Lamins A/C are essential for nesprin-2/ERK compartmentalisation at PML NBs

To investigate whether an intact nuclear lamina was necessary for complex formation, lamin A/C was depleted from VSMCs using siRNA (Figure 4a). Although the number of PML NBs per nuclei remained unaltered, they were larger in lamin A/C-depleted VSMCs compared with control (Figures 4b–d). Moreover, lamin A/C-depleted cells showed reduced nesprin-2 and ERK2 localisation at PML NBs (Figures 4e and f). To determine the long-term impact of lamin A/C depletion on VSMC proliferation, cells were subjected to three rounds of siRNA-mediated knockdown. Extended lamin A depletion had no effect on 53BP1 foci number per nuclei, PDT or SA-β-gal staining (Supplementary Figure 7). GST-pull down assays using a construct containing the C-terminal portion of lamin-A, which consists of an immunoglobulin domain-like region precipitated nesprin-2βΔKASH1, ERK2 and PML, and this was not observed with GST alone (Figure 4g), suggesting the complex could bind directly to lamin A. Several other nesprin-2 variants were also efficiently precipitated as expected, including nesprin-22 and p156KASH^nesp2 (Figure 4g and Supplementary Figure 4).

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Figure 4

The A-type lamins are essential for nesprin-2/ERK/PML complex organisation in VSMCs. (a) WB of control and lamin A-depleted VSMC lysates. (b) IF showing PML organisation (green) and DAPI (blue) in control and lamin A-depleted VSMCs; Quantification of (c) PML size (*P=<0.0001) and (d) PML number per nuclei in control and lamin A-depleted VSMCs. Graphs show the combined data of three independent experiments counting 300 cells. (e) IF showing nesprin-2 (green) and ERK2 (red) localisations in control and lamin A-depleted nuclei. (f) Quantification of nesprin-2 localisation at PML NBs in control and lamin A-depleted VSMC nuclei (***P=0.0009). Graph shows the combined data of three independent experiments counting 300 cells. (g) WB of GST-pull downs using bacterial expressed lamin A tail construct and VSMC lysates

Prelamin A-induced DNA damage triggers ERK1/2 compartmentalisation at DNA lesions

Next, we investigated whether nuclear lamina disruption induced by prelamin A was the triggering event for ERK1/2 compartmentalisation at PML NBs. SiRNA-mediated knockdown of the lamin A processing the enzyme FACE1 to induce prelamin A accumulation in proliferative VSMCs (Supplementary Figure 8) triggered an increase in cells with ERK2 localisation at PML NBs (Figures 5a and b). No nucleolar cap formation was observed; however, ERK2-associated PML NBs did redistribute toward the nucleolar periphery in FACE1-depleted cells (Figures 5a and b). As prelamin A accumulation promotes DNA damage, we also investigated whether DNA damage per se could induce complex formation. Treatment of proliferative VSMCs with the DNA damage inducer doxorubicin increased the levels of phosphorylated ERK1/2 in VSMC lysates (Supplementary Figure 9), and the association between nesprin-2βΔTM and both ERK2 and phosphorylated ERK1/2 was also enhanced (Figures 5c and d, and Supplementary Figure 9). Subcellular fractionation and WB showed that prelamin A also accumulated in the nuclear fraction in response to doxorubicin (Supplementary Figure 9) suggesting there is a potential feedback between ERK compartmentalisation and prelamin A accumulation in response to DNA damage.

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Figure 5

Prelamin A accumulation induces ERK1/2 compartmentalisation at PML NBs that abut DNA lesions. (a) IF showing nesprin-2 (green) and ERK2 (red) localisations in control and FACE1-depleted nuclei. Arrows mark PML NBs localised towards the nucleolar periphery. (b) Quantification of ERK2 localisation at PML NBs (*P=0.0278) and nucleolar periphery (**P=0.0202) in control and FACE1-depleted nuclei. Graph shows the combined data of three independent experiments counting 300 cells. IP of (c) ERK2 antibody to pellet 75kDa nesprin-2βΔKASH1 variant and (d) nesprin-2 antibody to pellet ERK2 from control and doxorubicin-treated VSMCs. (e) IF of doxorubicin-treated proliferative VSMCs; nesprin-2 (green), ERK2 (red). Arrowheads mark nucleolar caps. (f) Quantification of cells displaying ERK2-positive nucleolar caps after a 3-h doxorubicin treatment (*P=0.0081 and **P=0.0146). Graph shows the combined data of three independent experiments counting 300 cells

Confocal immunofluorescence showed that doxorubicin treatment caused a dose-dependent, relocalisation of both nesprin-2 and PML into nucleolar cap structures (Figures 5e and f, and Supplementary Figure 9). After a short-term treatment with doxorubicin (1h), ERK2 was lacking in these nucleolar caps (Figures 5e and f). However, prolonged doxorubicin (3h) treatment enhanced the association of ERK2 with the nesprin-2-positive nucleolar caps (Figures 5e and f). Pretreatment with the MEK inhibitor U0126 failed to disrupt nesprin-2 reorganisation into the nucleolar cap, but it dramatically reduced ERK2 association with the nucleolar cap suggesting that active ERK1/2 are components of this complex (Figures 5e and f).

Lamin A associates with nesprin-2, ERK and PML

Lamins A/C form a filamentous network throughout the nucleus that spatially organises DNA-repair and nuclear signalling events.⁶ We speculated that lamins A/C were good candidates to spatially regulate the nesprin-2/ERK/PML complex, and we confirmed that this complex interacts with the C-terminus of lamin A. Nesprin-2 and ERK1/2 have previously been shown to independently interact with lamin A; nesprin-2 binds to the lamin A tail via its SRs, whereas ERK1/2 bind to coil 2 of lamin A, suggesting that nesprin-2βΔKASH1 also couples ERK1/2 to the lamin A tail, independently of the coil 2/ERK interaction.^{21, 22, 23} Moreover, lamins A/C disruption resulted in increased PML NB size in VSMCs, and this is consistent with the larger PML NBs with reduced mobility observed in lamin A-deficient nuclei.¹⁸ Changes in PML size were also observed in VSMCs with prelamin A accumulation; however, whether these larger bodies have different functionality has not been determined. Lamins A/C disruption also resulted in disruption of ERK compartmentalisation, suggesting that lamins A/C perform a scaffolding role anchoring the nesprin-2/ERK complex at PML NBs during the DDR. However, despite the involvement of lamins A/C in nesprin-2/ERK compartmentalisation, lamins A/C depletion did not impair, ATM or Chk2 phosphorylation and DNA damage continued to be efficiently repaired. This suggests that lamins A/C and ERK compartmentalisation are not essential for DNA repair and that nesprin-2/ERK maintains signalling integrity even when diffusely localised in the nucleoplasm.

Efficient DNA repair does not require PML NBs in VSMCs

Although PML NBs have been implicated in DNA repair, their importance remains controversial.^{13, 24} Numerous DNA-repair proteins, including ATM and ATR, are known to localise at PML NBs in the absence of DNA damage.^{25, 26, 27} Genotoxic stress increases the PML NB number, stimulates their association at DNA lesions and induces the loss of these repair proteins from PML NBs.^{25, 26, 27} Post-translational modification is proposed to play a role in the relocalisation of these proteins raising the intriguing possibility that ERK1/2 recruitment to PML NBs may stimulate the redistribution of repair proteins. In addition, PML NBs may serve to store active ERK1/2 during the DDR, and further investigation is required to clarify the relationship between PML NBs and ERK1/2 signalling in the nucleus. DNA damage also stimulated organisation of PML-associated ERK1/2 into nucleolar caps. The function of these nucleolar caps within the DDR has yet to be fully elucidated, although PML has been shown to influence p53 activity by sequestering MDM2 at the nucleolus.^{14, 15, 28} Thus, nucleolar caps may serve as hubs to regulate the cell cycle during the DDR. However, despite these connections between PML NBs and DNA repair, we show that PML disruption did not ablate DNA repair in VSMCs (Supplementary Figure 5). This observation is consistent with a previous study demonstrating that PML NBs are nonessential for DNA repair and senescence in fibroblasts.²⁹ Importantly, depletion of lamins A/C or PML disrupted ERK2 compartmentalisation, suggesting that both serve as scaffolds to assist lesion repair. However, disrupted ERK compartmentalisation, either by lamins A/C or PML depletion, failed to ablate DNA repair, further suggesting that nesprin-2βΔKASH1 remains associated with ERK1/2 and signalling fidelity remains intact even when compartmentalisation is lost.

Antibodies, IF and flow cytometry

Antibodies used for WB, IF and IPs were the following: ATM (ab78), ATR (ab2905), nucleolin (ab13541; Abcam, Cambridge, UK); pH2AX (#9718), pSer15 p53, pSer1981 ATM (#5883), 53BP1 (#4937), pChk2 (T68) (#2661), Chk2 (#6334; Cell Signaling Technologies); Face-1 C-13 (sc-34777), prelamin-A C-20 (sc-6214), lamin A/C N-18 (sc-6215), PML PG-M3 (sc-966), ERK2 D-2 (sc-1647), pERK1/2 E-4 (sc-7383; Santa Cruz, Dallas, TX, USA); Vinculin (V9131), β-actin (A5316; Sigma); and nesprin-2 N3. Secondary antibodies for WB were horseradish peroxidase-conjugated antimouse (NA931) or antirabbit (NA94V) antibodies from GE Healthcare (Lafayette, CO, USA). ECL chemiluminescent kit (RPN2132, GE Healthcare) was used for the detection according to the manufacturer's instructions. Invitrogen (Paisley, UK) Anti-Mouse Alexa Fluor 568 (A11031) and Anti-Rabbit Alexa Fluor 488 (A11034, Invitrogen) were used as IF secondary antibodies. Cells were cultured on cover slips, fixed in 50% methanol/acetone and processed as described previously. SA-β-gal staining was performed as described previously.² For observing the DNA content of cells by propidium iodide staining, cells were trypsinised and washed three times in PBS. Cells were fixed in ice-cold 75% ethanol for 30min at 4°C. DNA staining was achieved by addition of PI and RNase for 45min at 37°C. For γH2AX staining cells were processed as per the manufacturer's instructions (Cell Signaling Technologies).

Confocal microscopy and data analysis

All images were captured at × 63 magnification using a Leica (Milton Keynes, UK) SP5 laser scanning confocal microscope. PML number and size was measured using the find object function in the Volocity software (PerkinElmer, Waltham, MA, USA).

IPs and subcellular fractionations

In vivo IPs, GST-pull downs and subcellular fractionations were performed as described previously.¹⁰

Comet assays

DNA damage was assessed using the Comet assays as described previously.² Protocols were obtained from the Comet Assay Interest Group website (www.cometassay.com).

This work was funded by a British Heart Foundation (BHF) program grant to CMS (program grant number RG/11/14/29056). DW holds a BHF IBSRF (FS/11/53/29020).