Figure 1.
4.1R80/135 and ICln interactions in HEK cells: co-immunoprecipitation and FRET.
(A) Co-immuprecipitation of FLAG-ICln and endogenous 4.1R in HEK cells. Anti-4.1R (4.1R) and anti-FLAG (FLAG) were respectively used to detect 4.1R and FLAGed proteins. Western blot showing immunoprecipitation of 4.1R with Flag-ICln, but not FLAG-BAP (control). (B) Images of an acceptor photobleaching FRET experiment using living cells over-expressing Y-4.1R80 and C-ICln. Pre-photobleaching (PRE pb) and post-photobleaching (POST pb) images are shown. Scale bar: 10 µm. (C) Quantification of FRET experiments with CFP-tagged ICln and YFP-tagged 4.1R80. The mean FRETeff ± SEM is plotted (*p<0.05 for Y-4.1R80+C-ICln vs Y-4.1R80+C; one-way ANOVA). The numbers inside the bars represent the number of cells analysed from at least 3 independent experiments. (D) Co-immunoprecipitation of Y-4.1R80 or (E) Y-4.1R135 with FLAG-ICln (ICln) or FLAG-tagged bovine alkaline peroxidase (BAP, control). The HEK cells were co-transfected with C-terminally FLAGed ICln and Y-4.1R80 or Y-4.1R135. FLAG-ICln was immunopurified using an anti-FLAG antibody. The 4.1R signal (anti-GFP antibody) and FLAG signal (anti-FLAG antibody) in cell lysates (L), and three sequential 40 µl eluates (E1-E3) are shown for all conditions.
Figure 2.
ICln over-expression affects 4.1R membrane localisation.
(A) The images in the first row show the intracellular localisation of the indicated proteins (single confocal planes); the unshown co-transfected protein is indicated in brackets. The images in the second row are enlargements of the insets indicated in the first row images. (B) Exemplificative images of HEK cells co-transfected with GFP-IRES-4.1R80 (4.1R80) or GFP-IRES-4.1R135 (4.1R135) and CFP (C) or CFP-ICln (C-ICln) vectors. The samples were immunolabelled with an anti-4.1R antibody to visualise the 4.1R signal. In the panels showing the endogenous 4.1R signal, the asterisks indicate the CFP or C-ICln transfected cells. Scale bar 10 µm. (C) Effect of ICln on endogenous 4.1R membrane localisation: Western blot of total membrane protein extracts (left) and total endogenous 4.1R (right) from HEK cells transfected with C-ICln or C (control). The histograms show the mean OD value of the 4.1R signal normalised for the corresponding cadherin (left) or tubulin (right) signal (n = 4). The values are percentages of the control. **p<0.01; *p<0.05.
Figure 3.
Downregulation of ICln by siRNA and 4.1R membrane localisation.
(A) Exemplificative images of HEK cells co-transfected with the ptdTOMATO-N1vector and scrambled (ctrl) or ICln siRNA (ICln). The samples were immunolabelled with an anti-4.1R antibody (4.1R) or anti-ICln antibody (ICln). Scale bar 20 µm. (B) Western blot of total protein extracts from HEK cells co-transfected with ICln siRNA (ICln) or scrambled siRNA (ctrl) and the fluorescent tdTomato protein. The histograms show the mean OD value of the ICln signal normalised for the corresponding GAPDH signal (n = 4). The values are percentages of the control. **p<0.01.
Figure 4.
FRET analysis of YFP-tagged 4.1R and CFP/β-actin (C- βactin) interaction.
(A) Example of an acceptor photobleaching FRET experiment in HEK cells over-expressing Y-4.1R135 and C-βActin. A FRET signal can be seen under the plasma membrane (asterisks). Scale bar 5 µm. FRETeff was measured in ROIs in the plasma membrane or cytoplasm. (B) Mean FRETeff ± SEM in cells over-expressing Y-4.1R135, C-βActin and IRES-DsRED (-ICln) or Y-4.1R135, C-βActin and ICln-IRES-DsRED (+ICln). ***p<0.001, -ICln vs +ICln. (C, D) Co-immuprecipitation of over-expressed 4.1R80 or 4.1R135 and actin. (C) HEK cells were transfected with GFP-IRES-4.1R80/135, and actin was immunoprecipitated using a goat anti-actin antibody (samples 135 or 80). Goat Ig-G (bovine alkaline peroxidase) was used as a negative control (ctr). (D) C-ICln (+ICln) or C (-ICln) was co-transfected with 4.1R to investigate the effect of ICln on 4.1R/actin interactions. The Western blots are representative of three (4.1R135) or two (4.1R80) independent experiments, all with comparable results. The 4.1R (anti-4.1R) and actin signals (anti-Act) in the cell lysates (L) and final eluates (E) are shown for all conditions. The additional bands in the 4.1R80 Western blot in D are probably residual incompletely denatured (anti-4.1R blot) or denatured (anti-actin blot) antibody chains as they were also recognised by the sole secondary anti-goat antibody.
Figure 5.
ICl,swell characterisation in cells over-expressing 4.1R80/135.
(A) Representative whole-cell traces recorded in control cells (over-expressing GFP) or cells over-expressing the 4.1R80 or the 4.1R135 protein exposed to hypertonic (Hyper) and hypotonic (Hypo) extracellular solutions. (B) Relationship between mean current density, d (pA/pF), and membrane voltage, V (mV), in cells over-expressing the indicated proteins and exposed to the hypotonic extracellular solution for 10 min (GFP: n = 22, 4.1R80: n = 15, 4.1R135: n = 14). (C) Chloride current activation during hypotonic exposure (GFP: n = 24, 4.1R80: n = 17, 4.1R135: n = 14. (D,E) Relationship between mean current density and membrane voltage in control cells or cells over-expressing the 4.1R80 (C) or the 4.1R135 protein (D) in the hypertonic extracellular solution (GFP: n = 43, 4.1R80: n = 38, 4.1R135: n = 27. *p<0.05; ***p<0.001. Two-way ANOVA.
Figure 6.
Hypotonic shock affects the membrane localisation of 4.1R, and promotes the 4.1R/ICln interaction.
(A) Single confocal plane of a cell expressing the membrane marker CFP-mem (Cm) and the Y-4.1R135 protein acquired in the hypertonic (hyper) and hypotonic (hypo 10′) extracellular solutions. The arrows indicate the membrane regions in which the 4.1R135 signal greatly decreases after hypotonic exposure. The mean Pearson (B) and Manders coefficients (C) between Cm and the YFP-tagged proteins indicated in the graphs were calculated from the Z-stacks. The Manders coefficient represents the fraction of the YFP signal overlapping the CFP signal. For each sample, the mean coefficients obtained in hypertonic extracellular solution were compared with the mean coefficients obtained under hypotonic conditions (ANOVA with Dunnett's post-hoc multiple comparison test; *p<0.05). Scale bar 10 µm. (D) Western blot of endogenous 4.1R in total membrane preparations of HEK cells under hypertonic (hyper) or hypotonic (hypo) conditions. The related graph shows the mean OD of the 4.1R bands normalised for those of the Na/K pump (OD4.1R/ODNaK) (n = 8) used to quantify the changes. The values are percentages of the hypertonic condition. *p<0.05. (E) Representative NFRET images of Y-4.1R80/C-ICln expressing cells in the hypertonic extracellular solution or after 10 min exposure to the hypotonic extracellular solution, and the NFRET time course during RVD. In comparison with the hypertonic condition, the NFRET signal in the Y-4.1R80/C-ICln expressing cells (but not the control Y-4.1R80/C expressing cells) significantly increased after five minutes in the hypotonic extracellular solution (one-way ANOVA with Dunnett's multiple comparison test). The NFRET values of the Y-4.1R80/C-ICln expressing cells in the hypertonic extracellular solution were statistically different from those of the control Y-4.1R80/C cells (t-test) after both 0 (hypertonic) and 10 min hypotonic solution exposure. Scale bar 10 µm. ***p<0.001; **p<0.01.
Figure 7.
Morphological analysis of HEK cells over-expressing 4.1R and ICln.
(A) The maximum projections of a number of optical sections over-expressing the indicated proteins and covering at least half of the cell (z-step size 0.25 µm). Only the YFP channel is shown. Scale bar: 20 µm. (B) The confocal plane of the cell attached to the coverglass acquired using the confocal or gated-STED (g-STED) module of a Leica TCS SP8. The cell over-expressed a membrane marker (YFP-mem) and the 4.1R135 protein. Only the YFP signal is shown. Scale bar is 5 µm, and 0.5 µm in 1 and 2. (C) Example of SEM acquisitions of cells transfected with GFP-IRES-4.1R135; the lower image is the magnified view of the inset, and makes it possible to see the mesh of cellular filopodia. Scale bar 10 µm (5 µm in the inset). (D) The histogram shows the mean cell area ± SEM (µm2) calculated from the SEM images. (E) The mean density of filopodia per cell (the number of filopodia per cell/cell perimeter (µm)). The numbers in the bars represent the number of analysed cells from three independent experiments. *p<0.05; ***p<0.001.