Mitochondria-derived nuclear ATP surge protects against confinement-induced proliferation defects

Introduction

Cancer cells are exposed to substantial mechanical forces arising from, for example, the growing pressure and restricted space inside a solid tumour or from trying to squeeze through tight spaces during metastasis (1). This confinement may cause changes to the cytoskeleton (2,3), the nucleus (4) and may lead to DNA damage (5) and mitotic defects (6). This preprint by Ghose, Pezzano and colleagues uncovers how cells can proliferate under such harsh conditions and highlights thus far unknown mechanisms of adaptation.

Key findings

Mitochondria localize close to the nucleus upon confinement

To study the consequences of confinement for cancer cell fitness, this preprint presents an experimental setup featuring an agarose disc which presses onto and deforms HeLa cells. To understand changes in nuclear vs cytoplasmic protein content upon confinement, the authors performed nucleo-cytoplasmic fractionation, followed by mass spectrometry. This analysis revealed the enrichment of mitochondrial and ATP-related proteins in the nuclear fractions of confined cells, which could be due to mitochondria being inside or in a physical proximity to the nucleus. To gain more information on mitochondrial localization, the authors observed these organelles in real-time via super-resolution microscopy. This approach revealed two interesting phenomena: First, mitochondria localized more close to the nucleus in confined HeLa cells. Second, the nucleus was deformed and exhibited indentations, enriched with mitochondria. Interestingly, mitochondrial relocalization happened within seconds upon applying the confinement and persisted for roughly half an hour after release.
To further understand how mitochondria are brought closer to the nucleus during confinement, the authors looked at the endoplasmic reticulum (ER) – a good candidate for facilitating the nucleus-mitochondria association. In agreement with previous work of the group demonstrating that the ER gets immobilized close to the nucleus in confined cells (7), super-resolution imaging in the current setup showed that mitochondria were entangled within the ER network. Next, the authors looked at actin and calcium levels, which are important for ER-mitochondria contacts and for mitochondrial activity respectively. Both actin depolymerization by Latrunculin A and interfering with actin elongation reduced the number of nucleus-associated mitochondria (NAMs), demonstrating a clear role for the actin cytoskeleton in mediating the confinement-related appearance of NAMs.

ATP surge in the nucleus protects DNA by increasing chromatin solubility

The preprint’s narrative then shifts its focus on attempts to try and understand the function of NAMs. Motivated by the enrichment of ATP-related proteins in the nuclear fraction upon mass spectrometry analysis, the authors hypothesized that NAMs may provide ATP to the nucleus to help it adapt to confinement. Using live imaging of an intracellular ATP sensor, the authors observed a rapid increase in nuclear ATP levels in HeLa cells during confinement. To test the potential mechano-protective role of this ATP surge in the nucleus, the authors assessed chromatin state. Using Hoechst images at different binning, they computed a nuclear coefficient of variation, which helped them assess how chromatin structures are spatially organized at different scales and, thus, served as an indicator of chromatic de-/condensation. The coefficient of variation of confined cells sharply dropped at high binning, suggesting an overall less heterogenous chromatin condensation pattern. By performing assays for transposase-accessible chromatin with sequencing (ATAC-Seq), the authors then showed that chromatin reorganization upon confinement does not serve a particular transcriptional program, but most likely protects DNA by promoting nuclear softening.

ATP surge serves DNA repair and enables cell cycle progression

The authors more closely examined the possible roles of the ATP surge in the nucleus, the first of which was DNA damage repair. Confinement increases DNA damage foci in HeLa cells, as evidenced by 53BP1 positive nuclei. Blocking mitochondrial function by oligomycin A treatment delayed the resolution of DNA damage, supporting a role of the ATP surge in DNA repair. Lastly, the authors explored the effects of confinement on cell cycle progression. They tracked the cell cycle progression of confined cells during the 36 h following their release. Both confinement and oligomycin A treatment alone affected the progression of G1 and G2 phases. A combination of the two led to a significant delay of S-phase progression, which could probably be attributed to delayed DNA damage repair.

Altogether, this preprint shows actin-dependent relocalization of mitochondria closer to the nucleus upon confinement, which leads to an ATP surge in the nucleus. This ATP surge decondensates chromatin to soften the nucleus as a mechanical protection mechanism and allows for timely DNA repair to aid S-phase completion and cell cycle progression.

What I like about this preprint

I attended a talk by one of the preprint authors who presented this work at the conference ‘Nanoengineering for Mechanobiology’ (M4M) in Camogli, Italy, earlier this year. The simplicity of the concept stuck with me – the observed phenomenon makes so much sense and the preprint explains a lot. At the same time, this preprint is extremely thorough, and every claim is solidly backed by multiple experiments and all possible controls. The idea that chromatin decondensation can also merely serve a protective function deeply fascinated me. Since I work on ageing, this work made me curious whether age-related chromatin states may also have an additional, mechanoadaptive function. Finally, this preprint is very well-written. I enjoyed following the thought process behind the experiments – it made the reading very enjoyable.

Questions for the authors

Besides the relocalization of mitochondria close to the nucleus, do you see any changes in the total mitochondrial number and/or their shapes, pointing to possibly altered fission-fusion dynamics?

I was wondering if there are any long-term epigenetic or transcriptional consequences of the sudden chromatin decondensation upon confinement? Have you looked at transcriptomics or chromatin accessibility long after confinement release?

Lastly, are cells in your confinement setup static? In the hypothetical case of metastasis where a cell would need to migrate through a confined space, how would the mitochondria be located to provide ATP both for DNA repair and for migration at the leading edge? Or would one of these processes have a metabolic privilege?

References

1. Massey, A. et al. Mechanical properties of human tumour tissues and their implications for cancer development. Nat. Rev. Phys. 6, 269–282 (2024).
2. Li, X. et al. Tensile force-induced cytoskeletal remodeling: Mechanics before chemistry. PLoS Comput. Biol. 16, e1007693 (2020).
3. Walker, M., Rizzuto, P., Godin, M. & Pelling, A. E. Structural and mechanical remodeling of the cytoskeleton maintains tensional homeostasis in 3D microtissues under acute dynamic stretch. Sci. Rep. 10, 7696 (2020).
4. Hoffman, L. M. et al. Mechanical stress triggers nuclear remodeling and the formation of transmembrane actin nuclear lines with associated nuclear pore complexes. Mol. Biol. Cell 31, 1774–1787 (2020).
5. Irianto, J., Xia, Y., Pfeifer, C. R., Greenberg, R. A. & Discher, D. E. As a Nucleus Enters a Small Pore, Chromatin Stretches and Maintains Integrity Even with DNA Breaks. Biophys. J. 112, 373a (2017).
6. Tse, H. T. K., Weaver, W. M. & Carlo, D. D. Increased Asymmetric and Multi-Daughter Cell Division in Mechanically Confined Microenvironments. PLoS ONE 7, e38986 (2012).
7. Venturini, V. et al. The nucleus measures shape changes for cellular proprioception to control dynamic cell behavior. Science 370, eaba2644 (2020).

 

doi: https://doi.org/10.1242/prelights.37433

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