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First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00801

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Visualization of Rab5 activity in living cells by FRET microscopy and influence of plasma-membrane-targeted Rab5 on clathrin-dependent endocytosis

Department of Pharmacology, University of Colorado Health Sciences Center, Denver, CO 80262, USA

View larger version (62K): [in a new window]   Fig. 1. Schematic representation and immunodetection of fusion proteins. (A) Depicted are Rab5, and fragments of Rabaptin5 and EEA.1 proteins fused to CFP/YFP at the N- or C-terminus. The first and the last amino acid residues in Rabaptin 5 (R5RB) and EEA.1 (EEA.1sh) fragments are numbered according to the full-length sequences. The FYVE domain of EEA.1 is indicated. PAE/EGFR cells transiently expressing wild-type (wt), Q79L or S34N CFP-Rab5 mutants or YFP-EEA.1 and R5BD-YFP were lysed, and CFP/YFP-fusion proteins present in lysates were detected by western blotting with anti-Rab5 or anti-GFP antibodies, respectively. All fusion proteins generated in this work migrated on SDS-PAGE according to the predicted molecular masses. (B and C) PAE/EGFR cells transiently expressing R5BD-YFP alone (B) or together (C) with CFP-EEA.1sh were incubated with 1 ng/ml EGF-Rh for 10 minutes at 37°C and fixed. EGF-Rh, CFP and YFP were detected using Cy3, CFP and YFP filter channels. Insets in (C) show an enlargement of the outlined regions. Bars, 10 µm.

View larger version (121K): [in a new window]   Fig. 2. Detection of GTP-bound Rab5 in living cells. CFP-Rab5, CFP-Rab5(Q79L) or CFPRab5(S34N) were co-expressed with R5BDYFP or YFP-R5BD in PAE/EGFR cells. YFP, CFP and FRET images were obtained from living cells at RT. FRETC was calculated as described in Materials and Methods, and presented as pseudocolor images. Insets show an enlargement of the outlined regions. A.l.u.f.i., arbitrary linear units of fluorescence intensity. Bar, 10 µm.

View larger version (71K): [in a new window]   Fig. 3. Specificity of R5BD-YFP and YFPEEA.1sh sensors for Rab5. (A) CFP-Rab4 was co-expressed with R5BD-YFP or YFP-EEA.1sh in PAE/EGFR cells. (B) CFP-Rab5 or CFPRab5(S34N) were co-expressed with YFPEEA.1sh in PAE/EGFR cells. YFP, CFP and FRET images were obtained from living cells at RT. FRETC images were calculated as described in Materials and Methods, and presented as pseudocolor images. A.l.u.f.i., arbitrary linear units of fluorescence intensity. Bars (A and B), 10 µm. (C) Gallery of high-magnification images shows individual endosomes or tethered endosomes in cells co-expressing CFPRab5(Q79L) and YFP-EEA.1sh or R5BD-YFP. FRET images are presented as pseudocolor intensity-modulated images (FRETC/CFP). Bar, 2 µm.

View larger version (90K): [in a new window]   Fig. 4. Effect of Rab5 mutant overexpression on EGF receptor internalization. (A) Wild-type CFP-Rab5 (wt) and CFP-Rab5(S34N) mutant were transiently co-expressed with ß2-YFP in PAE/EGFR cells. The cells were incubated with 1 ng/ml EGF-Rh for 10 minutes at 37°C and fixed. EGF-Rh, YFP and CFP were detected using Cy3, YFP and CFP filter channels. Optical sections from the bottom of the cells are shown. 'Yellow' signifies colocalization of YFP and rhodamine fluorescence. 'Cyan' signifies colocalization of CFP and rhodamine fluorescence. Insets show an enlargement of the outlined regions. An additional inset (lower panel, ß2-YFP image) shows the merge image of EGF-Rh and ß2-YFP, in which the EGF-Rh image (red) was left-shifted 4 pixels (250 nm) to allow better visualization of colocalization of red and green dots. The arrow points to a cell that does not express CFP-Rab5(S34N) and displays typical endosomal localization of EGF-Rh. The images are representative of at least four independent experiments. Bar, 10 µm. (B) GFP-dynamin mutant (DynK44A) was transiently expressed in PAE/EGFR cells. Cells were incubated with 1 ng/ml EGF-Rh for 10 minutes at 37°C. EGF-Rh and GFP-dynamin were detected using Cy3 and GFP filter channels. Insets show an enlargement of the outlined regions. 'Yellow' signifies colocalization of GFP and rhodamine fluorescence. The images are representative of at least four independent experiments. Bar, 10 µm.

View larger version (78K): [in a new window]   Fig. 5. Detection of Rab5-CAAX activity in the plasma membrane. (A) Schematic representation and immunodetection of fusion proteins. Depicted is CFP-Rab5 protein fused to the 20-residue CAAX module of K-Ras4B at the C-terminus (CFP-Rab5-CAAX). PAE/EGFR cells transiently expressing CFP-Rab5-CAAX or its mutants, Q79L and S34N, were lysed, and the CFP-fusion proteins present in lysates were detected by western blotting with anti-Rab5 antibodies. All fusion proteins migrated on SDS-PAGE according to predicted molecular masses. (B) CFPRab5(Q79L)-CAAX was co-expressed with R5BD-YFP or YFP-EEA.1sh in PAE/EGFR cells. YFP, CFP and FRETC images were obtained as in Fig. 2. Calculated FRET images presented as pseudocolor intensity-modulated images (FRETC/CFP) (see Materials and Methods). Insets show an enlargement of the outlined regions. Arrows point to the absence of YFP-EEA.1sh associated with CFP-Rab5(Q79L)-CAAX at the plasma membrane. A.l.u.f.i, arbitrary linear units of fluorescence intensity. Bars, 10 µm.

View larger version (91K): [in a new window]   Fig. 6. Effect of CFP-Rab5-CAAX on EGF receptor endocytosis. CFP-Rab5-CAAX, CFP-Rab5(Q79L)-CAAX and CFPRab5(S34N)-CAAX were transiently cotransfected with ß2-YFP in PAE/EGFR cells. The cells were incubated with 1 ng/ml EGF-Rh for 10 minutes at 37°C and fixed. EGF-Rh, YFP and CFP were detected using Cy3, YFP and CFP filter channels. Optical sections close to the bottom of the cell are shown. 'Yellow' signifies colocalization of YFP and rhodamine fluorescence. 'Cyan' signifies colocalization of CFP and rhodamine fluorescence. Insets show an enlargement of the outlined regions. Additional insets (lower panel, ß2-YFP image) show the merge images of EGF-Rh and ß2-YFP, in which the EGF-Rh image (red) was right-shifted 4 pixels (250 nm) to allow better visualization of colocalization of red and green dots. The arrows point to a cell that does not express CFP-Rab5(S34N)CAAX and that displays typical endosomal localization of EGF-Rh. Note colocalization of EGF-Rh with CFP-Rab5(Q79L)-CAAX and CFP-Rab5-CAAX in endosomes is seen as 'donut-shape' structures. Bar, 10 µM.

View larger version (66K): [in a new window]   Fig. 7. Colocalization of clathrin and EGFRh in cells expressing the dominant-negative Rab5-CAAX mutant. CFP-Rab5-CAAX and CFP-Rab5(S34N)-CAAX were transiently co-transfected with YFP-CLA in PAE/EGFR cells. The cells were incubated with EGF-Rh as in Fig. 6. EGF-Rh, YFP and CFP were detected using Cy3, YFP and CFP filter channels. 'Yellow' signifies colocalization of YFP and rhodamine fluorescence, whereas 'cyan' signifies colocalization of CFP and rhodamine fluorescence. Insets show an enlargement of the outlined regions. Additional insets below the YFP image show the merge image of EGF-Rh and ß2-YFP, in which the EGF-Rh image (red) was right-shifted 4 pixels (250 nm) to allow better visualization of colocalization of red and green dots. The arrows show an example of EGF-Rh-containing endosomes in a cell that does not express CFP-Rab5(S34N)-CAAX.

View larger version (70K): [in a new window]   Fig. 8. The effect of CFP-Rab5-CAAX proteins on transferrin endocytosis. (A) CFP-Rab5-CAAX or CFPRab5(S34N)-CAAX were transiently co-expressed with ß2-YFP in PAE/EGFR cells. The cells were incubated with 5 µg/ml Trf-TR for 10 minutes at 37°C and fixed. Tfr-TR, YFP and CFP were detected using Cy3, YFP and CFP filter channels. 'Yellow' signifies colocalization of YFP and rhodamine fluorescence, whereas 'cyan' signifies colocalization of CFP and rhodamine fluorescence. Insets show an enlargement of the outlined regions. Additional inset (low panel, YFP image) shows the merge image of Trf-TR and ß2-YFP, in which the Trf-TR image (red) was right-shifted 4 pixels (250 nm) to allow better visualization of the overlap of red and green dots. The rhodamine image (low panel) is shown overexposed to visualize low levels of Trf-TR fluorescence in the cell expressing CFPRab5(S34N)-CAAX. This resulted in saturation of intense endosomal signals of Trf-TR in neighboring cells not expressing CFP-Rab5(S34N)-CAAX. Bar, 10 µM. (B) Gallery of high-magnification images from several cells expressing CFP-Rab5(S34N)-CAAX and treated as in (A) that show Tfr-TR colocalization with ß2-YFP.