QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online October 27, 2005
doi: 10.1242/10.1242/jcs.02628

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Giese, B. Articles by Müller-Newen, G. Search for Related Content PubMed PubMed Citation Articles by Giese, B. Articles by Müller-Newen, G. Social Bookmarking                         What's this?

Dimerization of the cytokine receptors gp130 and LIFR analysed in single cells

Bernd Giese^*, Christoph Roderburg^*, Michael Sommerauer, Saskia B. Wortmann, Silke Metz, Peter C. Heinrich and Gerhard Müller-Newen

Institut für Biochemie, Universitätsklinikum RWTH Aachen, Pauwelsstraße 30, 52074 Aachen, Germany

View larger version (54K): [in a new window]   Fig. 1. Characterization of YFP-IL-6 and binding to gp130. (A) YFP-IL-6 consists of the signal sequence for secretion (black, 29 amino acids) followed by the YFP moiety (yellow, 238 amino acids) and mature human IL-6 (grey, 184 amino acids). (B) Ba/F3-gp130-IL-6R cells were left unstimulated or were stimulated for 15 minutes with 20 ng/ml IL-6 or 40 ng/ml purified YFP-IL-6. Phosphorylation of STAT3 was analysed by immunoblotting of cellular lysates using an antibody against tyrosine-phosphorylated STAT3. Equal loading was controlled using a STAT3 antibody. (C) COS-7 cells were transfected with an expression vector encoding gp130-CFP. 48 hours after transfection, confocal images from living cells incubated in a perfusion chamber at 37°C were taken using the YFP and CFP channels of the confocal microscope. Cells were stimulated with YFP-IL-6 (40 ng/ml) and sIL-6R (1 µg/ml) (upper row) or with YFP-IL-6 alone (lower row). Confocal images were taken at the time points indicated. Bar, 10 µm.

View larger version (69K): [in a new window]   Fig. 2. Binding of YFP-IL-6 to gp130 deletion mutants. (A) COS-7 cells were transfected with an expression vector encoding human gp130/id-CFP, gp130/D1id-CFP or gp130/D2,3id-CFP (lower schemes). The cytoplasmic parts of the gp130 constructs are deleted after Pro668. Therefore, the constructs lack the dileucine internalization motif resulting in enhanced surface expression (id, internalization deficient). Cells were fixed 48 hours after transfection. Non-permeabilized cells were stained for immunofluorescence using the antibody B-T2 against D1 (upper row), the antibody B-P8 against CBM/D2,D3 (middle row) or the antibody B-P4 against D4 of gp130 (lower row) followed by a PE-coupled secondary antibody. In the confocal images PE fluorescence is red and CFP fluorescence is blue. COS-7 cells were transfected with an expression vector encoding gp130/id-CFP (B), gp130/D1id-CFP (C) or gp130/D2,3id-CFP (D). 48 hours after transfection, living cells were incubated in a perfusion chamber at 37°C. Confocal images were taken 10 minutes after addition of YFP-IL-6 (40 ng/ml) and sIL-6R (1 µg/ml). Bar, 20 µm.

View larger version (42K): [in a new window]   Fig. 3. Detection of FRET at the plasma membrane by acceptor photobleaching. Hek293T cells were transfected with an expression vector encoding gp130/id-C/YFP. 48 hours after transfection cells were fixed and FRET between CFP and YFP was measured using a confocal microscope. (A) YFP (yellow) and CFP (blue) fluorescence of a cell expressing gp130/id-C/YFP selected for qualitative FRET detection is shown on the left. Rows of ten consecutive images represent the fluorescence intensities of YFP and CFP in a plasma membrane region of the selected cell in false colour mode showing highest and lowest fluorescence intensities in yellow and black, respectively. Within the rectangular ROI, YFP is bleached after recording of image number 5. (B) For quantitative FRET analysis, fluorescence intensities of CFP (blue) and YFP (yellow) during a time series of ten images in a ROI at the plasma membrane of a Hek293T cell expressing gp130/id-C/YFP was recorded. YFP was bleached after image 5. Fluorescence of CFP and YFP was recorded simultaneously as described in Materials and Methods. CFP fluorescence intensities of images 5 and 6 were used for quantification of FRET efficiency. (C) Comparison of individual FRET efficiencies at the plasma membrane of cells expressing gp130/id-C/YFP (red squares) or gp130/id-CFP (blue diamonds). The FRET efficiencies are given as a function of the average fluorescence intensity of CFP and YFP. A different cell was selected for each FRET measurement. (D) Mean FRET efficiencies (±s.d.) at the plasma membrane of cells expressing gp130/id-CFP (white bar) or gp130/id-C/YFP (grey bar). Gp130/id-CFP is termed `negative control' and gp130/id-C/YFP `positive control' for further evaluations. Bar, 10 µm.

View larger version (23K): [in a new window]   Fig. 4. Analysis of gp130 homodimerization and gp130/LIFR heterodimerization by FRET. (A) Hek293T cells were transfected with expression vectors encoding gp130/id-CFP and gp130/id-YFP. 48 hours after transfection, cells were stimulated with IL-6 (20 ng/ml) and sIL-6R (1 µg/ml) (red squares) or left unstimulated (blue diamonds). 15 minutes after stimulation, cells were fixed and analysed by quantitative FRET at the confocal microscope. Individual FRET efficiencies at the plasma membrane of cells expressing gp130/id-CFP and gp130/id-YFP are given as a function of the excess of YFP (acceptor) fluorescence (left) or as a function of the average fluorescence intensity of CFP and YFP (right). Individual cells were selected for each FRET measurement. (B) Mean values of the individual FRET efficiencies at the plasma membrane of unstimulated (light grey) or stimulated (black) cells expressing gp130/id-CFP and gp130/id-YFP in comparison to FRET negative and positive control (±s.d.). Significant differences (***P<0.001) in FRET efficiency were observed between experimental groups and the two control groups as indicated. Dashed bars represent the calculated FRET efficiencies for exclusive heterodimerization between CFP and YFP. (C) Hek293T cells were transfected with expression vectors encoding human LIFR/id-CFP and gp130/id-YFP. 48 hours after transfection, cells were stimulated with LIF (20 ng/ml) (red squares) or left unstimulated (blue diamonds). 15 minutes after stimulation, cells were fixed and analysed by quantitative FRET at the confocal microscope. Individual FRET efficiencies at the plasma membrane of cells expressing LIFR/id-CFP and gp130/id-YFP are given as a function of the excess of YFP (acceptor) fluorescence (left diagram) or as a function of the average fluorescence intensities of CFP and YFP (right diagram). Individual cells were selected for each FRET measurement. (D) Mean values of the individual FRET efficiencies at the plasma membrane of unstimulated (light grey) or stimulated (black) cells expressing LIFR/id-CFP and gp130/id-YFP in comparison to FRET negative and positive control (±s.d.). Significant differences in FRET efficiency (***P<0.001) were observed between groups as indicated.

View larger version (63K): [in a new window]   Fig. 5. Analysis of gp130 homodimerization and gp130/LIFR heterodimerization by BiFC with confocal microscopy. (A) Hek293T cells were transfected with expression vectors encoding gp130/id-YC173 and gp130/id-YN173. Cells were incubated for 30 hours at 37°C, subsequently incubated for 18 hours at 30°C and afterwards analysed by FACS. 4 hours before temperature reduction, the cells were stimulated with IL-6 (20 ng/ml) and sIL-6R (1 µg/ml) or left unstimulated. Histograms represent unstimulated cells (dark grey), stimulated cells (black) or cells that expressed only gp130/id-YC173 (light grey). The scheme (right) depicts the complementation between the fragments of the fluorophores fused to gp130. (B) Hek293T cells were transfected and stimulated as described in A. Images of unstimulated (left) and stimulated cells (right) with high (upper row) and low magnification (lower row) were taken by using the YFP channel of the confocal microscope. (C) Proportion of the area of cells with BiFC fluorescence (ABiFC) in the total cell covered area (Acells) in low magnification images (n=30) of unstimulated cells (light grey), stimulated cells (dark grey) and cells that had expressed only gp130/id-YC173 (white) as a BiFC negative control (±s.d.). Significant differences in fluorescence ratio (***P<0.001) were observed between groups as indicated. (D) Hek293T cells were transfected with expression vectors encoding gp130/id-YC173 and LIFR/id-CN173. Cells were incubated for 30 hours at 37°C, subsequently incubated for 18 hours at 30°C and afterwards analysed by FACS. 4 hours before temperature reduction, the cells were stimulated with LIF (20 ng/ml) or left unstimulated. Histograms represent unstimulated cells (dark grey), stimulated cells (black) or cells that expressed only gp130/id-YC173 (light grey).The scheme (right) represents the complementation between the fragments of the fluorophores fused to LIFR and gp130. (E) Hek293T cells were transfected and stimulated as described in D. Images of unstimulated (left) and stimulated cells (right) under high (upper row) and low magnification (lower row) were taken using a channel of the confocal microscope adapted to the characteristics of the new fluorophore formed by YC173 and CN173 complementation. (F) Proportion of the cell area with BiFC fluorescence (ABiFC) in the total cell covered area (Acells) in low magnification images (n=30) of unstimulated cells (light grey), stimulated cells (dark grey) and cells that had expressed only gp130/id-YC173 (white) as a BiFC negative control (±s.d.). Significant differences in fluorescence ratio (*P<0.05 and ***P<0.001) were observed between groups as indicated. Bar, 10 µm (upper images in E,B); 100 µm (lower images in E,B).

View larger version (18K): [in a new window]   Fig. 6. Model for the dimerization of gp130 and LIFR. According to our data, a large portion of gp130 exists as a transient homodimer in the plasma membrane (a). This preformed homodimer is stabilized upon binding of IL-6/IL-6R complexes (b). Monomeric gp130 binds IL-6/IL-6R complexes less efficiently (c) and therefore only marginally contributes to IL-6-signalling. A gp130/LIFR heterodimer is formed mainly in response to LIF stimulation (d).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter