Confinement plus myosin-II suppression maximizes heritable loss of chromosomes, as revealed by live-cell ChReporters

Abstract

A cell's mechanical environment can have many effects, but whether it impacts a cell's DNA sequence has remained unclear. To investigate this, we developed a live-cell method to measure changes in chromosome numbers. We edited constitutive genes with GFP/RFP-tags on single alleles and discovered that cells that lose Chromosome-reporters (ChReporters) become non-fluorescent. We applied our new tools to confined mitosis and to inhibition of the putative tumor suppressor Myosin-II. We quantified compression of mitotic chromatin in vivo and demonstrated that similar compression in vitro resulted in cell death, but also rare and heritable ChReptorter loss. Myosin-II suppression rescued lethal multipolar divisions and maximized ChReporter loss in 3D-compression and 2D-confinement, but not in standard 2D-culture. ChReporter loss associated with chromosome mis-segregation, rather than just the number of divisions, and loss in vitro and in mice was selected against in subsequent 2D-cultures. Inhibition of the spindle assembly checkpoint (SAC) caused ChReporter loss in 2D, as expected, but not in 3D-compression, suggesting a SAC perturbation. Thus, confinement and myosin-II affect DNA sequence and mechano-evolution, and ChReporters enable diverse studies of viable genetic changes.

Mitosis sets nuclear homeostasis of cancer cells under confinement

“…During cell division, actomyosin contractility participates in cleavage furrow formation (37). A recent study pointed out that myosin-II has indeed a role of mechano-protection, with increase of abnormal mitosis and genetic changes upon myosin-II suppression (38). We report here for the first time a role of myosin-II also in mechano-adapatation of nuclear volume and tension: contractility is required to set nuclear volume and nuclear envelope folding, mostly during nuclear envelope reformation at the end of mitosis.…”

Differences in cell shape, motility, and growth reflect chromosomal number variations that can be visualized with live-cell ChReporters

“…The DNA sequence in a viable cell can change as a result of physical stressors that range from radiation to mechanical strain ( Hayes et al. , 2023 ).…”

“…To address our hypothesis, we developed a general “ChReporter” method that allows us to see a specific Chr loss in a living cell ( Hayes et al. , 2023 ).…”

“…As one particular example, the constitutive gene Lamin-B1 on Chr-5 was gene-edited as a monoallelic RFP or GFP fusion such that visible loss of the fluorescent signal signifies loss of one copy of Chr-5. Detecting an increase in RFP signal can in principle be used to detect a Chr gain, but levels of Lamin-B1 happen to increase about two-fold from G1 to G2/M ( Hayes et al. , 2023 ) whereas complete loss of signal is more obvious.…”

“…In standard cultures of many cell lines, micronuclei are frequently seen and contain chromosomal DNA ( Figure 1A image, arrow) before loss or gain of a Chr in daughter cells of subsequent cell generations. Indeed, we have recently shown that rare RFP-negative cells emerging from pure RFP-positive cells ( Figure 1A ) not only reflect loss of one allele of Chr-5 but also relate to abnormal mitosis on a range of substrate stiffnesses; these included collagen-coated soft gels on which cells exhibited more frequent mitotic errors and significantly higher Chr-5 loss than cells on plastic despite reduced proliferation ( Hayes et al. , 2023 ).…”