(E-164) Matrix stiffness drives drop-like nuclear deformation and correlated YAP nuclear translocation in cancer cells

Introduction:: Many solid tumors are characterized by a general stiffening of the tissue. Cancer cells adapt to this change in mechanical stiffness through increased proliferation and migration. In addition to the canonical differences between soft and stiff matrices in adhesion assembly and cytoskeletal organization, it is increasingly appreciated that nuclear morphology is significantly different on soft matrices compared to stiff matrices. Because nuclear deformations can induce a host of secondary effects including alterations to chromatin conformation and nucleoplasmic shuttling of transcription factors, there is much interest in understanding how mechanical cues from the extracellular matrix impact the nucleus. We have recently reported that the nucleus deforms like a liquid drop against slender obstacles in migrating cells. In this “nuclear drop model”, surface tension is conferred on the nuclear drop by the nuclear lamina due to its resistance to extensional deformation. This surface tension balances the intranuclear pressure that likely arises from chromatin and osmotic pressure. The drop model of the nucleus asserts that the nuclear lamina has an excess area in it compared to the area of a sphere of the same volume. The excess area in the nuclear lamina, combined with liquid-like deformation of the nuclear interior, allows the nucleus to offer little resistance to deformation. When the nuclear lamina becomes smooth at extreme deformations, the nucleus resists any further deformation to cellular forces owing to the inextensible nuclear lamina and the incompressible nuclear volume.


Materials and Methods:: We cultured different cancer cell lines (oral cancer, breast cancer, fibrosarcoma) on soft and stiff polyacrylamide hydrogels conjugated with collagen. High resolution 3D confocal fluorescence microscopy of lamin B1 stained cells or GFP lamina A expressing cells was performed. Nuclear irregularity was analyzed using our newly developed Elliptical Fourier analysis. Fluorescence photobleaching methods were used to quantify strain relaxation in the nuclear lamina. Micropatterning of cell shape using collagen coated islands of different sizes and circular shapes was used to control cell shape and 3D confocal imaging of nuclear shapes performed.


Results, Conclusions, and Discussions:: It is not known if the drop model of nuclear deformation is applicable in contexts other than cell migration. In this paper, we set out to test key features of the drop model in the context of sensing of matrix stiffness by cancer cells. Specifically, we asked the following questions: a) Is volume conserved between the diverse nuclear shapes on soft and stiff hydrogels, b) Is excess area present in the nuclear lamina, c) is the lamina essentially inextensible once fully unfolded in spread cells, and d) can these features of the nuclear drop be controlled by constraining cell shape and spreading area. We found across different cell lines from different cancer origin that nuclei contain excess area in the form of folds and wrinkles in the nuclear lamina. Folds in the lamina were present in rounded cells on soft hydrogels but were absent in spread cells on stiff hydrogels. Importantly, patterned fluorescence photobleaching experiments revealed that the nuclear lamina in spread cells is not elastically strained, because rounding nuclei produced no measurable relaxation in the curved regions of the lamina between fiduciary photobleached markers. Rapid rounding of the nucleus induced irregularities in the nuclear lamina without any changes in volume. Micropatterning of cell shape could be used to control nuclear laminar folds/wrinkles, and the irregularities correlated inversely with YAP nuclear localization. Imaging of GFP tagged lamins post mitosis revealed that the source of excess area is the irregular coating of chromosomes during laminar reformation. These data strongly support a model in which matrix stiffness drives drop-like nuclear deformation and correlated YAP nuclear translocation in cancer cells.


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