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1.
J Cell Biol ; 218(8): 2545-2563, 2019 08 05.
Article in English | MEDLINE | ID: mdl-31239284

ABSTRACT

Migration through 3D constrictions can cause nuclear rupture and mislocalization of nuclear proteins, but damage to DNA remains uncertain, as does any effect on cell cycle. Here, myosin II inhibition rescues rupture and partially rescues the DNA damage marker γH2AX, but an apparent block in cell cycle appears unaffected. Co-overexpression of multiple DNA repair factors or antioxidant inhibition of break formation also exert partial effects, independently of rupture. Combined treatments completely rescue cell cycle suppression by DNA damage, revealing a sigmoidal dependence of cell cycle on excess DNA damage. Migration through custom-etched pores yields the same damage threshold, with ∼4-µm pores causing intermediate levels of both damage and cell cycle suppression. High curvature imposed rapidly by pores or probes or else by small micronuclei consistently associates nuclear rupture with dilution of stiff lamin-B filaments, loss of repair factors, and entry from cytoplasm of chromatin-binding cGAS (cyclic GMP-AMP synthase). The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for DNA misrepair and genome variation.


Subject(s)
Cell Cycle , Cell Movement , DNA Damage , Mechanotransduction, Cellular , Animals , Antioxidants/metabolism , Cell Line, Tumor , DNA Repair , Exodeoxyribonucleases/metabolism , Humans , Ku Autoantigen/metabolism , Lamin Type B/metabolism , Mice , Mutagenesis , Myosin Type II/metabolism , Nuclear Pore/metabolism , Nuclear Pore/ultrastructure , Nucleotidyltransferases/metabolism , Phosphoproteins/metabolism
2.
Dev Cell ; 49(6): 920-935.e5, 2019 06 17.
Article in English | MEDLINE | ID: mdl-31105008

ABSTRACT

Whether cell forces or extracellular matrix (ECM) can impact genome integrity is largely unclear. Here, acute perturbations (∼1 h) to actomyosin stress or ECM elasticity cause rapid and reversible changes in lamin-A, DNA damage, and cell cycle. The findings are especially relevant to organs such as the heart because DNA damage permanently arrests cardiomyocyte proliferation shortly after birth and thereby eliminates regeneration after injury including heart attack. Embryonic hearts, cardiac-differentiated iPS cells (induced pluripotent stem cells), and various nonmuscle cell types all show that actomyosin-driven nuclear rupture causes cytoplasmic mis-localization of DNA repair factors and excess DNA damage. Binucleation and micronuclei increase as telomeres shorten, which all favor cell-cycle arrest. Deficiencies in lamin-A and repair factors exacerbate these effects, but lamin-A-associated defects are rescued by repair factor overexpression and also by contractility modulators in clinical trials. Contractile cells on stiff ECM normally exhibit low phosphorylation and slow degradation of lamin-A by matrix-metalloprotease-2 (MMP2), and inhibition of this lamin-A turnover and also actomyosin contractility are seen to minimize DNA damage. Lamin-A is thus stress stabilized to mechano-protect the genome.


Subject(s)
Cell Cycle Checkpoints , Cell Nucleus/metabolism , DNA Damage , Heart/embryology , Lamin Type A/metabolism , Mechanotransduction, Cellular , Nuclear Lamina/metabolism , Animals , Cell Differentiation , Chick Embryo , Chickens , DNA Repair , Extracellular Matrix , Heart/physiology , Humans , Organogenesis , Phosphorylation
3.
J Cell Biol ; 217(11): 3796-3808, 2018 11 05.
Article in English | MEDLINE | ID: mdl-30171044

ABSTRACT

The nucleus is physically linked to the cytoskeleton, adhesions, and extracellular matrix-all of which sustain forces, but their relationships to DNA damage are obscure. We show that nuclear rupture with cytoplasmic mislocalization of multiple DNA repair factors correlates with high nuclear curvature imposed by an external probe or by cell attachment to either aligned collagen fibers or stiff matrix. Mislocalization is greatly enhanced by lamin A depletion, requires hours for nuclear reentry, and correlates with an increase in pan-nucleoplasmic foci of the DNA damage marker γH2AX. Excess DNA damage is rescued in ruptured nuclei by cooverexpression of multiple DNA repair factors as well as by soft matrix or inhibition of actomyosin tension. 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. Additional stresses likely play a role, but the data suggest high curvature promotes nuclear rupture, which compromises retention of DNA repair factors and favors sustained damage.


Subject(s)
Cell Nucleus/metabolism , DNA Repair , Histones/metabolism , Lamin Type A/metabolism , A549 Cells , Cell Nucleus/genetics , Histones/genetics , Humans , Lamin Type A/genetics
4.
Mol Biol Cell ; 29(16): 1948-1962, 2018 08 08.
Article in English | MEDLINE | ID: mdl-29742017

ABSTRACT

Cell migration through dense tissues or small capillaries can elongate the nucleus and even damage it, and any impact on cell cycle has the potential to affect various processes including carcinogenesis. Here, nuclear rupture and DNA damage increase with constricted migration in different phases of cell cycle-which we show is partially repressed. We study several cancer lines that are contact inhibited or not and that exhibit diverse frequencies of nuclear lamina rupture after migration through small pores. DNA repair factors invariably mislocalize after migration, and an excess of DNA damage is evident as pan--nucleoplasmic foci of phosphoactivated ATM and γH2AX. Foci counts are suppressed in late cell cycle as expected of mitotic checkpoints, and migration of contact-inhibited cells through large pores into sparse microenvironments leads also as expected to cell-cycle reentry and no effect on a basal level of damage foci. Constricting pores delay such reentry while excess foci occur independent of cell-cycle phase. Knockdown of repair factors increases DNA damage independent of cell cycle, consistent with effects of constricted migration. Because such migration causes DNA damage and impedes proliferation, it illustrates a cancer cell fate choice of "go or grow."


Subject(s)
Cell Cycle , Cell Movement , DNA Damage , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Proliferation , DNA Repair , Gene Knockdown Techniques , Histones/metabolism , Humans
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