasma membrane (PM), small cytoplasmic vesicles (SV) and tubules (T). (C) Detail of the juxta-nuclear region showing multivesicular body (MVB)-like structures (MV). (D) Endogenous PtdIns4KII{alpha} in fixed HT1080 cells and (E) detail. (F) Cells co-immunostained for endogenous PtdIns4KII{alpha} and lamp-1. Staining for (G) GFP-PtdIns4KII{alpha} and lamp-1, (H) CD63 and (I) LBPA. Arrows in G,H indicate colocalisation of GFP-PtdIns4KII{alpha} and marker in punctate structures. Arrows in I indicate the localisation of GFP-PtdIns4KII{alpha} to the boundary membrane of MVB-like structures. All zoom boxes are 20 µm square. Bars, 10 µm.

 

Figure 2
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  		Fig. 2. PtdIns4KII{alpha} is a component of highly dynamic membranes. (A) A confocal image of a live GFP-PtdIns4KII{alpha}-expressing HT1080 cell. (B) Frames from a time-series experiment were projected in the Z-axis to reveal the paths of moving vesicles. These paths and the direction of movement (arrows) are detailed in C and D. (E) Frames from the same cell showing the progress of a vesicle (arrows) along a tubule toward a multivesicular body (also see supplementary material Movie 1). Bar, 10 µm.

 

Figure 3
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  		Fig. 3. GFP-PtdIns4KII{alpha} colocalises with endocytosed Rh-EGF. (A) Time course of Rh-EGF internalisation in HT1080 GFP-PtdIns4KII{alpha} cells. Detail from each time point is shown in the zoom boxes (10 µm square). (B) Anti-GFP western analysis of total cell lysates from HT1080 GFP-PtdIns4KII{alpha} cells stimulated with EGF for the times shown was also performed to rule out the possibility that the endosomal localisation could be due to degradation of the fusion protein. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. Internalised EGF is trafficked in rapidly moving PtdIns4KII{alpha}-containing vesicles. Time-lapse images were obtained from live HT1080 GFP-PtdIns4KII{alpha} cells that had been serum-starved and stimulated with Rh-EGF for 120 seconds. Frames from the boxed region show the movement of a single vesicle marked with an arrow. Also see supplementary material Movie 2. Bar, 10 µm.

 

Figure 5
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  		Fig. 5. Inhibition of endogenous PtdIns4KII activity results in abnormal trafficking of internalised EGF. HT1080 cells were transfected with control monoclonal antibody 9E10 (A) or 4C5G (B) and, after serum starvation, stimulated with Rh-EGF then fixed at the time points indicated. All images are extended focus views representing 12 confocal sections collected 0.12 µm apart. Bars, 10 µm.

 

Figure 6
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  		Fig. 6. Inhibition of PtdIns4KII{alpha} mRNA expression results in the abnormal trafficking of internalised EGF. (A) siRNA with two independent duplex oligonucleotides (oligo-1 and oligo-2) effectively depletes HT1080 cells of PtdIns4KII{alpha} but not the closely related isoform PtdIns4KII{alpha} (the doublet in the PtdIns4KII{alpha} blot is frequently observed). A time course of Rh-EGF internalisation in HT1080 cells in mock (B) or PtdIns4KII{alpha} siRNA (C) transfected cells. (D) Detail of the boxed sub-plasma membrane region from the 900 seconds time point from C. Arrows indicate the retention of Rh-EGF in sub-plasma membrane punctae. All images are extended focus views representing 12 confocal sections collected 0.12 µm apart. Bars, 10 µm.

 

Figure 7
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  		Fig. 7. The effects of in vivo PtdIns4KII inhibition on the endosomal distribution of internalised EGF. Z-series data from time course experiments were reconstructed in three dimensions and the mean diameter of Rh-EGF-labelled punctae measured as described in the Materials and Methods. Punctae were analysed in single cells (300-800 objects per cell) from at least three cells from three independent time course experiments (i.e. 2.7x103-7.2x103 objects per time point). (A) Analysis of mean vesicle diameter (± s.e.m.) in 9E10 and 4C5G-transfected cells. The same cells were also scored for scattered punctae (B) and sub-plasma membrane vesicle clusters (C). (D) Analysis of mean vesicle diameter (± s.e.m.) in mock-transfected and PtdIns4KII{alpha} siRNA-transfected cells. Rh-EGF treated cells were also manually scored for the presence of scattered punctae (E) and the persistence of small vesicles clustered beneath the plasma membrane (F). For B,C and E,F, a minimum of 63 cells per time point were scored from three independent experiments.

 

Figure 8
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  		Fig. 8. Degradation of the EGF receptor is impaired in RNAi-treated HeLa cells. (A) Mock, lamin, oligo-1 or oligo-2 RNAi-transfected cells were treated with 100 ng/ml EGF for the times indicated in the presence of 10 µg/ml cycloheximide and total cell lysates analysed by western blotting with anti-EGFR antibodies to monitor degradation of the EGFR and anti-{alpha}-tubulin antibodies to control for protein levels. (B) Densitometric analysis of western blot signals derived from three independent experiments (mean ± s.e.m.).

 

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