4. Results
4.6 Regulation of membranous structures by p63RhoGEF overexpression in adult mouse
Co-localization analysis of p63RhoGEF and caveolin-3 in confocal center view images revealed (Fig. 22 A) that when p63RhoGEF is relatively low expressed, its correlation with caveolin-3 is higher than vice versa. This is mainly due to its predominant perinuclear localization and the lacking sarcolemmal localization. In highly expressing cells both proteins co-localize to a similar extent to each other, which can be explained by the increasing localization of p63RhoGEF at the sarcolemma (Fig. 22 B). However, by analyzing surface view images of both proteins, the obtained correlation coefficients were remarkable lower as those detected in center view images (Fig. 22 C). This is mainly based on the missing signal resolution at the sarcolemma in center view images. In addition to the degree of co-localization of both proteins, the impact of p63RhoGEF on the distribution of caveolin-3 was assessed.
Interestingly, the accumulation of p63RhoGEF in the perinuclear region, which is independent of its expression height, led to an increased accumulation of caveolin-3 in the same area. The observed increase of p63RhoGEF at the sarcolemma in highly transduced cells did not result in higher caveolin-3 levels, instead its intracellular amount was decreased (Fig. 22 D).
Fig. 22: Analysis of the co-localization of p63RhoGEF and of caveolin-3 in AMCM.
Isolated adult mouse cardiomyocytes (AMCM) were transduced for up to 48 h with recombinant adenoviruses encoding for EGFP only (Ad-EGFP), or for EGFP and for full-length, c-myc-tagged p63RhoGEF (Ad-p63RhoGEF).
The localization of p63RhoGEF and caveolin-3 were visualized by immunofluorescence with help of a c-myc antibody (red) and an anti-caveolin-3 antibody (green), respectively. A) Representative center view images of p63RhoGEF and caveolin-3 are given of control transduced (Ad-EGFP) and Ad-p63RhoGEF-transduced cells showing low and high p63RhoGEF expression. The merges are presented below. Scale bar 20 µm. B) Co-localization of p63RhoGEF and caveolin-3 was analyzed by the ImageJ plugin Coloc2 in center view images. M1 reflects the co-localization of p63RhoGEF with caveolin-3 and M2 of caveolin-3 with p63RhoGEF. In total 20 to 25 cells were analyzed from 3 independent transduction experiments. Given are the means ± SEM, *p<0.05.
C) Representative magnifications of surface view images are given of cells highly expressing p63RhoGEF. Co-localization of p63RhoGEF and caveolin-3 was analyzed by the ImageJ plugin Coloc2 in 4 cells in three different areas. The single values are shown together with their mean. D) The localization of caveolin-3 (left) and p63RhoGEF (right) was analyzed in control transduced cells (for caveolin-3 analysis, p63RhoGEF-no) and cells expressing low (p63RhoGEF-low) and high (p63RhoGEF-high) amounts of p63RhoGEF. In total 20 to 25 cells of 3 independent transduction experiments were analyzed. Given are the percentages as means ± SEM of the respective proteins in each compartment, *p<0.05.
As p63RhoGEF expression influenced the distribution of the membrane protein caveolin-3, its impact on the intracellular secretory membrane system was analyzed.
First, the co-localization of both p63RhoGEF constructs with proteins containing the ER retention and targeting motif KDEL (lysine - aspartic acid - glutamic acid - leucine) was investigated. Surprisingly, this revealed a very strong overlap between full-length p63RhoGEF and KDEL-containing proteins as well as between p63∆N and KDEL-proteins (Fig. 23 A).
Quantification of the localization demonstrated that p63RhoGEF and KDEL-proteins co-localized to a similar extent to each other, whereas the KDEL-proteins showed a higher correlation to p63∆N than vice versa (Fig. 23 B). Although a certain degree of co-localization was also found for caveolin-3, the surprising increase in KDEL-signal intensity in cells overexpressing the two p63RhoGEF constructs compared to control transduced cells was unusual. To further investigate this phenomenon, immunoblot studies were performed.
However, with the used antibody no difference in the detected KDEL-proteins were found (data not shown), but instead the p63RhoGEF constructs could be detected. This is exemplarily shown for p63∆N in Fig. 23 C. Thus, it can be assumed that the observed co-localization results from an experimental artefact. The reason for this artefact could lay in the recognition of the antibody of the C-terminal EDEL sequence of p63RhoGEF.
Fig. 23: Analysis of the co-localization of p63RhoGEF and of KDEL-proteins in AMCM.
Isolated AMCM were transduced for up to 48 h with recombinant adenoviruses encoding EGFP only (Ad-EGFP), EGFP and full-length tagged p63RhoGEF (Ad-p63RhoGEF), or EGFP and N-terminally truncated c-myc-tagged p63RhoGEF (Ad-p63ΔN). The localization of c-myc c-myc-tagged p63RhoGEFand p63∆N were detected with an anti-c-myc antibody, KDEL proteins were detected with an antibody against the KDEL-motif. Immunostained AMCM were visualized by confocal microscopy and confocal images were used for co-localization analyses. A) The representative images of complete cells show an overlay of all four channels. Magnifications (below) show all individual channels and overlay excluding the EGFP (gray scale) signal. Scale bar: 25 µm or 10 µm. B) Co-localization of p63RhoGEF constructs and KDEL-proteins was analyzed by the ImageJ plugin Coloc2 in center view images. M1 reflects the co-localization of the respective p63RhoGEF construct with KDEL and M2 of KDEL with the p63RhoGEF construct. In total 31 cells per condition were analyzed from 3 independent transduction experiments.
Given are the means ± SEM, *p<0.05. C) Representative Immunoblots of c-myc and KDEL in cell lysates from
AMCM transduced either with Ad-EGFP or with Ad-p63∆N. For immunoblotting, the same antibodies were used as for the immunostains shown in A) The arrows indicate the specific p63∆N bands.
Second, localization analysis of the cis-Golgi matrix protein GM-130 was performed together with the p63RhoGEF constructs. Independent of the used adenoviral constructs, GM-130 was found in discrete speckles in the perinuclear region and in speckles distributed over the cells (Fig. 24 A). Furthermore, GM-130 was located close to p63RhoGEF accumulations in the in the Ad-p63RhoGEF-transduced cells. Quantification of the particle size revealed a significant decrease from 0.57 µm² to 0.52 µm² for p63RhoGEF and p63∆N compared to control AMCM expressing EGFP only. The average particle number per cell in one center view imaging plane changed significantly from 42 particles in AMCM overexpressing EGFP only, to 45 particles for p63∆N and to 58 particles per AMCM in p63RhoGEF transduced AMCM. Moreover, p63RhoGEF expression led to a slight, but significant reduction of GM-130 in the perinuclear region. Instead more GM-130 could be detected in the cell periphery (Fig. 24 B).
Fig. 24: Analysis of the co-localization of p63RhoGEF and of the cis-Golgi matrix protein GM-130 in AMCM.
Isolated adult mouse cardiomyocytes (AMCM) were transduced for up to 48 h with recombinant adenoviruses encoding for EGFP only (Ad-EGFP), or for EGFP and for full-length, c-myc-tagged p63RhoGEF (Ad-p63RhoGEF), or for EGFP and for N-terminally, c-myc-tagged truncated p63RhoGEF (Ad-p63ΔN). The localization of p63RhoGEF was visualized by immunofluorescence with the help of a c-myc antibody (red) and the Golgi apparatus with an antibody against the cis-Golgi matrix protein GM-130. Transduced cells were identified by EGFP (gray scale) expression. Co-staining of nuclei with DAPI (blue) was performed. A) Representative center view confocal images are shown as an overlay of all four channels. Magnifications (below) show all individual channels and overlay excluding the EGFP signal. Scale bar: 25 µm. B) Analysis of the Golgi particle size (upper) and the particle number (middle) was performed with the help of the particle analysis tool of ImageJ. In total 35 to 45 cells per group were analyzed. The distribution of GM-130 was analyzed by its fluorescence in the perinuclear region (selection of the nuclei with 2.5 µm enlargement) and in the rest of the cells. In total 20 to 25 cells per group were analyzed. All values are given as means ± SEM, n=4, *p<0.05.