Epithelial mesenchymal transition (EMT) is a cellular transformation of epithelial cells that entails the loss of apical-basal cell polarity and intercellular adhesion in combination with a gain of mesenchymal cell traits, see figure 1(a) [1–4]. EMT was linked to the initiation of metastasis and bad cancer prognosis through the acquisition of aggressive traits in cancer cells of epithelial origin [1–4]. In particular, EMT was reported to be connected to enhanced cell migration and cell proliferation in metastatic cancer cells [1–5].

Figure 1. EMT induces characteristic changes of the cortical abundance of Rho GTPases RhoA, Rac1 and RhoC in MCF-7 cells. (a) Schematic of EMT-induced changes of cell morphology and adhesion. (b), (c) image analysis of confocal images of immunostained cell cross sections to extract the cortex-to-cytoplasm ratio. (b) Exemplary picture of RhoC immunostaining fluorescence profile of the equatorial cross-section of an EMT-induced mitotic cell including elements of image analysis. Scale bar: 10 µm. (c) Mean radial fluorescence intensity profile of picture (blue curve) along radial lines shown in panel (c). The fitted intensity profile, is shown in orange, see section 5. (d) Representative confocal images of suspended interphase cells and STC-arrested mitotic cells in control and EMT-induced conditions. Cells were fixed and DAPI-stained for DNA (blue) and immunostained for Rac1/RhoA/RhoC (green), see section 5. Scale bar: 10 µm. (e)–(g) Cortex-to-cytoplasm ratio of RhoA, Rac1 and RhoC inferred from immunofluorescence staining as shown in panel (b) before and after EMT. (h) Schematic of changes of cortical association of Rac1, RhoA and RhoC upon EMT. (i)–(l) RhoC knockdown elicits cortical softening and tension reduction in the actin cortex in pre-EMT interphase MCF-7 cells ((i), (j), white boxplots) and post-EMT mitotic MCF-7 cells ((k), (l), blue-shaded boxplots). Post-EMT cells are referred to as modMCF-7. Number of cells analyzed: (e): MCF-7 interphase n = 34, modMCF-7 interphase n = 32, MCF-7 mitosis n = 31, modMCF-7 mitosis n = 31. (f): MCF-7 interphase n = 36, modMCF-7 interphase n = 28, MCF-7 mitosis n = 31, modMCF-7 mitosis n = 31. (g): MCF-7 interphase n = 44, modMCF-7 interphase n = 43, MCF-7 mitosis n = 30, modMCF-7 mitosis n = 32. (i), (j): MCF-7 n = 39, esiRhoC n = 37, modMCF-7 n = 37, esiRhoC n = 39, (k), (l): MCF-7 n = 27, esiRhoC n = 29, modMCF-7 n = 29, esiRhoC n = 28. Measurements represent at least two independent experiments. n.s.: p > 0.05, : p < 0.05, : p < 0.01, : p < 0.001.

Download figure:

Standard image High-resolution image

The actin cytoskeleton is a major regulator of cell mechanics, cell shape and cellular force generation. Thereby, the actin cytoskeleton constitutes a key player in cancer-related changes of cell migration and cell division [6–8]. Consistent with this, it was found that EMT causes major changes in the actin-cytoskeleton [5, 9, 10].

Rho GTPases are known to be essential regulators of the actin cytoskeleton. We and others showed that EMT is associated with characteristic changes in the activation of Rho GTPases as judged by the abundance of its active GTP-bound forms [5, 11–13]. In particular, we reported a decrease of total RhoA-GTP and an increase of total Rac1-GTP upon EMT in MCF-7 breast epithelial cells. Furthermore, the increased expression of the Rho GTPase RhoC was associated to enhanced metastasis in several cancer types [14]. In addition, RhoC signaling was featured to be essential for EMT [13–16].

Previously, we reported characteristic EMT-induced changes of actin cortex mechanics in rounded cells of diverse epithelial cancer cell lines originating from breast, lung, prostate and skin tissue indicating that this EMT-induced cell-mechanical change is a widely conserved feature in cells of diverse tissue origin [5, 17, 18]. Cortex-mechanical changes were entailing cortical softening and contractility reduction in interphase but a cortical stiffening and contractility increase upon EMT in mitosis. Concomitantly, we found EMT-induced changes of cortical actin and myosin II with reduced cortical myosin in interphase and increased cortical actin in mitosis [5].

While associated EMT-induced changes in Rho GTPases signaling provide a viable hypothesis for downstream changes of cortical actin and myosin, the details of EMT-induced changes in cortical signaling remain elusive. In particular, it is unclear how cortical mechanics is affected in an opposite way in interphase and mitosis, as none of the downstream actomyosin effectors of Rho GTPases are known to induce mechanical changes in the cortex that depend on the cell cycle stage [19, 20].

With this work, we aim to deepen our understanding of EMT-induced changes in cortical signaling, cortical composition and cortical mechanics with a focus on the differences between interphase and mitosis. To this end, we quantify EMT-induced changes of Rho GTPases RhoA, RhoC and Rac1 which were previously linked to EMT. Furthermore, we investigate as actin-regulating downstream targets formin, Arp2/3 and cofilin. In particular, we provide a quantitative analysis of EMT-induced changes of cortical protein localization in non-adherent cells in combination with changes of cortical mechanics and protein expression. In light of our results, we propose that two hitherto unappreciated signaling mechanisms at the cortex are at the heart of EMT-induced cell-mechanical changes—i) an interaction between Rac1 and RhoC and ii) an inhibitory effect of Arp2/3 activity on myosin II cortical localization.

To investigate the effects of EMT on actin-cortical signaling and mechanics, we chose to work with the breast epithelial cancer cell line MCF-7 which exhibits epithelial cell traits in control conditions. We induced EMT in these cells via an established method (see e.g. [5, 21–25]) that entails a 48 h treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 100 nM, see section 5. We and others showed previously that in response to this treatment, MCF-7 cells display an EMT-characteristic protein expression change, corresponding cell-morphological changes towards a mesenchymal-like phenotype, as well as increased proliferation and migration, see e.g. [5, 24, 25]. In particular, we showed earlier that the epithelial marker E-cadherin is downregulated through the treatment, while the mesenchymal markers N-cadherin and Vimentin are upregulated, see figures S1(b) and (c) in the supporting information of [5]. Further, cells acquire a more spindle-shaped morphology and grow more isolated from each other, see figure S1(a) in the supporting information of [5]. We note that EMT-transformed cells will be referred to as modMCF-7 cells throughout this manuscript.

Previous research has shown that activation of Rho GTPases is connected to their association with the plasma membrane [26]. In addition, their immediate downstream signaling is inherently local as direct downstream effectors such as mDia1, Rock and Wasp and Wave require persistent binding for activation [27–29]. Furthermore, the activation of downstream actin effectors cofilin, formin and Arp2/3 was shown to be linked to f-actin binding [28, 30–32]. Correspondingly, association of these proteins to cortical f-actin is a measure of their activity at the actin cortex. Therefore, one prevalent strategy of this study is to quantify changes of the relative amount of cortex-associated cortical regulators upon EMT as a readout of EMT-induced changes in cortical signaling. Following previous studies [5, 18, 19, 33, 34], we worked with rounded, non-adherent cells since this has the advantage that cell shapes are spherical in both epithelial and EMT-transformed conditions with a largely uniform actin cortex. In this way, a meaningful comparative analysis of cortical protein association between epithelial and the mesenchymal-like cells becomes possible.

For the measurement of cortical protein association, immunostaining of the cortical regulator under consideration was combined with fluorescent DNA staining (DAPI or Hoechst) which allowed to identify cells to be in an interphase or mitotic stage, see section 5. For the measurement of cortical regulators in mitotic cells, the fraction of mitotic cells was enriched through mitotic arrest induced by co-incubation with S-trityl-L-cysteine (STC), see section 5. Using a previously established image analysis scheme, we analyzed confocal images of immunostaining fluorescence intensities to infer the cellular outline and the averaged cortical fluorescence profile along the radial coordinate, i.e. orthogonal to the cell boundary, see figure 1(b) and [5, 18]. The averaged radial fluorescence intensity was then used to derive the cortex-to-cytoplasm ratio of protein localization in the cells by calculating the ratio of the integrated cortical fluorescence normalized by the cytoplasmic fluorescence intensity, see figure 1(c), section 5 and [5, 18].

2.1. Rho GTPases change their cortical association upon EMT in interphase and mitosis

To investigate whether cortical association of Rho GTPases changes through EMT, we quantified the cortex-to-cytoplasm ratio of RhoA, RhoC and Rac1 in cells with and without EMT induction. To this end, we performed confocal imaging of the equatorial cross section of suspended interphase or STC-arrested mitotic cells which were immunostained for either of the Rho GTPases under consideration, see figure 1(d) and section 5. Quantitative analysis shows that the cortex-to-cytoplasm ratio of Rac1 increases upon EMT both in interphase and mitosis (figures 1(d) and (e)). By contrast, the cortex-to-cytoplasm ratio of RhoA decreases through EMT (figures 1(d) and (f)). We conclude that cortical association of Rac1 and RhoA follows the EMT-induced quantitative change of GTP-bound Rac1 and RhoA in whole-cell-lysates of MCF-7 cells [5]. For RhoC, EMT-induced changes of the cortex-to-cytoplasm ratio are distinct in interphase and mitosis. While cortical RhoC goes down in interphase, we see an increase of cortical RhoC in mitosis (figures 1(d) and (g)). Our results on the effect of EMT on cortical signaling of Rho GTPases is summarized in figure 1(h).

We further asked about the influence of Rho GTPases on cortical mechanics. For this purpose, we relied on cortex-mechanical measurements with an established cell confinement setup based on oscillatory cell-squishing with the cantilever of an atomic force microscope (AFM). We chose a deformation frequency of 1 Hz. This assay was previously shown to provide a readout of cortical stiffness, cortical tension as well as a characterization of the viscoelastic nature of the cortex quantified by the phase shift between stress and strain [5, 17, 18, 35]. (The phase shift takes values between 0∘–90∘ with lower values corresponding to a more solid-like response). In particular, we previously showed that Rac1 signaling was linked to a decrease in cortical stiffness and contractility in interphase cells but to an increase of cortical stiffness and contractility in mitotic cells with a stronger effect in post-EMT cells [5]. This is in agreement with our here reported finding of increased cortical Rac1 association post-EMT (figure 1(e)). On the other hand, we previously found that RhoA signaling increases cortical stiffness and contractility in particular in pre-EMT cells [5]. Again, the bigger mechanical effect pre-EMT is in agreement with our current observation of higher cortical RhoA association pre-EMT (figure 1(f)).

The effect of RhoC on cortical mechanics has to our knowledge not been reported previously. Using the AFM-based cell confinement assay, we measured cortical mechanics with and without RhoC knock-down via RNA interference in pre- and post-EMT conditions, see figures 1(i)–(l), S1 and section 5. We find that similar to RhoA, RhoC signaling increases cortical contractility and stiffness, see figures 1(i)–(l). However, this effect is restricted to pre-EMT interphase cells and post-EMT mitotic cells. It is plausible that the absence of an effect in these conditions is linked to low abundance of RhoC at the cortex (figure 1(g)). Furthermore, RhoC knock-down increases the phase shift in pre-EMT interphase conditions indicating that RhoC signaling contributes to the solid-like nature of the cortex in interphase, see figure S1(b).

2.2. Rac1 and RhoC mutually affect their cortical association

The previously reported finding that Rac1 activity affects cortical mechanics opposite in interphase and mitosis provides a clue that the signaling of Rac1 might be at the heart of the cell-cycle dependence of cytoskeletal changes upon EMT. However, currently it is unclear how cortical Rac1 signaling can act in a manner that is qualitatively different in interphase and mitosis. In particular, it surprised us that Rac1 would make a strong contribution to cortical contractility in mitosis in post-EMT conditions given that Arp2/3 activity increase downstream of Rac1 is expected to diminish cortical contractility, see figure 6 and [20, 36]. Furthermore, previous reports showed that RhoA is at the heart of cortical contractility in mitosis [37]. While RhoA activity and cortical association is low in post-EMT MCF-7 cells (figure 1(f) and [5]), we note that RhoC signaling is similar to RhoA. Therefore, RhoC signaling might step in for RhoA signaling after EMT during mitosis.

Following this line of thought, we asked whether Rac1 might increase cortical contractility in post-EMT mitosis via (direct or indirect) activation of RhoC. To test this hypothesis, we monitored changes in cortical association of RhoC upon knock-down of Rac1 in pre- and post-EMT conditions judged by fluorescence intensity of RhoC immunostaining (figure 2(a)). Obtained confocal images of equatorial cross-sections were used for image analysis in all conditions. We find that inferred cortex-to-cytoplasm ratios of RhoC increase upon knock-down of Rac1 in rounded interphase cells with a stronger effect in post-EMT conditions (figures 2(a) and (c)). By contrast, the cortex-to-cytoplasm ratio of RhoC decreases upon knock-down of Rac1 in mitosis with a stronger effect in post-EMT conditions (figures 2(a) and (d)). We conclude that Rac1 signaling increases cortical association of RhoC in mitosis but diminishes cortical association in interphase in MCF-7 cells (figure 2(g)).

Figure 2. Rho-GTPases Rac1 and RhoC mutually affect their abundance at the cortex. (a) Representative confocal images of RhoC (immunostained, green) and DNA (DAPI, blue) with and without Rac1 knock-down in suspended interphase cells and STC-arrested mitotic cells in pre-EMT (MCF-7) and post-EMT (modMCF-7) conditions. Scale bar: 10 µm. (b) Representative confocal images of Rac1 (immunostained, green) and DNA (DAPI, blue) with and without RhoC knock-down in suspended interphase cells and STC-arrested mitotic cells in pre-EMT (MCF-7) and post-EMT (modMCF-7) conditions. Scale bar: 10 µm. (c)–(f) Changes of cortical association of RhoC and Rac1 upon knock-down of the other protein, i.e. Rac1 or RhoC, respectively. Cortical association of either protein was quantified by its cortex-to-cytoplasm ratios which was inferred from immunofluorescence staining as shown in panels (a) and (b) before and after EMT. (g) Schematic summary of Rac1 and RhoC mutual interactions in interphase and mitosis. Post-EMT cells are referred to as modMCF-7. Number of cells analyzed: (c): MCF-7 n = 20, esiRac1 n = 20, modMCF-7 n = 20, esiRac1 n = 20, (d): MCF-7 n = 19, esiRac1 n = 22, modMCF-7 n = 19, esiRac1 n = 19, (e): MCF-7 n = 25, esiRhoC n = 21, modMCF-7 n = 20, esiRhoC n = 24, (f): MCF-7 n = 24, esiRhoC n = 26, modMCF-7 n = 22, esiRhoC n = 24. Measurements represent at least two independent experiments. n.s.: , , .

Download figure:

Standard image High-resolution image

To test whether in turn also RhoC signaling influences Rac1, we performed immunostaining of Rac1 with and without RhoC knock-down in pre- and post-EMT conditions in interphase and mitosis, see figure 2(b). We find that inferred cortex-to-cytoplasm ratios of Rac1 decrease upon knock-down of RhoC in pre-EMT interphase cells and in post-EMT mitotic cells (figures 2(e) and (f)). In all other conditions, there is no significant effect on Rac1 cortical association (figures 2(e) and (f)). We conjecture that the signaling from RhoC to Rac1 is restricted to pre-EMT interphase and post-EMT mitosis due to the low cortical representation of RhoC in post-EMT interphase and pre-EMT mitotic conditions, see figures 1(g) and (h). With this explanation approach, our data are consistent with an in general activating effect of RhoC on Rac1 (figure 2(g)).

In previous work, the active forms of RhoA and Rac1 were shown to affect each other through mutually inhibitory interactions in breast epithelial cells [38]. This is consistent with the EMT-induced switch-like change from a state of high RhoA and low Rac1 activation to a state of low RhoA and high Rac1 activation [5]. The interaction between Rac1 and RhoC has been to our best knowledge unknown so far.

2.3. Cortical cofilin association increases through EMT

We went on to ask how EMT-induced changes in Rho GTPases signaling affect downstream cortical regulators. We first investigated EMT-induced changes of cofilin cortical association. Cofilin is known to promote the depolymerization of the actin cortex through severing of actin fibers [39]. In the context of cancer, cofilin activity at the cortex has been suggested to be a main factor in f-actin turnover thus playing a key role in cancer cell migration and invasion [40]. Cofilin becomes deactivated through phosphorylation mediated by Lim kinases [40] and phosphorylated cofilin was shown to not interact with f-actin [30]. Correspondingly, cortex-bound cofilin can be interpreted as active cofilin.

We quantified total amounts of cofilin (CFL1) and phospho-cofilin (phospho-CFL1 (Ser3)) via western blotting from lysates of adherent cells with or without EMT-induction, see figures 3(a), (b) and section 5. Calculating fold changes upon EMT, we find a trend of a shallow increase of total cofilin (only interphase) and a decrease of phospho-cofilin upon EMT, see figure 3(c). Taken together, this points at an increase of the active non-phosphorylated form of cofilin upon EMT in MCF-7.

Figure 3. Actin cortical regulators LIMK1 and cofilin (CFL1) influence actin cortical mechanics and change their cortical representation upon EMT. (a) Exemplary western blots for p-LIMK1, p-CFL1 and CFL1 expression in MCF-7 cells in control and EMT-induced conditions. (b) Bar charts of normalized quantities of the p-LIMK1 (active form), p-Cofilin (inactive form) and total cofilin western blots. Normalization was done against GAPDH bands. Error bars represent standard error of the mean. (c) Fold changes of normalized protein amounts of p-LIMK1, p-CFL1 and total CFL1 upon EMT from western blots. Individual data points are depicted in black, see section 5. Error bars represent standard error of the mean. Significance was tested with a one-sample t-test against the null hypothesis that the data comes from a normal distribution with mean equal to zero (p-values from left to right: 0.04, 0.82, 0.034, 0.03, 0.04, 0.013). (d) Representative confocal images of suspended interphase cells and STC-arrested mitotic cells upon EMT, fixed and stained for DAPI (blue) and cofilin (green). Scale bar: 10 µm. (e) Cortex-to-cytoplasm ratio of cofilin inferred from immunofluorescence staining as shown in panel (d) before and after EMT. (f)–(i) CFL1 knockdown elicits cortical stiffening and tension rise in the actin cortex in interphase (f), (g) and mitotic (h), (i) MCF-7 cells. For panels (e)–(i), significance was tested with a Mann-Whitney U-test (two tailed). Post-EMT cells are referred to as modMCF-7. Number of cells analyzed: (e): MCF-7 interphase n = 38, modMCF-7 interphase n = 38, MCF-7 mitosis n = 46, modMCF-7 mitosis n = 44. (f), (g): MCF-7 n = 49, siCFL1 n = 49, (h), (i): MCF-7 n = 46, siCFL1 n = 50. Measurements represent at least two independent experiments. n.s.: p > 0.05, : p < 0.05, : p < 0.01, : p < 0.001.

Download figure:

Standard image High-resolution image

To assess cortical association of cofilin, we performed also cofilin-immunostaining of rounded cells, see figure 3(d). We find that the cortex-to-cytoplasm ratio of cofilin is elevated upon EMT indicating an EMT-mediated increase of cortical cofilin activity, see figures 3(d) and (e). This finding is in agreement with an increase of active cofilin as suggested by western blotting as described above, see figure 3(c).

Immunostaining of the cofilin upstream regulator phospho-Limk1 (phospho-LIMK1 (Thr508)) shows no cortical association but cytoplasmic localization in agreement with previous findings, see figure S3(a) and [41]. Quantifying phospho-Limk1 abundance in whole-cell lysates via western blotting, we find an EMT-induced decrease in interphase (asynchronous cell population) in accordance with the observed concomitant decrease of phospho-cofilin, see figures 3(a)–(c) and section 5. In mitosis, phospho-Limk1 amounts are very low and show a trend of decrease upon EMT which is, surprisingly, in disagreement with the EMT-induced trend of phospho-cofilin (figures 3(a)–(c)). We speculate that this apparent inconsistency may be attributed to the previously reported modified activation scheme of Limk1 in mitosis, where hyperphosphorylation rather than phosphorylation at Thr508 is at the heart of Limk1 activation [42]. This observation features phospho-Limk1 (Thr508) as an unsuitable readout of cofilin phosphorylation activity in mitosis.

Investigating the effect of cofilin on cortical mechanics, we find that cofilin knock-down through RNA interference changes cortical mechanics, see figures 3(f)–(i) and S3(e)–(h). Both cortical tension and stiffness increase in interphase and mitosis, see figures 3(f)–(i). In addition, the phase shift and therefore the fluid-like nature increased mildly upon knock-down in interphase cells, see figure S3(f). We conclude that increased cortical association of cofilin in EMT-induced cells contributes to a trend of decreased cortical contractility and stiffness.

We note that Chugh et al [19] previously reported a tension increase upon CFL1-knockdown in interphase HeLa cells in agreement with our findings. However, the authors reported by contrast a tension decrease upon CFL1 knock-down in mitosis opposite to our findings in MCF-7. This apparent discrepancy might be rooted in the non-monotonous dependence of cortical tension on actin filament length as was proposed by the same study [19]. According to this idea, increased actin filament length through cofilin knock-down can either increase or decrease cortical tension depending on the initial state of the cortex. Large differences in cortical tension values between mitotic MCF-7 cells and mitotic HeLa cells make different cortical configurations in mitosis for the two cell lines additionally plausible.

2.4. The actin nucleator mDia1 shows distinct changes of cortical association upon EMT in interphase and mitosis

In order to further understand EMT-induced changes of cortical composition and mechanics [5], we addressed how actin nucleators are affected upon EMT downstream of Rho GTPases. Cortical actin is polymerized by formins and Arp2/3. We will first focus on the influence of the former. Previous studies have shown that formin activity has a major influence on cortical mechanics [19, 20, 43, 44]. We confirmed this finding in rounded MCF-7 cells with our AFM-based cell confinement setup showing that formin inhibition via 40 µM SMIFH2 reduced cortical stiffness and contractility in MCF-7 cells in interphase and mitosis, see figure S4.

To further investigate how formin-mediated polymerization changes at the cortex upon EMT, we decided to focus on the formin representative mDia1 (also Diaph1) which together with the actin nucleator Arp2/3 polymerizes the majority of f-actin in the actin cortex [45]. mDia1 is activated downstream of RhoA, RhoB or RhoC through binding to the active form of the respective Rho GTPase [28]. The active form of mDia1 associates with f-actin [28, 32] and thus with the actin cortex.

Performing quantification of protein amounts in whole-cell lysates via western blots, we find that there is no significant change of expression of mDia1 upon EMT (figures 4(a)–(c)). We then went on to monitor cortical association of mDia1 via immunostaining and quantification of the cortex-to-cytoplasm ratio, see figures 4(d) and (e). We find clear EMT-induced changes; in interphase cells, the cortex-to-cytoplasm ratio of mDia1 is reduced, see figure 4(e), blue boxes. We conclude that cortical association of mDia1 is decreased upon EMT in agreement with our finding of reduced presence of RhoA and RhoC at the cortex. In mitotic cells, on the other hand, the cortex-to-cytoplasm ratio of mDia1 is increased, see figure 4(e), yellow boxes. This finding points at an increase of cortical mDia1 activity upon EMT in mitosis. We suggest that this effect is due to an EMT-induced rise of cortical activity of RhoC in mitosis overcompensating the effect of reduced RhoA activity in post-EMT mitotic cells, see figure 1(h).

Figure 4. The actin nucleator and RhoA/C downstream effector mDia1 changes its cortical association upon EMT with opposite trend in interphase and mitosis. (a) Exemplary western blots for mDia1 expression in MCF-7 cells in control and EMT-induced conditions. (b) Bar charts of normalized quantities of mDia1 western blots. Normalization was done against GAPDH bands. Error bars represent standard error of the mean. (c) EMT-induced fold changes of normalized protein amounts of mDia1 upon EMT induction from western blots, see section 5. Individual data points are depicted in black. Error bars represent standard error of the mean. Changes are not significant from zero according to a two-tailed one-sample t-test. (d) Representative confocal images of suspended interphase cells and STC-arrested mitotic cells upon EMT, fixed and stained for DAPI (blue) and mDia1 (green). Scale bar: 10 µm. (e) Cortex-to-cytoplasm ratio of mDia1 inferred from immunofluorescence staining as shown in panel (d) before and after EMT. Significance was tested with a Mann-Whitney U-test (two tailed). Post-EMT cells are referred to as modMCF-7. Number of cells analyzed: (e): MCF-7 interphase n = 72, modMCF-7 interphase n = 32, MCF-7 mitosis n = 33, modMCF-7 mitosis n = 36. Measurements represent at least two independent experiments. n.s.: p > 0.05, : p < 0.05, : p < 0.01, : p < 0.001.

Download figure:

Standard image High-resolution image

Taken together, our results suggest that the observed characteristic EMT-induced changes of cortical mDia1 likely make an essential contribution to EMT-induced changes of cortex-associated actin and cortical mechanics in interphase and mitosis.

2.5. The actin nucleator Arp2/3 increases its cortical association upon EMT

To further increase our understanding of changes in cortex-associated actin upon EMT as reported in [5], we also investigated EMT-induced changes of the second major actin nucleator beyond mDia1, namely the Arp2/3 complex [6, 45]. The Arp2/3 complex becomes activated by the Wasp family of proteins [31, 46]. Activated Wasp proteins promote binding of the Arp2/3 complex to f-actin [31, 47, 48] and thus its association to the actin cortex. Wasp proteins (WAVE) are activated downstream of Rac1 [29]. We therefore expect that our finding of EMT-induced increase of cortical Rac1 association (figure 1(h)) should give rise to a downstream increase of cortical Arp2/3 association.

Performing protein quantification in whole-cell lysates, we find that there are no significant expression changes of Arp2 upon EMT, see figures 5(a)–(c). This indicates that there are no significant changes of the amount of the Arp2/3 complexes in MCF-7 cells upon EMT induction. To assess cortical association of the Arp2/3 complex, we performed immunostaining of Arp2 in rounded cells in interphase and mitosis, see figure 5(d). Interestingly, in spite of a direct interaction between Arp2/3 and f-actin, a cortical enrichment of Arp2 is only visible in interphase cells, see figure 5(d), lower row. Quantification of corresponding cortical association in interphase via the cortex-to-cytoplasm ratio indicates that Arp2/3 signaling at the cortex is enhanced through EMT, see figure 5(e).

Figure 5. The actin nucleator and Rac1 downstream effector Arp2/3 is elevated at the cortex upon EMT in interphase. (a) Exemplary western blots for Arp2 expression in MCF-7 cells in control and EMT-induced conditions. (b) Bar charts of normalized quantities of Arp2 western blots. Normalization was done against GAPDH bands. Error bars represent standard error of the mean. (c) Fold changes of normalized protein amounts of Arp2 upon EMT from western blots, see section 5. Individual data points are depicted in black. Error bars represent standard error of the mean. Changes are not significant from zero according to a two-tailed one-sample t-test. (d) Representative confocal images of suspended interphase cells and STC-arrested mitotic cells upon EMT, fixed and stained for DAPI (blue) and Arp2 (green). Scale bar: 5 µm. (e) Cortex-to-cytoplasm ratio of Arp2 inferred from immunofluorescence staining as shown in panel (d) before and after EMT. Mitotic cells did not show a clear cortical Arp2 association and were therefore not quantified. (f)–(i) Arp2 inhibition using 50 µM CK666 elicits cortical stiffening and tension increase in the actin cortex in interphase (f), (g) and mitotic MCF-7 cells (h), (i). In panels (e)–(i), significance was tested with a Mann–Whitney U-test (two tailed). Post-EMT cells are referred to as modMCF-7. Number of cells analyzed: (e): MCF-7 interphase n = 47, modMCF-7 interphase n = 48. (f), (g): MCF-7 n = 24, CK666 n = 24. (h), (i): MCF-7 n = 27, CK666 n = 24. Measurements represent at least two independent experiments. n.s.: p > 0.05, : p < 0.05,