1. Introduction

2. Material and methods

3. Results

3.1. Septin 9 is essential to orient apico-basal axis and its knock down inverts polarity

To decipher the role that septin 9 could play in the establishment of apico-basal polarity, we used MDCK cells cultured in 3D in Matrigel as a model system. After 4 days in culture, the control cells form cysts with a monolayer of polarized cells surrounding an open central lumen. Endogenous septin 9 was essentially presented at BM. However, knock down of septin 9 led to the formation of an important proportion of cysts with multiple lumens and inverted polarity (Figure 1A,B). Furthermore, septin 9 filamentous structure which is required to perform its biological activities. Importantly the filaments are formed by an octameric complex that included septin 2, septin 6, septin 7 (Kim et al. 2011) (Figure S1A). Subsequently we analyzed septin 7 and septin 2 and we found that the two proteins are located at BM as observed for septin 9 and depletion of septin 9 decreased their expressions as shown by immunofluorescence analysis (Figure S1B) and by immunoblot (Figure S1C). Together these data strongly suggested that septin 9 form filament structures at BM in a complex with septin 2 and septin 7.

Figure 1. Basolateral membrane localized Septin 9 is required for the orientation of apico-basal axis in MDCK cysts. (A). MDCK cells were transfected or not with siRNA of septin 9 for 24 h and then transferred to the Matrigel-covered Lab-Tek. The cells were incubated for 4 days to form cysts. Cells were stained for Septin 9 (green), GP135 (red) and Hoechst (blue). A single confocal section through the middle of a cyst is shown. Scale bar 10 µm. (B). Cysts presented different phenotype, including single open lumen, multiple-lumens and inverted polarity. Color for each phenotype in column is corresponding the color below pictures. (C). For another experiment as in (A), MDCK cysts sections labeled for the Golgi marker GM130 (green), actin (red) and Hoechst (blue). Higher magnifications of the boxed areas are shown. (D). Line profiles showing Golgi distribution from apical to basal area, using Image J. (E). Quantification of Golgi distribution: the ratio of maximal fluorescence intensity at the apical side versus the maximal fluorescence intensity at the basal side. (F). The schematic graph shows that Golgi distributions associated with different cyst phenotypes including cell polarity and lumen formation. Data information: Data are at least two times replicates. n=69 (Control), n=32 (siRNA) (B). n=10 (E). The statistics values are means ±s.e.m. Student’s t-test was used. ***P < 0.001.

Figure 1. Basolateral membrane localized Septin 9 is required for the orientation of apico-basal axis in MDCK cysts. (A). MDCK cells were transfected or not with siRNA of septin 9 for 24 h and then transferred to the Matrigel-covered Lab-Tek. The cells were incubated for 4 days to form cysts. Cells were stained for Septin 9 (green), GP135 (red) and Hoechst (blue). A single confocal section through the middle of a cyst is shown. Scale bar 10 µm. (B). Cysts presented different phenotype, including single open lumen, multiple-lumens and inverted polarity. Color for each phenotype in column is corresponding the color below pictures. (C). For another experiment as in (A), MDCK cysts sections labeled for the Golgi marker GM130 (green), actin (red) and Hoechst (blue). Higher magnifications of the boxed areas are shown. (D). Line profiles showing Golgi distribution from apical to basal area, using Image J. (E). Quantification of Golgi distribution: the ratio of maximal fluorescence intensity at the apical side versus the maximal fluorescence intensity at the basal side. (F). The schematic graph shows that Golgi distributions associated with different cyst phenotypes including cell polarity and lumen formation. Data information: Data are at least two times replicates. n=69 (Control), n=32 (siRNA) (B). n=10 (E). The statistics values are means ±s.e.m. Student’s t-test was used. ***P < 0.001.

The structure, organization, and positioning of the Golgi apparatus are implicated in the apico-basal polarization process (Omrane et al. 2019b). Given that septin 9 regulates Golgi asymmetrically assembly (Omrane et al. 2019b), we assessed whether the disrupted apical-basal polarization seen in septin 9-depleted cysts also affected Golgi organization. Subsequently we stained for Golgi using an antibody against the Golgi matrix protein GM130. In control cells, the Golgi was localized above the nucleus in the apical part of the cytoplasm toward the cyst lumen as expected (Figure 1C). By contrast, in inverted polarized cysts formed by cells treated with siRNA septin 9, the Golgi was disorganized and was localized behind the nucleus in the basal part of the cytoplasm beneath the intense actin signal at cell periphery (Figure 1C–F). Together, these data suggested a specific role of septin 9 in the orientation of Golgi positioning and the orientation of apico-basal polarity axis.

3.2. Septin 9 regulates cell-cell junctions and BM stability

The proper localization and assembly of cell-cell junctions are essential to establishment of apico-basal polarity and the BM stability (Figure 2A). At the BM, β-catenin links the cytoplasmic domain of E-cadherin with the actin-binding proteins such as cortactin to organize the cortical actin networks and maintain therefore the adherens junctions (Sroka et al. 2016) (Figure 2A). According to the septin 9 localization at BM membrane and its effects on apico-basal polarity, we investigated the consequences of septin 9 depletion on the cell-cell junctions. We also analyzed the tight junctions protein, Zonula occludens-1 (ZO-1) which plays a crucial role in the conversion of AJs to belt-like structures in polarized epithelial cells (Ikenouchi et al. 2007). The results showed that knock-down of septin 9 expression using siRNA decreased the expression of both E-cadherin and β-catenin (Figure 2B). Loss of cortactin by septin 9 depletion was also validated in 3D culture by immunofluorescence analysis (Figure 2B). These immunofluorescence data were validated by immunoblots (Figure 2C).

Figure 2. Septin 9 regulates impacts cell-cell adhesion and BM stability. (A). The schematic representation of the cell-cell adhesion structure. In the cytoplasm, e-cadherin/catenin complex link to ZO-1 and actin, and actin binding protein cortactin. (B). MDCK cells were transfected or not with siRNA of septin 9 for 24h and plated on Matrigel for 4 days to form cysts, and then stained for E-cadherin (green) and actin (red), β-catenin (green) and ZO-1 (red), and cortactin (green) and actin (red). A single confocal section through the middle of a cyst is shown. Quantification of the fluorescence intensity of each protein expression, E-cadherin (green), and β-catenin (green) and ZO-1 (red). Scale bar 10 µm. (C). MDCK cells transfected or not with siRNA of septin9 for 24 h then grown on plates for 3 days. Cells were lysed and analyzed by western blot for septin 9, E-cadherin, β-catenin, and cortactin proteins expressions. (D). For another experiment as in (C), qRT-PCR analysis of the expression of mRNA encoding septin 9, vimentin, N-cadherin and ZEB1 in cells with Control and septin 9 knock down in 2D condition. Data information: Data are at least two times replicates and cysts (n>10) for 3D staining, three replicates for immunoblot and qRT-PCR. The statistics values are means ±s.e.m. Student’s t-test was used. *P<0.05, **P<0.01, ***P < 0.001.

Figure 2. Septin 9 regulates impacts cell-cell adhesion and BM stability. (A). The schematic representation of the cell-cell adhesion structure. In the cytoplasm, e-cadherin/catenin complex link to ZO-1 and actin, and actin binding protein cortactin. (B). MDCK cells were transfected or not with siRNA of septin 9 for 24h and plated on Matrigel for 4 days to form cysts, and then stained for E-cadherin (green) and actin (red), β-catenin (green) and ZO-1 (red), and cortactin (green) and actin (red). A single confocal section through the middle of a cyst is shown. Quantification of the fluorescence intensity of each protein expression, E-cadherin (green), and β-catenin (green) and ZO-1 (red). Scale bar 10 µm. (C). MDCK cells transfected or not with siRNA of septin9 for 24 h then grown on plates for 3 days. Cells were lysed and analyzed by western blot for septin 9, E-cadherin, β-catenin, and cortactin proteins expressions. (D). For another experiment as in (C), qRT-PCR analysis of the expression of mRNA encoding septin 9, vimentin, N-cadherin and ZEB1 in cells with Control and septin 9 knock down in 2D condition. Data information: Data are at least two times replicates and cysts (n>10) for 3D staining, three replicates for immunoblot and qRT-PCR. The statistics values are means ±s.e.m. Student’s t-test was used. *P<0.05, **P<0.01, ***P < 0.001.

The loss of cell-cell junctions is a fundamental event during the EMT, a process by which epithelial cell loss polarity to acquire mesenchymal and invasive features which characterized cancer cells (Nieto et al. 2016). Thus, using qRT-PCR analysis, we showed that the decrease of septin 9 significantly increased the gene expression of mesenchymal cell markers such as vimentin, EMT-inducing transcriptional factor (ZEB1) and N-cadherin (Figure 2D). Together, these results suggest a potential function of septins in driving the assembly, maintenance, and remodeling of adhesion junctions, impairing the cells to undergo EMT. Thus septin 9 regulates cell-cell junctions at BM and appeared as a gatekeeper against EMT.

3.3. The two PB domains of septin 9 are required for its basolateral localization and apico-basal polarity

We reported that septins have a second Polybasic Domain (PB2) that forms with PB1 a basic cluster at the NC Interface. In particular, we found that septin 9 PB domains control its filamentous structure formation and assembly and functionality of the Golgi apparatus (Omrane et al. 2019b:9) (Figure 3A). Accordingly, we sought for the potential requirement of the two PB domains of septin 9 in its role in apico-basal polarity. For that, we established different MDCK cells that stably expressed either empty vector (EV) or the V5 tagged-septin 9_i1 (septin 9_i1) or the mutants deleted from PB1 (septin 9_del1) or the mutants deleted from PB2 (septin 9_del2) or deleted from both PB1 and PB2 (septin 9_del1.2). The expression of the septin 9 protein in these cells was validated first by immunoblot using the V5 tag antibody (Figure 3B). Then, all the different cell lines were cultured in 3D to form cysts and stained for V5 tag, and actin (Figure 3C). The EV and septin 9_i1 expressing cells form cysts with an open central lumen and septin 9_i1 was enriched at BM as showed for endogenous septin 9 (Figure 1A). However, the lumen likely to be filled with septin 9_i1 cells compared to EV cells (Figure 3C). By contrast the mutants septin 9_del1 and del2 formed multi-lumen cysts while the septin 9_del1.2 cells formed in majority cysts with inverted phenotype (Figure 3C). The quantification of these different phenotypes was presented in the Figure 3D. Importantly, we observed a cytoplasmic localization of septin 9_del1, septin 9_del2 and septin 9_del1.2 (Figure 3C). The increase of septin 9 del1.2 cytoplasm accumulation was confirmed by cell fractionation (Figure 3E) and thus, confirmed that PB domains are required for septin 9 assembly (Omrane et al. 2019b:9). Together, these data indicated that the PB domains of septin 9 regulated its basolateral localization at the cell cortex, affecting cell shape and single lumen formation. We also validated that the Golgi was localized above the nucleus facing the lumen in EV and septin_i1 cysts whereas, in septin 9_del1.2 cells which formed cysts with an inverted phenotype, the Golgi was localized at the cell periphery as (Figure 3F–H). Furthermore, deletion of the two PB domains induced the presence of ZO-1 at the periphery of del1.2 cysts with the inverted polarity phenotype (Figure S2). In conclusion, preventing the assembly of septin 9 by the deletion of its two PB domains inverted apico-basal polarity.

Figure 3. Deletion of septin 9 PB domains impact different cyst phenotype and septin 9 distribution in the cells. (A). The schematic graph shows the organization of septin 9, in which the two PB domains are highlight in pink and green. And the two PB domains are near the NC interface of the protein. (B). The lysed MDCK stable cells which transfected with septin 9_i1 (I1), septin 9_del1 (Del1), septin 9_del2 (Del2) and septin 9_del1.2 (Del1.2) or empty vector (EV) are tested by western blot with septin 9-v5 tag antibody. (C). The MDCK stable cell lines plated on Matrigel for 4 days to form cysts, and then stained for septin 9-V5 tag (green) and actin (red). Septin 9_del1.2 (Del1.2) MDCK cell lines presented inverted phenotype. A single confocal section through the middle of a cyst is shown. Scale bar 10 µm. (D). Color for each phenotype in column is corresponding the color below pictures. (E). The MDCK stable cell lines were grown overnight before being subjected to a subcellular fractionation assay and analyzed with western blot for septin 9-v5 tag and actin. The graph besides shows septin 9-v5 tag densitometry analysis of the subcellular fractionation assay. (F). As experiment in (C), the cysts from MDCK stable cell lines of EV, i1 and Del1.2 were stained for Golgi (green) and actin (red). A single confocal section through the middle of a cyst is shown. Scale bar 10 µm. (G). Line profiles showing the Golgi distribution from the basal to the apical areas, using Image J. (H). Quantification of Golgi distribution: the ratio of maximal fluorescence intensity at the apical side versus the maximal fluorescence intensity at the basal side. Data information: Data are at least two times replicates and cysts (n>10) for 3D staining, three replicates for immunoblot. For D, n=23 (EV), n=29 (i1), n=38 (Del1), n=20 (Del2), n=44 (Del1.2). The statistics values are means ±s.e.m. Student’s t-test was used. *P<0.05, **P<0.01, ***P < 0.001.

Figure 3. Deletion of septin 9 PB domains impact different cyst phenotype and septin 9 distribution in the cells. (A). The schematic graph shows the organization of septin 9, in which the two PB domains are highlight in pink and green. And the two PB domains are near the NC interface of the protein. (B). The lysed MDCK stable cells which transfected with septin 9_i1 (I1), septin 9_del1 (Del1), septin 9_del2 (Del2) and septin 9_del1.2 (Del1.2) or empty vector (EV) are tested by western blot with septin 9-v5 tag antibody. (C). The MDCK stable cell lines plated on Matrigel for 4 days to form cysts, and then stained for septin 9-V5 tag (green) and actin (red). Septin 9_del1.2 (Del1.2) MDCK cell lines presented inverted phenotype. A single confocal section through the middle of a cyst is shown. Scale bar 10 µm. (D). Color for each phenotype in column is corresponding the color below pictures. (E). The MDCK stable cell lines were grown overnight before being subjected to a subcellular fractionation assay and analyzed with western blot for septin 9-v5 tag and actin. The graph besides shows septin 9-v5 tag densitometry analysis of the subcellular fractionation assay. (F). As experiment in (C), the cysts from MDCK stable cell lines of EV, i1 and Del1.2 were stained for Golgi (green) and actin (red). A single confocal section through the middle of a cyst is shown. Scale bar 10 µm. (G). Line profiles showing the Golgi distribution from the basal to the apical areas, using Image J. (H). Quantification of Golgi distribution: the ratio of maximal fluorescence intensity at the apical side versus the maximal fluorescence intensity at the basal side. Data information: Data are at least two times replicates and cysts (n>10) for 3D staining, three replicates for immunoblot. For D, n=23 (EV), n=29 (i1), n=38 (Del1), n=20 (Del2), n=44 (Del1.2). The statistics values are means ±s.e.m. Student’s t-test was used. *P<0.05, **P<0.01, ***P < 0.001.

3.4. The two PB domains of septin 9 are critical to maintain cell-cell junctions and BM stability

According to data presented above (Figure 2B,C), we explore the effects of the deletion of the two PB domains on E-cadherin expression (Figure 4A–C). We didn’t observe a striking decrease of E-cadherin expression rather E-cadherin decreased from the membrane and became more cytoplasmic as highlighted by line plots (Figure 4B,C). Furthermore, the cells were grown in 2D on semi-permeable filters. Even though this system did not allow lumen formation it permitted a different analysis of apico-basal axis. After 3 days of culture, the EV cells form a monolayer as seen by X-Y and X-Z sections from staining of β-catenin (Figure 4D). In cells expressing septin 9_i1, the β-catenin was well organized and the septin 9 is enriched in the BM clearly visible on X-Z section and formed filaments mostly at the basal part (Figure 4D). By contrast the septin 9_del1.2 lost its filamentous structure and was delocalized on the apical domain as better seen on X-Z section (Figure 4D). Remarkably in septin 9_del1.2 cysts, the signal of β-catenin was diffused and strongly reduced at the zonula adherens (ZAs) as indicated by the line plot and the 3D reconstruction of the septin 9 and β-catenin signals in a single cell from each group (Figure 4E,F). These results were supported by the quantification of the β-catenin cellular signal (Figure 4G). Together the data demonstrated that the two PB domains of septin 9 are required for its filaments and basolateral localization and to maintain cell-cell adhesion.

Figure 4. Deletion of septin 9 PB domains impacts cell-cell adhesion (A). The MDCK stable cell lines plated on Matrigel for 4 days to form cysts, and then stained for E-cadherin (green) and actin (red). A single confocal section through the middle of a cyst is shown. Scale bar 10 µm. (B). The distribution of the E-cadherin signal in cysts presented in (A) was analyzed by ImageJ software in an individual cell from a cyst. A circle was defined at the periphery of each cell and the plugin produced a profile plot of normalized integrated intensities in concentric circles as a function of distance from a point in the image, considered here as the center of the cell. The circle was divided into three bands (1, 2, 3) with an equal radius. (C). Quantification of the fluorescence intensity of E-cadherin from each band is represented as a histogram. (D). The MDCK stable cell lines of EV, i1 and Del1.2 plated on transwell filters for 24h. Cells were fixed and stained for V5 tag (green) and β-catenin (red). Confocal images of XZ sections are presented at the top. A single confocal section of the apical and basal part of the monolayer is shown in the middle. At the bottom of each column, the fluorescence of V5 tag (green) and β catenin (red) signals was scanned along the pink line drawn at a random position and presented in pink squares. Scale bar: 10 µm. (E). Arrowheads indicate the Zonula adherens. Bar graphs in red presents the fluorescence intensity of β-catenin in Zonula adherens. (F). The 3D reconstruction of the septin 9 and β-catenin signals in a single cell from each group. (G). Bar graphs in red present the percentage of β-catenin intensity in the basolateral and apical zones. Data information: Data are at least two times replicates and cysts (n=10) for 3D staining and cysts (n=30) for 2D staining. The statistics values are means ±s.e.m. Student’s t-test was used. *P<0.05, **P<0.01, ***P < 0.001.

Figure 4. Deletion of septin 9 PB domains impacts cell-cell adhesion (A). The MDCK stable cell lines plated on Matrigel for 4 days to form cysts, and then stained for E-cadherin (green) and actin (red). A single confocal section through the middle of a cyst is shown. Scale bar 10 µm. (B). The distribution of the E-cadherin signal in cysts presented in (A) was analyzed by ImageJ software in an individual cell from a cyst. A circle was defined at the periphery of each cell and the plugin produced a profile plot of normalized integrated intensities in concentric circles as a function of distance from a point in the image, considered here as the center of the cell. The circle was divided into three bands (1, 2, 3) with an equal radius. (C). Quantification of the fluorescence intensity of E-cadherin from each band is represented as a histogram. (D). The MDCK stable cell lines of EV, i1 and Del1.2 plated on transwell filters for 24h. Cells were fixed and stained for V5 tag (green) and β-catenin (red). Confocal images of XZ sections are presented at the top. A single confocal section of the apical and basal part of the monolayer is shown in the middle. At the bottom of each column, the fluorescence of V5 tag (green) and β catenin (red) signals was scanned along the pink line drawn at a random position and presented in pink squares. Scale bar: 10 µm. (E). Arrowheads indicate the Zonula adherens. Bar graphs in red presents the fluorescence intensity of β-catenin in Zonula adherens. (F). The 3D reconstruction of the septin 9 and β-catenin signals in a single cell from each group. (G). Bar graphs in red present the percentage of β-catenin intensity in the basolateral and apical zones. Data information: Data are at least two times replicates and cysts (n=10) for 3D staining and cysts (n=30) for 2D staining. The statistics values are means ±s.e.m. Student’s t-test was used. *P<0.05, **P<0.01, ***P < 0.001.

3.6. The PB domains of septin 9 regulate lumen formation at the different stages of the polarization process

To further decipher the importance of PB domains during the polarization process, the different MDCK cell lines were cultured on 3D and analyzed for β-catenin and the apical marker GP135 at different time points, starting from apical membrane initiation site (AMIS) formation after 1 day, the preapical patch (PAP) after 2 days, open lumen formation after 4 days (Bryant et al. 2010) and then we follow the cyst cultured up to 6 days. In EV and septin 9_i1cysts, AMIS was clearly visible, and the PAP was formed, and lumen continued to grow until 6 days (Figure 5A). Here again as observed in Figure 3C, the lumen started to fill in septin 9_i1 cysts which suggested overgrowth of the cells. However, no AMIS was presented in the mutant cells and the multiple lumens were already present at the PAP stage. Then at day 4 and day 6 the cysts from both del1 and del2 displayed multiple lumens phenotype. The del1.2 cysts presented the inverted polarity phenotype as expected (Figure 5A). Although the phenotype was exacerbated at day 6 and the cysts displayed invasive features which are different from the round shape of the cyst at day 4. The apical marker presented asymmetric localization which recalled, the front-rear polarization in migrating cells (Bryant et al. 2014) (Figure 5A). The quantification of the cyst forming AMIS (Figure 5B,C) and those with the different phenotype were performed for each condition and at the different time points (Figure 5D). The schematic representation of the study was presented in Figure 5E.

Figure 5. Deletion of septin 9 PB domains impact cyst phenotype from the AMIS formation to the lumen formation. (A). All the MDCK stable cell lines plated on Matrigel for 1 day, 2 days, 4 days and 6 days to form cysts, and then stained for β-catenin (green) and GP135 (red). A single confocal section through the middle of a cyst is shown. For the AMIS stage, β-catenin (green), scale bar is 20 µm, others are all 10 µm. (B). The analysis of AMIS formation of each stable cell lines after 1 days is showed in the histogram. (C). Bars represented the size of AMIS. (D). The phenotype of open lumen, multi-lumen, inverted polarity and filled lumen are counted during the culture times. Color for each phenotype in pie is corresponding the color below pictures. (E). Schematic representation of the septin 9 situation and its effects on the phenotype of the MDCK cell cysts during the different stage. Data information: Data are at least two times replicates and cysts (n>20) for 3D staining. The statistics values are means ±s.e.m. Student’s t-test was used. ***P < 0.001.

Figure 5. Deletion of septin 9 PB domains impact cyst phenotype from the AMIS formation to the lumen formation. (A). All the MDCK stable cell lines plated on Matrigel for 1 day, 2 days, 4 days and 6 days to form cysts, and then stained for β-catenin (green) and GP135 (red). A single confocal section through the middle of a cyst is shown. For the AMIS stage, β-catenin (green), scale bar is 20 µm, others are all 10 µm. (B). The analysis of AMIS formation of each stable cell lines after 1 days is showed in the histogram. (C). Bars represented the size of AMIS. (D). The phenotype of open lumen, multi-lumen, inverted polarity and filled lumen are counted during the culture times. Color for each phenotype in pie is corresponding the color below pictures. (E). Schematic representation of the septin 9 situation and its effects on the phenotype of the MDCK cell cysts during the different stage. Data information: Data are at least two times replicates and cysts (n>20) for 3D staining. The statistics values are means ±s.e.m. Student’s t-test was used. ***P < 0.001.

3.7. The deletion of the two PB domains of septin 9 promotes invasive features through the regulation of src and cortactin

To get more mechanistic insights on how deletion of the two PB domains of septin 9 induced the invasive phenotype we studied the cysts formed from EV, septin 9_i1 and septin 9_del1.2 after 6 days of culture in 3D. We found that septin 9 was cytoplasmic at day 6 in each cell cysts (Figure S4) similarly to data observed at day 4 (Figure 3C). Interestingly, septin 9 was enriched in the front of cysts with the invasive phenotype (Figure S4). We analyzed cortactin which was distributed uniformly on the BM membrane and especially on the basal part in EV cysts. A similar staining was observed for septin 9_il cysts. By contrast, cortactin signal strongly increased and colocalized with actin in septin 9_del1.2 cysts (Figure 6A). Furthermore, the phosphorylation of cortactin (p-cortactin) which reflected its activity showed much more striking signal in septin 9_del1.2 cells (Figure 6A). As cortactin is the substrate of src (Head et al. 2003; Huang et al. 1998; Patel et al. 1998), and src phosphorylation of cortactin enhances actin assembly (Tehrani et al. 2007), we then analyzed src. Interestingly we found that src and p-src expression increased in septin 9_del1.2 cysts (Figure 6B). Src and cortactin expressions increased were confirmed by immunoblot (Figure 6C). Thus, these data indicated that the deletion of two PB domains of septin 9 induces activation and recruitment of the src/cortactin signal allowing the cells to acquire invasive features.

Figure 6. Deletion of septin 9 PB domains induces collective migration by regulating src/cortactin signal pathway in day 6. (A). and (B). The MDCK stable cell lines of EV, i1 and Del1.2 plated on Matrigel for 6 days to form cysts, and then stained for cortactin (green) and Actin (red) and p-cortactin (green) and Actin (red), src (green) and Actin (red), p-src (green) and Actin (red). A single confocal section through the middle of a cyst is shown. The scale bar is 10 µm. The quantification of the fluorescence intensity of each marker is showed beside the images. (C)The immunoblotting of the src and cortactin protein and the densitometry analysis. The data are means ±s.e.m. Student’s t-test was used. **P<0.01. Data information: Data are at least two times replicates and cysts (n=10) for 3D staining and immunoblotting three times. The statistics values are means ±s.e.m. Student’s t-test was used. *P<0.05, **P<0.01, ***P < 0.001.

Figure 6. Deletion of septin 9 PB domains induces collective migration by regulating src/cortactin signal pathway in day 6. (A). and (B). The MDCK stable cell lines of EV, i1 and Del1.2 plated on Matrigel for 6 days to form cysts, and then stained for cortactin (green) and Actin (red) and p-cortactin (green) and Actin (red), src (green) and Actin (red), p-src (green) and Actin (red). A single confocal section through the middle of a cyst is shown. The scale bar is 10 µm. The quantification of the fluorescence intensity of each marker is showed beside the images. (C)The immunoblotting of the src and cortactin protein and the densitometry analysis. The data are means ±s.e.m. Student’s t-test was used. **P<0.01. Data information: Data are at least two times replicates and cysts (n=10) for 3D staining and immunoblotting three times. The statistics values are means ±s.e.m. Student’s t-test was used. *P<0.05, **P<0.01, ***P < 0.001.

3.8. Inhibition of RhoA and TGF-β type I receptor rescues the polarity of del1,2 cysts

RhoA is the first family member of Rho family of GTPases which control the actin dynamic and is regulated by cortactin (Cox et al. 2001; Liang et al. 2017). Furthermore the activation of RhoA in the cysts with inverted polarity phenotype was reported and inhibition of Rho-associated kinase (ROCK) prevented the inversion (Bryant et al. 2014; Yu et al. 2008). Therefore, we asked whether the inverted polarity and the invasive phenotype of septin 9_del1.2 cysts were dependent on RhoA. We then examined the effect of ROCK inhibitor (Y27632) on control (EV) and septin 9_del1.2 cells (Del1,2) (Figure S5; Figure 7A). Interestingly treatment of del1.2 cells with Y27632 at 30 and 50 μM, reverted the polarity and the apical domain was found in the interior either as single or multiple lumens (Figure 7A,B) and represented 47.7% and 61% of the cysts respectively for the doses of Y27632 at 30 and 50 μM (Figure 7A,B). Subsequently, we assessed RhoA activity which significantly increased in septin 9_del1.2 cells compared to EV cells as expected (Figure 7C). Thus, these data indicated that RhoA dependent-actomyosin contractility is involved in the formation of the inverted polarity phenotype of del1,2 cysts.

The transforming growth factor-β (TGF-β), is a master regulator of epithelial cell plasticity and a driver of EMT (Nieto et al. 2016:2016). Furthermore, TGF-β mediated activation of RhoA to maintain basal RhoA–ROCK signaling (Fleming et al. 2009; Zhang 2009). Therefore, we analyzed the TGF-β signaling in cysts from depletion of the two PB domains of septin 9 with invasive phenotype. First, we treated the cells with TGF-β type 1 receptor inhibitor (SB431542) at two different concentrations (Figure 7D). Strikingly, this treatment hampered the inversion of polarity and allowed the formation of single monolayer of cells and a recovery of β-catenin at cell-cell junctions (Figure 7D,E). Subsequently, we showed a decrease of E-cadherin and the increase of vimentin by immunoblot performed on EV and del1,2 cells (Figure 7F). Next, using qRT-PCR, we showed the increase of the transcripts of other mesenchymal markers such as ZEB1 and N-Cadherin (Figure 7G). Interestingly we also observed an increase of the TGF-β (Figure 7G). Together, these data revealed the role of TGF-β driving plasticity in the invasiveness feature induced by depletion of the two PB domains of septin 9. Thus, it strongly suggested the involvement of the EMT process regulated by the TGFβ/RhoA/cortactin pathway (Figure 7H).

Figure 7. Deletion of septin 9 PB domains induces collective migration by regulating src/cortactin signal pathway in day 6. (A). MDCK cells expressing EV and septin 9_del1.2 were plated on Matrigel for 6 days and treated with 30µ M and 50 µM of Y27632. All the cysts were stained with β-catenin (green) for basolateral membrane, actin (red) for apical surface and Hoechst (blue) for nuclei. The representative confocal images are show in merge. (B). Quantification of polarized (lumen inside the cysts) and inverted polarity phenotypes in septin 9_EV and septin 9_del1.2 cysts. (C). RhoA activation fold change in septin 9_EV and septin 9_del1.2 cysts. (D). MDCK cells expressing EV and septin 9_del1.2 were plated on Matrigel for 6 days and treated with 2 µM and 5µM of SB431542. All the cysts were stained with β-catenin (green) actin (red). The representative confocal images for the merge signals are shown. (E). Quantification of cysts with polarized phenotype and the inverted polarity phenotype was represented. (F). Western blot analysis of E-cadherin, vimentin, in cells with EV and septin 9_del1.2 cultured in 3D condition. Actin is shown as a loading control. Quantifications of western blot data of E-cadherin and vimentin protein levels. (G). qRT-PCR analysis of the expression of mRNA encoding septin 9, N-cadherin, ZEB1 and TGFβ in cells with EV and septin 9_del1.2 cultured in 2D condition. (H). Schematic diagram illustrating the inverted polarity phenotype induced upon deletion of the two PB domains of septin 9_i1 in 3D culture. These plastic processes are EMT, and the process is regulated by the TGFβ/RhoA/Src/Cortactin pathway. Data information: Data are at least two times replicates and cysts (n>10) for 3D staining, and three times for RhoA activity test and immunoblotting and qRT-PCR. The statistics values are means ±s.e.m. Student’s t-test was used. *P<0.05, **P<0.01, ***P < 0.001.

Figure 7. Deletion of septin 9 PB domains induces collective migration by regulating src/cortactin signal pathway in day 6. (A). MDCK cells expressing EV and septin 9_del1.2 were plated on Matrigel for 6 days and treated with 30µ M and 50 µM of Y27632. All the cysts were stained with β-catenin (green) for basolateral membrane, actin (red) for apical surface and Hoechst (blue) for nuclei. The representative confocal images are show in merge. (B). Quantification of polarized (lumen inside the cysts) and inverted polarity phenotypes in septin 9_EV and septin 9_del1.2 cysts. (C). RhoA activation fold change in septin 9_EV and septin 9_del1.2 cysts. (D). MDCK cells expressing EV and septin 9_del1.2 were plated on Matrigel for 6 days and treated with 2 µM and 5µM of SB431542. All the cysts were stained with β-catenin (green) actin (red). The representative confocal images for the merge signals are shown. (E). Quantification of cysts with polarized phenotype and the inverted polarity phenotype was represented. (F). Western blot analysis of E-cadherin, vimentin, in cells with EV and septin 9_del1.2 cultured in 3D condition. Actin is shown as a loading control. Quantifications of western blot data of E-cadherin and vimentin protein levels. (G). qRT-PCR analysis of the expression of mRNA encoding septin 9, N-cadherin, ZEB1 and TGFβ in cells with EV and septin 9_del1.2 cultured in 2D condition. (H). Schematic diagram illustrating the inverted polarity phenotype induced upon deletion of the two PB domains of septin 9_i1 in 3D culture. These plastic processes are EMT, and the process is regulated by the TGFβ/RhoA/Src/Cortactin pathway. Data information: Data are at least two times replicates and cysts (n>10) for 3D staining, and three times for RhoA activity test and immunoblotting and qRT-PCR. The statistics values are means ±s.e.m. Student’s t-test was used. *P<0.05, **P<0.01, ***P < 0.001.

4. Discussion

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