bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.197517; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. DNA damage alters nuclear mechanics through chromatin reorganisation Ália dos Santos1, Alexander W. Cook1, Rosemarie E Gough1, Martin Schilling2, Nora Aleida Olszok2, Ian Brown3, Lin Wang4, Jesse Aaron5, Marisa L. Martin-Fernandez4, Florian Rehfeldt2,6* and Christopher P. Toseland1* 1Department of Oncology and Metabolism, University of Sheffield, Sheffield, S10 2RX, UK.2University of Göttingen, 3rd Institute of Physics – Biophysics, Göttingen, 37077, Germany. 3School of Biosciences, University of Kent, Canterbury, CT2 7NJ, UK. 4Central Laser Facility, Research Complex at Harwell, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Harwell, Didcot, Oxford OX11 0QX, UK. 5Advanced Imaging Center, HHMI Janelia Research Campus, Ashburn, USA. 6University of Bayreuth, Experimental Physics 1, Bayreuth, 95440, Germany. *Corresponding Authors: Florian Rehfeldt [email protected] & Christopher P. Toseland [email protected] Key words: Mechanics, DNA damage, DNA organisation, Nucleus ABSTRACT Cisplatin, specifically, creates adducts within the DNA double-strand breaks (DSBs) drive genomic double helix, which then lead to double-strand instability. For efficient and accurate repair of breaks (DSBs) in the DNA during replication, these DNA lesions, the cell activates DNA through replication-fork collapse3. damage repair pathways. However, it remains DSBs can result in large genomic aberrations and unknown how these processes may affect the are, therefore, the most deleterious to the cell. In biomechanical properties of the nucleus and what the event of a DNA break, the cell activates DNA role nuclear mechanics play in DNA damage and Damage Response (DDR) pathways that allow repair efficiency. detection and repair of this lesion. Failure to Here, we used Atomic Force Microscopy (AFM) repair damage leads to cell death through to investigate nuclear mechanical changes, apoptosis, or to the propagation of mutations that arising from externally induced DNA damage. We drive genomic instability and cancer development found that nuclear stiffness is significantly 4-6. reduced after cisplatin treatment, as a It is well known that the ataxia-telangiectasia consequence of DNA damage signalling. This mutated (ATM) kinase localises to regions of softening was linked to global chromatin damage, where it phosphorylates histone H2AX, decondensation, which improves molecular producing γH2AX. This, in turn, promotes the diffusion within the organelle. We propose that recruitment of multiple repair factors to foci of this can increase recruitment for repair factors. damage7,8. However, it is not yet known if DNA Interestingly, we also found that reduction of damage, or DDR itself, could lead to alterations nuclear tension, through cytoskeletal relaxation, in nuclear mechanics. has a protective role to the cell and reduces It is established that chromatin and the lamina are accumulation of DNA damage. Overall, these major determinants of nuclear mechanics 9, and changes protect against further genomic we have previously shown that a thicker nuclear instability and promote DNA repair. We propose lamina correlate with higher nuclear stiffness 10. that these processes may underpin the In the context of DDR, local changes to the development of drug resistance. condensation state of the chromatin are associated to efficiency of DNA repair, and INTRODUCTION chromatin remodelling factors are known to be Cells are known to respond to external stimuli recruited to foci of damage 11,12. As with DNA through changes to their biomechanical damage, it is still unknown how these alterations properties, such as cellular stiffness. Some impact the physical properties of the nucleus. studies have previously related chemotherapy Here, we investigated the relationship between treatments to changes in the stiffness of cells and DNA damage and nuclear mechanics. We used tissues1,2. Chemotherapy agents such as Atomic Force Microscopy (AFM) to probe cisplatin induce DNA damage, and therefore their mechanics of the nucleus of mammalian cells main mechanism of action occurs within a and monitor changes that occur after treatment nuclear context. However, little is known about with cisplatin. We found that, following DNA the biomechanical changes that might occur in damage, large-scale mechanical alterations to the organelle following DNA damage. the nucleus arise, caused by global bioRxiv preprint doi: https://doi.org/10.1101/2020.07.10.197517; this version posted July 11, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. decondensation of chromatin. This is dependent the nucleus and the cytoskeleton are to whole- on DDR activation and increases molecular cell mechanics. diffusion in the nucleus, potentially resulting in To understand if the mechanical changes higher accessibility for repair factors. observed were a result of alterations to the Surprisingly, we also found that mechanical cytoskeleton, we labelled cells with phalloidin relaxation of the nucleus, independently of after cisplatin treatment (Supp Fig.1). Our data chromatin, protects against genomic instability. show that there is no actin cytoskeleton Collectively, our data reveal how changes to impairment, indicating that the observed chromatin architecture following DNA damage mechanical effect is not a result of the both suppress further genomic instability and dissociation of actin filaments in the cell. We provide an environment to promote repair. These therefore postulated that the change in stiffness findings may be harnessed to promote could be the result of altered biophysical therapeutic approaches (e.g. resistance within a properties in the nucleus. tumour environment). To understand the individual contribution of the nucleus to global mechanics, we decided to RESULTS perform measurements on cells at initial Whilst the biochemical responses to DNA adhesion stages, isolated nuclei (Fig. 2A, Supp damage are well investigated 4,13,14, their effects Fig 2) and cells treated with two different on the biophysical properties of the nucleus are cytoskeletal destabilizing drugs - Blebbistatin and still poorly understood. To investigate this, we Latrunculin B (Supp. Fig. 1). used cisplatin, a chemotherapy drug that At initial adhesion stages, the nucleus is the crosslinks DNA, thereby inducing DSBs within largest contributor to whole-cell mechanics. In DNA, and AFM to measure mechanical changes this case, the cells are given enough time to in nucleus. adhere to the surface (2-3 hours for HeLa cells) but the cytoskeleton and in particular stress Cisplatin treatment reduces nuclear stiffness. fibres, are not fully established (Supp Fig.1). As a Before AFM measurements, fully-adhered cells result, AFM measurements taken above the on glass were treated with 25 µM cisplatin for 4 nucleus only reflect the mechanical properties of hours, or 24 hours, which induced DNA damage, the organelle. Alternatively, Blebbistatin, a recorded as fluorescent foci of gH2AX (Fig. 1A). Myosin II inhibitor, and Latrunculin B, an actin AFM measurements were performed at a central depolymerising drug, relax the cytoskeleton 15-17, point above the nucleus of a selected cell, up to and therefore measurements in these cells are a maximal force of 10 nN (Fig. 1B and C). Force- mainly directed at the nucleus, without a distance curves were then fitted using the Hertz contribution from the cytoskeleton. model, to determine the effective Young’s elastic Our AFM data show that, as expected, fully- moduli E. Figure 1D shows representative adhered cells (4.4±0.7 kPa) are stiffer than experimental curves of deflection versus probe- Latrunculin B and Blebbistatin treated cells sample separation and, in the insets, the fitted (2.6±1.2 kPa and 2.1±0.8 kPa, respectively), curves. initially-adhered cells (2.9±0.3 kPa) and isolated We found that long treatment with cisplatin (24 nuclei (2.6±0.4 kPa), whilst there is no significant hours), but not short treatment (4 hours), difference between these four latter conditions significantly reduced cellular stiffness (Fig. 1E). (Fig. 2B). Young’s modulus values changed from 4.4 ± 0.6 From these data we confirmed that nuclear kPa (mean ± SEM) in non-treated cells to 3.2 ± measurements performed in initially-adhered 0.4 kPa after cisplatin 4-hours and 1.9 ± 0.2 kPa cells are relatively free of cytoskeletal in cells, following 24-hour cisplatin treatment. contributions and that the nucleus remains as the These results suggest that the observed only large structural variable. Therefore, we mechanical change is temporally separated from decided to use this approach to investigate the DNA damage itself. Therefore, breaks in DNA changes to nuclear mechanics, as this allows for do not directly change the mechanical properties the physical properties of nucleus to be probed of the nucleus, but rather trigger events that whilst maintaining the organelle in its further downstream affect the nuclear stability. physiological environment and without the