2. Results
2.4. Tension on the nuclear envelope regulates cyclin B1 nuclear translocation
2.4. Tension on the nuclear envelope regulates cyclin B1
which was reverted upon confinement (Fig. 4F, G; ***p<0.001). This confinement-generated decrease in NII likely reflects an unfolding of the NE, which is evident from the images of confined nuclei (Fig. 4F, L) as well as the increased NPC-NPC distance (Fig. S1; ***p<0.001). Overall, this suggests that actomyosin activity transmits forces to the NE through the LINC complex, leading to nuclear unfolding. To further characterize the mechanism behind nuclear unfolding and cPLA2 recruitment, we then inhibited cPLA2 activity with AAOCF3 and analysed the changes in NII. Indeed, interfering with cPLA2 activity significantly increased NII (Fig. 4H; ***p<0.001), although to a lesser extent than actomyosin inhibition (0.024±0.019 for AAOCF3, 0.035±0.015 for Y27632;
p=0.007 and 0.033±0.016 for p-N-blebb; p=0.019). As expected, inhibition of cPLA2 also led to a decrease of its association with the NE, even after confinement (Fig. 4I, J;
***p<0.001). Importantly, confinement was able to induce nuclear unfolding and increase NPC distance, even after cPLA2 inhibition (Fig. 4I-K; ***p<0.001). It should be noted that AAOCF3 treatment alone does not decrease NPC distance below those observed for control cells (Fig. S1K and Fig. 4K; 0.350±0.05ìm for controls vs 0.364±0.08ìm for
AAOCF3-treated cells).
So far, our results indicate that actomyosin activity leads to increased nuclear unfolding and cPLA2 NE recruitment. Whether this is reflective of increased nuclear tension, remained unknown. For that purpose, we sought to interfere with nuclear tension, independently of actomyosin activity, by overexpressing lamin B receptor (LBR).
Overexpression of LBR is known to cause NE folding (Gravemann et al., 2010), decrease nuclear tension and significantly affect cytoplasmic calcium levels and production of arachidonic acid, key components of the cPLA2 signalling pathway (Lomakin et al., 2020). As expected, overexpression of LBR resulted in a significant increase in NII (Fig.
4H, I, ***p<0.001) and a consequent decrease in the levels of cPLA2 on the NE (Fig. 4I, J; p<0.001). Collectively, these data indicate that actomyosin contractility triggers NE unfolding and increased tension during prophase. This was further confirmed by blocking actomyosin activity or expressing DN-KASH, which significantly decreased cPLA2 accumulation on the NE (Fig. 4F, L and M; ***p<0.001), and could be rescued by confinement (Fig. 4L, M; ***p<0.001). Taken together, this indicates that an intact connection between the cytoskeleton and nucleus is required to set off a tension-dependent signal that results in cPLA2 recruitment to the NE.
Overall, we concluded that an increase in actomyosin contractility during prophase is required to unfold the NE, increasing nuclear tension, and leading to cPLA2 recruitment.
The question remains of the functional relevance of cPLA2 NE recruitment. If cPLA2 is functionally important to facilitate cyclin B1 translocation, inhibiting its activity should
with AAOCF3 led to a significant decrease in cyclin B1 nucleoplasmic shuttling (Fig. 5A, B; **p<0.01; ***p<0.001). To further understand how cPLA2 interfered with cyclin B1 translocation, we analysed the levels of CDK1 Y15 phosphorylation and cyclin B1, following cPLA2 inhibition. Phosphorylation of CDK1 on T14/Y15 is well known to prevent mitotic entry (Parker, Atherton-Fessler and Piwnica-Worms, 1992; Kornbluth et al., 1994; Mueller et al., 1995) We reasoned that if cPLA2 affected the pathway controlling CDK1 activation, we should see an increase in this inhibitory phosphorylation, following treatment with AAOCF3. Accordingly, treatment with AAOCF3 increased the levels of CDK1 Y15, when compared to controls for both synchronized (1 for controls vs.
1.7 for AAOCF3-treated cells) and asynchronous cells (1 for controls vs. 1.7 for AAOCF3-treated cells), but not for CDK1i cells (1 for CDK1i vs 0.9 for CDK1i+AAOCF3), without affecting total CDK1 levels (Fig S4D). These results suggest that inhibition of cPLA2, through a yet unknown mechanism, delays activation of the cyclin B1-CDK1 complex, which could partly explain the delay in nuclear translocation of cyclin B1
observed upon treatment with AAOCF3 (Fig. 5A, B).
Since the release of internal Ca2+ stores triggers cPLA2 NE recruitment and activation (Enyedi, Jelcic and Niethammer, 2016), we next decided to acutely interfere with the release of Ca2+ by using BAPTA-AM + 2APB. Strikingly, this treatment also decreased cyclin B1 nuclear translocation (Fig.5C, D; ***p<0.001), as anticipated. While we cannot rule out that interfering with calcium release might affect other cellular processes, the use of BAPTA to disrupt internal calcium release during prophase has been previously described (Kao et al., 1990) and should not affect NEP at the concentrations used in our study. Moreover, we sought to minimize possible side-effects by adding the drugs acutely in late G2. Similarly to the BAPTA treatment, decreasing nuclear stiffness by overexpression of LBR also significantly delayed cyclin B1 nuclear translocation (Fig.
5E, F; ***p<0.001) and decreased cPLA2 loading on the NE (Fig. 4I, J). Remarkably, confinement was able to stimulate cyclin B1 translocation, when cPLA2 activity or calcium release were inhibited (Fig. 5A-D; ***p<0.001). This likely occurs due to confinement-induced unfolding of the NE (Fig. S1), that is sufficient to bypass the pharmacological inhibition of contractility and still induce an increase in NPC distance (Fig. 5G, H; ***p<0.001). Together, these observations support a working model for the mechanical regulation of mitotic entry based on actomyosin activity, that triggers nuclear unfolding and increases tension on the nucleus, leading to cPLA2 activation. This facilitates cyclin B1 transport across the NPCs, increasing its nuclear accumulation.
Figure 22: Nuclear unfolding during prophase recruits cPLA2 to the NE (A) Representative images of nuclei of interphase and prophase cells immunostained for Lamin A/C and DAPI.
Please note the irregularities on the NE surface in interphase cells. Scale bars, 10μm.
Figure 22 (cont.): (B) Nuclear irregularity index (NII) in interphase (black; n=34) versus mitotic cells (green;
n=20; ***p<0.001; Rank Sum test). (C) Distribution of NII values in interphase (black) and mitotic cells (green).
(D) Representative immunofluorescence images of parental RPE- 1, non-confined interphase cells (control interphase; n=32), non-confined prophase cells (control prophase; n=28) and confined interphase cells (interphase confiner; n=19), stained for cPLA2, Lamin A/C and DAPI. Scale bars, 10μm. White arrowheads indicate the NE limit. (E) Quantification of the NE/nucleoplasm integrated fluorescence intensity for cPLA2 in control interphase cells (black), control prophase cells (green; ***p<0.001) and confined interphase cells (magenta; ***p<0.001). Comparison between experimental groups was performed using a One-way ANOVA test. (F) Representative immunofluorescence images of parental RPE-1 cells in non-confined prophase cells (control prophase; n=14), prophase cells expressing the KASH (n=26) and prophase cells expressing DN-KASH under confinement (n=27), stained with cPLA2, DAPI and Lamin A/C. (G) Nuclear irregularity index (NII) in control, prophase cells (n=15), prophase cells expressing DN-KASH without (n=14) or with (n=13) confinement, prophase cells treated with Y-27632 without (n=17) or with (n=23) confinement and cells treated p-Nitro-blebb without (n=20) or with confinement (n=16) (***p<0.001; n.s. – not significant). (H) Nuclear irregularity index (NII) in control prophase cells (green; n=20), cells treated with AACOCF3 without (black;
n=30; ***p<0.001) or with confinement (magenta; n=30; n.s.) and cells over- expressing LBR (LBR o.e.; black;
n=30; ***p<0.001). (I) Representative immunofluorescence images of parental RPE-1 cells in prophase, non-confined conditions, prophase cells treated with AACOCF3 without (n=34) or with (n=29) confinement and stained with cPLA2, DAPI and Lamin A/C. The panel on the right corresponds to a representative image of cells stained with cPLA2 and DAPI and over-expressing LBR. As cam be seen both the treatment with AACOCF3 and over-expression of LBR increase NE folding during prophase. Scale bars, 10μm. (J) Quantification of the NE/nucleoplasm integrated fluorescence intensity for cPLA2 in control, prophase cells (green; n=20), AAOCF3-treated prophase without (black; n=30; ***p<0.001) or with confinement (magenta;
n=30; ***p<0.001) and cells prophase over-expressing LBR (black; n=30; ***p<0.001). Note how both the treatment with AACOCF3 and LBR over-expression impair the accumulation of cPLA2 at the NE during prophase. Comparison between experimental groups was performed using a One-way ANOVA when samples had a normal distribution. Otherwise, comparisons were done using a Kruskal-Wallis One Way ANOVA on Ranks. (K) Quantification of the NPC-NPC distance in cells treated with AACOCF3 without (black; n=41) and with mechanical confinement (magenta; n=47; ***p<0.001). Note how confinement increases NPC-NPC distance even with cPLA2 inhibition. This measurement was done using the custom MATLAB algorithm, as described in the Materials and Methods section. (L) Representative immunofluorescence images of parental RPE-1 cells in prophase treated with Y-27632 without (n=17) or with (n=23) confinement and cells treated with p-N-blebb without (n=20) or with (n=16) confinement. Cells were stained for cPLA2, DAPI and Lamin A/C.
Note how confinement restores the accumulation of cPLA2 on the NE, even with actomyosin inhibition. Scale bars, 10μm (M) Quantification of the NE/nucleoplasm integrated fluorescence intensity for cPLA2 in control prophase cells, prophase cells expressing DN- KASH (n=26), treated with Y-27632 or treated with p-Nitro-blebb, without or with confinement (***p<0.001; n.s. – not significant). Note how interfering with the LINC complex or the actomyosin cytoskeleton prevents cPLA2 recruitment to the NE and is reverted by confinement.
For panels (B), (E), (G), (H), (J), (K) and (M), the box size represents 75% of the population and the line inside the box represents the median of the sample. The size of the bars (whiskers) represents the maximum (in the upper quartile) and the minimum (in the lower quartile) values. All experiments were replicated at least three times and n represents the number of cells analysed.
Figure 23: cPLA2 is required for cyclin B1 nuclear translocation (A) RPE-1 cell expressing cyclinB1-Venus/tubulin-mRFP dividing on an FBN- coated substrate treated with AACOCF3 in non-confined conditions (top panel; n=12) and confined conditions (lower panel; n=15). Time frame in seconds and scale bar corresponds to 10μm. Images were acquired with 20sec interval and time zero corresponds to NEP. (B) Normalized nuclear cyclin B1 fluorescence over time for cells treated with DMSO (magenta; n=16; ***p<0.001) and the cPLA2 inhibitor (AACOCF3) in non-confined (black; n=15) or confined (green; n=12) conditions.
2.5. Premature nuclear entry of cyclin B1 increases the frequency of mitotic errors
Nuclear translocation of cyclin B1 sets the time for the G2-M transition (Strauss et al., 2018) and is essential for preventing untimely mitotic entry, which results in chromosome segregation errors (Furuno, Elzen and Pines, 1999). Similarly, confining cells throughout mitosis also contributes to the occurrence of segregation errors (Tse, Weaver and Carlo, 2012; Lancaster et al., 2013). Whether a short confinement during prophase only, which is sufficient to induce premature cyclin B1 translocation and NEP, results in chromosome
Figure 23 (cont.): C) RPE-1 cell expressing cyclin B1-Venus/tubulin-mRFP dividing on an FBN-coated substrate treated with BAPTA-AM + 2APB in non- confined (top panel; n=17) or confined (lower panel;
n=11) conditions. Time frame in seconds and scale bar corresponds to 10μm. Images were acquired with 20sec interval and time zero corresponds to NEP. (D) Normalized nuclear cyclin B1 fluorescence over time for cells treated with DMSO (magenta; n=16) and cells treated with BAPTA-AM + 2APB in non-confined (black; n=17) or confined (green; n=11) conditions (***p<0.001; n.s. – not significant). Statistical analysis of cyclin B1 accumulation was performed for the entire time course using an ANOVA Repeated Measures test when samples had a normal distribution. Otherwise, analysis was done using a Repeated Measures ANOVA on Ranks. (E) RPE-1 cell expressing cyclin B1-Venus/LBR-mRFP dividing on an FBN-coated substrate (n=30). Time frame in seconds and scale bar corresponds to 10μm. Images were acquired with 20sec interval and time zero corresponds to NEP. White arrowhead indicates the presence of LBR on the NE. (F) Normalized nuclear cyclin B1 fluorescence over time for mock- transfected cells (black; n=11) and cells with LBR-RFP overexpression (green; n=16; ***p<0.001). Note how LBR over-expression delays the nuclear accumulation of cyclin B1. Statistical analysis of cyclin B1 accumulation was performed for the entire time course using an ANOVA Repeated Measures test when samples had a normal distribution.
Otherwise, analysis was done using a Repeated Measures ANOVA on Ranks. In panels B, D, F, quantifications of cyclin B1 fluorescence were normalized to the lowest fluorescence intensity inside the nucleus and aligned relative to that value (which we defined as time zero), to obtain a measure of cyclin B1 translocation rate. Note that the DMSO treated group is the same for panels B and D. (G) Representative images of parental RPE-1 cells stained for TPR (magenta) and an NPC mix (green) in non-confined (top panel; n=62) and confined conditions (bottom panel; n=59), after treatment with p-N-blebb, acquired with CH-STED. Scale bar corresponds to 10μm. Note in the inset how mechanical confinement increases NPC-NPC distances (scale bar on inset corresponds to 2μm). (H) Distance between neighbouring nuclear pore complexes (NPC-NPC distance) in cells treated with p-N-blebb without (black) or with confinement (green;
***p<0.001). This measurement was done using the custom MATLAB algorithm, as described in the Materials and Methods section. For panels (B), (D) and (F), the lines correspond to the average value and the shaded area corresponds to SEM. All experiments were replicated at least three times and n represents the number of cells analysed.
short confinement, which was released shortly after NEP (Fig. 6A). This approach should induce mitotic entry, while still providing enough volume for the spindle to assemble unconstrained (Lancaster et al., 2013). Cells were then allowed to progress through mitosis unperturbed, so that we could determine mitotic timings, as well as the rate of chromosome missegregation. Notably, a significant proportion of cells that were subjected to short confinement entered mitosis with incomplete centrosome separation (Fig. 6A- D). This condition has been shown, by us and others, to increase the frequency of mitotic errors, in particular the occurrence of lagging chromosomes (Silkworth et al., 2012; Nunes et al., 2020), by favouring the establishment of erroneous kinetochore-microtubule attachments. Importantly, our acute confinement resulted in increased chromosome segregation errors (Fig. 6E; *p<0.05) and a slight mitotic delay (Fig. 6F;
24±7 min for controls vs. 36±20 min for confined cells; *p<0.05), when compared to unconfined cells. We propose these mitotic errors are triggered by the acute confinement that accelerates NEP, before cells had time to organize a mitotic spindle. To test this, we decided to generate an artificial rupture of the NE with laser microsurgery (Schweizer et al., 2015), allowing cyclin B1 and tubulin to enter the nuclear space and anticipating mitotic entry (Fig. 6G). Using this approach, we triggered immediate mitotic entry, which was sufficient to increase chromosome missegregation events (white arrowhead, Fig.
6G, H; **p=0.02) and induce a slight mitotic delay (Fig. 6I). Together, these experiments demonstrate that untimely mitotic entry through acute mechanical confinement during the G2-M transition, can have deleterious downstream consequences for chromosome segregation.
Figure 24: Premature mitotic entry potentiates chromosome segregation errors RPE-1 cell expressing H2B-GFP/tubulin-mRFP dividing on an FBN-coated substrate without (A) or with short-term confinement (B). Time frame in min:sec and scale bars correspond to 10μm. Images were acquired with a 2min interval and time zero corresponds to NEP. White arrowhead in panel B is pointing to a lagging chromosome which is generated after a short confinement. Centrosome positioning relative to the shortest nuclear axis at the moment of NEP in cells dividing without (C) or with confinement (D).
(E)
Figure 6 (cont.): Percentage of mitotic errors in control, non-confined cells (n=26) and cells under a short period of confinement (n=23; *p<0.05; Z-score test). (F) Mitotic timings (NEP-metaphase and NEP- anaphase) in control, non-confined cells (black) and cells under a short period of confinement (green;
*p<0.05). (G) RPE-1 cells expressing H2B-GFP/tubulin- mRFP dividing on an FBN-coated substrate, with a mock laser microsurgery performed on the cytoplasm (top panel; n=12), and NE rupture induced by laser microsurgery (lower panel; n=15). Time frame in seconds and scale bar corresponds to 10μm. Images were acquired with 2min interval and time zero corresponds to NEP. White arrowhead indicates the presence of lagging chromosomes. (H) Percentage of mitotic errors in control cells and cells where the NE was ruptured using laser microsurgery (**p<0.01). (I) Mitotic timings (NEP- metaphase and NEP-anaphase) in control cells (black) and cells where the NE was ruptured using laser microsurgery (green; *p<0.05; **p<0.01).
Comparison between groups was performed using a two-sided t test when the sample had normal distribution. Otherwise, the comparison was performed using a Rank Sum test. For panels (F) and (I), the box size represents 75% of the population and the line inside the box represents the median of the sample.
The size of the bars (whiskers) represents the maximum (in the upper quartile) and the minimum (in the lower quartile) values. All experiments were replicated at least three times and n represents the number of cells analysed.
3. Discussion
The biochemical regulation of the G2-M transition has been extensively studied (Pines and Hunter, 1989; Li, Meyer and Donoghue, 1997; Hagting et al., 1998, 1999; Toyoshima et al., 1998; Gavet and Pines, 2010). A master regulator of this transition is the complex composed of cyclin B1-CDK1, whose activity must be tightly regulated. During interphase, cyclin B1 is expressed at low levels and localizes mainly to the cytoplasm (Hagting et al., 1998; Toyoshima et al., 1998). As cells transit from S to G2, cyclin B1 levels progressively increase (Akopyan et al., 2014; Feringa et al., 2016; Pines and Hunter, 1989), leading to cyclin binding to CDK1 in late G2. Once this occurs, the complex is inactivated by Myt1- and Wee1-mediated phosphorylation of CDK1 on residues T14 and Y15 (Lindqvist, 2010). When these inhibitory phosphorylations are removed by Cdc25 phosphatases, the complex becomes activated, rapidly stimulating its own nuclear import (Gavet and Pines, 2010). In addition to the removal of inhibitory phosphorylations on CDK1, additional mechanisms ensure the nuclear translocation of cyclin B1. These involve phosphorylation of the CRS sequence in cyclin B1 (Li, Meyer and Donoghue, 1997; Hagting et al., 1999) and binding to importin â in a Ran-independent manner (Moore et al., 1999; Takizawa, Weis and Morgan, 1999). Once inside the nucleus, the cyclin B1-CDK1 complex then triggers NPC disassembly by phosphorylating nucleoporin Nup53 (Linder et al., 2017), and is involved in the phosphorylation and subsequent disassembly of the nuclear lamina (Heald and McKeon,
1990). This mechanism allows a fast redistribution of the cyclin B1-CDK1 complexes between the cytoplasm and the nucleus, ensuring the spatiotemporal coordination of all
necessary steps leading up to mitotic entry.
So far, the contribution of mechanical forces for this essential step of the cell cycle was little explored. Recent evidence proposed that during the G2-M transition, mechanical stretch activated Piezo 1, ultimately leading to cyclin B1 transcription and consequent mitotic entry (Gudipaty et al., 2017). Here, we propose a nongenetic, mechanical pathway based on nuclear tension that acts during the G2-M transition, impacting cyclin B1 translocation and NEP. In agreement with this model, we showed that this process requires force transmission to the nucleus through the LINC complex (Fig. 3A, B, I).
Interestingly, the LINC complex was previously proposed to play a role in early spindle assembly, by facilitating chromosome alignment in a myosin II- dependent manner (Booth et al., 2019) and assisting in centrosome positioning (Stiff et al., 2020). Our work now proposes an additional role for the LINC complex- and actomyosin activity in facilitating cyclin B1 translocation (Fig. 3). In addition, we also demonstrated that decreasing nuclear tension by overexpression of LBR significantly impaired nuclear cyclin B1 uptake. Taken together, these observations suggest that nuclear force transmission and sensing could be an important player for early mitotic events. One key aspect that remains to be determined is how these forces crosstalk with the biochemical pathways that control cyclin B1 translocation. Our data clearly shows that both importin â (Fig. 2G) and CRS phosphorylation (Sup. Fig. 3C, D) are essential for force-mediated cyclin B1 translocation. Moreover, inhibition of cPLA2, increases the levels of inactive CDK1 (Sup. Fig. 3L). Therefore, while forces seem to accelerate nuclear entry of cyclin B1, they cannot bypass the biochemical requirements for cyclin B1-CDK1 translocation.
How and when physical forces might affect the biochemical pathways regulating the G2-M transition is an interesting question for future studies.
Physical forces acting on the nucleus can trigger NE unfolding and increase nuclear tension (Lomakin et al., 2020; Venturini et al., 2020). Here, we demonstrate that nuclear unfolding is a process that normally occurs in prophase cells (Fig. 4; Supplementary Fig.
1), similarly to previous observations in G2 cells (Lomakin et al., 2020). This unfolding increases cPLA2 recruitment to the NE (Fig. 4D, E), indicative of cPLA2 activation (Enyedi, Jelcic and Niethammer, 2016; Lomakin et al., 2020; Venturini et al., 2020) and likely reflects higher nuclear tension during the G2-M transition. So how does this increased tension affect cyclin B1 transport? Recent work showed that imposing forces on the nucleus is sufficient to drive nuclear import (Andreu et al., 2022) and decrease the restriction to nuclear transport, due to NPC deformation (Elosegui-Artola et al., 2017).
could be sufficient to deform the nucleus and NPCs, leading to faster cyclin B1 transport across the NE, as was proposed for YAP or MyoD (Elosegui-Artola et al., 2017; Jacchetti et al., 2021). In fact, a recent report showed that NPCs can deform in vivo, and facilitate transport across the NE (Zimmerli et al., 2021). Such a model, based on force- induced modifications in the stringency of NPCs could also explain how cyclin B1 enters the nucleus even when CDK1 is inhibited (Gavet and Pines, 2010) and why CDK1-inhibited cells show only partial rescue of nuclear cyclin B1 under confinement (Fig. 2F). Overall, we propose this mechanical pathway cooperates with the classical cyclin B1 transport machinery to fine tune NEP according to the cellular tension state, thus ensuring timely
and accurate cell division.
Establishing mechanical forces as an important player in cyclin B1 nuclear translocation and mitotic entry raises the interesting possibility that the nucleus might act as a sensor (Lomakin et al., 2020; Venturini et al., 2020) for external forces, regulating cell cycle progression and cell division to control tissue growth and avoid over-proliferation.