Graduate student University of Pennsylvania Philadelphia, Pennsylvania, United States
Introduction:: Mechanics of the cellular microenvironment, including matrix stiffness, has been shown to play a role in gene expression and the structure of cells in interphase – as modulated by myosin-II. Additionally, myosin-II has roles in cell division where it drives mitotic rounding to work against compression and to facilitate cytokinesis. Blebbistatin is a widely used, reversible inhibitor of myosin-II that has provided such insights. Some studies have since suggested that myosin-II inhibition perturbs mitotic spindles. Sequencing of many cancer types shows that solid tumors generally exhibit far greater aneuploidy than their soft or liquid-tissue counterparts. Myosin-II is also reported to be a tumor suppressor in skin, which is a stiff tissue, with its depletion reportedly leading to cancer. Such observations lead us to hypothesize that myosin-II suppression can increase aneuploidy, not in 2D, but only when cell division is 3D-confined.
Materials and Methods:: Gene fusions were made by inserting GFP/RFP sequences to native, constitutive genes found on chromosomes 5, 9, 12, and 19 of a solid tumor derived line, A549 lung adenocarcinoma. Thus, the loss of the GFP/RFP signal (quantified by microscopy and flow cytometry) allows us to track chromosome loss in living cells. To study mechanically induced chromosome loss, we physically confined A549 cells to perturb the mitotic rounding necessary for proper chromosome segregation. The myosin-II inhibitor, blebbistatin, was used to further study genome stability under confinement, particularly examining mitotic rounding and spindle formation.
Results, Conclusions, and Discussions:: Confinement of A549 cells leads to an increase in Chr-5 reporter-negative populations compared to 2D cultures, suggesting that perturbation of the spindles during division by compression can lead to an increase in mis-segregation events. For myosin-II inhibition studies, we see no effect on reporter-negative populations in 2D culture relative to the control, but a significant increase of the population when confined (Fig. 1A). This result suggests a protective role in maintaining genome stability for myosin-II, but only in confined, stiff microenvironments.
To understand the lack of effect in 2D cultures, we measured the percentage of abnormal anaphase and multipolar anaphase events at varying compression levels with myosin-II inhibition. Across all compression levels, abnormal anaphase events appear to not be affected by blebbistatin treatment. However, with increasing levels of compression, we notice a significant percentage decrease in multipolars with blebbistatin treatment (Fig. 1B). Previous papers suggest multipolar events under compression are often lethal. As such, this result suggests blebbistatin treatment rescues multipolar spindle formation during confinement, leading to an increase in the more tolerable, bipolar mis-segregation events.
