A Side-View On Nuclear Mechanics: Combined Atomic Force Microscopy And Light Sheet Microscopy Inform Chromatin'S Role In Regulating Nuclear Morphology

The nucleus is a mechanosensor that dynamically responds to external stimuli. Nuclear morphology and chromatin stretching have been correlated to varying levels of gene expression; nuclear rupture and subsequent DNA damage have been monitored during constricted migration. Understanding how nuclear substructures regulate mechanics and morphology is then critical to discern the full mechanosensation pathway. To study the mechanical and morphological properties of the nucleus, we utilize a combined single-objective, side-view, light-sheet microscope and an atomic force microscope (AFM). This provides us the capability to apply forces (up to 100 nN with 20 pN resolution) while simultaneously monitoring nuclear morphology in the plane of applied force. We can also dynamically scan the light sheet and image plane to collect multi-color, 3D images during deformation with the AFM. Here, we show that by analyzing the fixed, x-z cross-section image, that the 2D nuclear cross-sectional area and the perimeter begin to deform at different degrees of full cell deformation, and result in a distinct two-regime force time series. This allows us to independently determine the contributions from the volume of the nucleus and the nuclear envelope. Chromatin decompaction with Trichostatin A (TSA) reduces the nucleus’ ability to resist changes in volume as well as lowers the local curvature at the site of indentation. This implies that chromatin provides a rigid architecture to the nucleus, complementary to the nuclear lamina, that aids in maintaining its steady-state morphology. Further work will investigate Lamin A/C interventions to study its role in regulating nuclear morphology under compression.