Date of Award
Doctor of Philosophy (PhD)
Chemical and Biomolecular Engineering
Dennis E. Discher
Cell migration through narrow interstitial space in tissues is commonly seen in both physiological and pathological conditions. The bulky stiff nucleus in the cell becomes a barrier during constricted migration making such process pore-size dependent with smaller pores exponentially alleviate the passage capability. However, in some cases, cells actively deform their nuclei to squeeze through constrictions, during which nuclear envelope can rupture. As key intracellular processes such as DNA replication and transcription take place in the nucleus, the consequences of nuclear envelope rupture become intriguing.
In the past five years, we have observed increased DNA damage, cell cycle delay and permanent genomic variation after repetitive nuclear envelope rupture as they migrate through 3µm pores, but a detailed mechanism that can connect all pieces of evidence was missing. Therefore, we have dissected the problem into three parts here (1. causality of nuclear envelope rupture, 2. how nuclear envelope rupture causes DNA damage, and 3. propagation of DNA damage to genomic variation) to reveal the mechanism step by step.
We showed that, from 2D reductionist approach, nuclear envelope rupture correlates with high nuclear envelope curvature imposed by an external AFM probe or by cell attachment to either aligned collagen fibers or stiff matrix, consistent with the shape of the nucleus when it is entering a stiff narrow pore. Mis-localization of multiple DNA repair factors are seen quickly after nuclear envelope rupture, and it is greatly enhanced by lamin-A depletion which reduces nuclear mechano-protection. The mis-localized DNA repair factors require hours for nuclear re-entry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker, γH2AX. Excess DNA damage is rescued in ruptured nuclei by co-overexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension that also rescues rupture. Increased contractility has the opposite effect, and stiff tumors with low lamin-A indeed exhibit increased nuclear curvature, more frequent nuclear rupture, and excess DNA damage. Therefore, high nuclear envelope bending dramatically facilitate nuclear envelope rupture similar to the situation when a nucleus is entering a constricting space. Mis-localization of DNA repair factors after rupture breaks the equilibrium of DNA damage and repair, resulting in a temporary elevation of DNA damage.
Benefit from 2D reductionist study, we would like to further rescue cell cycle delay after constricted migration. Myosin-II inhibition rescues nuclear envelope rupture and partially rescues the DNA damage consistent with our 2D observation, but an apparent delay in cell cycle is surprisingly unaffected. Co-overexpression of multiple DNA repair factors and antioxidant inhibition of break formation also have only partial effects, independent of rupture. Complete rescue of both DNA damage and cell cycle delay requires myosin inhibition plus antioxidant, and such result reveals a bimodal dependence of cell cycle on DNA damage. Migration through custom-etched pores yields the same damage threshold, with ~4µm being critical. Nuclear envelope rupture consistently associates with high curvature, whether imposed by pores, probes or small micronuclei, with lamin-B dilution, entry of chromatin-binding cGAS (cyclic-GMP-AMP-synthase) from cytoplasm, and loss of DNA repair factors. The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for mis-repair of DNA damage leading to genomic variations.
Finally, to investigate the propagation of DNA damage to genomic variation, we have developed a method to track chromosomal copy number changes in live cells. Our fluorescence-based reporter system functions properly in terms of both genomic characterizations and under known biological perturbations. Applying such system to constricted migration is one of our future plans and we certainly believe that the system can help us to further advance our knowledge on genomic variation after constricted migration.
In this dissertation, only first author publications are included in the chapters. Chapters 1, 2, and Appendix D are published respectively in: Acta Mechanica Sinica (2019), Journal of Cell Biology (2018), and Methods (2019). Chapter 3 has been recently accepted by Journal of Cell Biology (2019). Chapter 4 is a preprint on bioRxiv (2018), and Chapter 5 has some comments on ongoing/future work relevant to completing and submitting Chapter 4 to a peer-reviewed journal. Additional 10+ co-authored publications ranging from tumor immunotherapy to cell differentiation will not be discussed in detail here but they can be found in google scholar.
Xia, Yuntao, "Nuclear Envelope Rupture And Genomic Instability In Cancer" (2019). Publicly Accessible Penn Dissertations. 3503.