UNC-CH Physics and Astronomy Thesis Defense

Chad Hobson

“On the Biomechanics of Cell Nuclei: Insights from Combined Force and Light Microscopy”

Cell nuclei are multifunctional. Not only do they house and protect the genome, but they additionally provide mechanical stability to the surrounding cell and convert extracellular mechanical cues into biochemical responses. For example, forces exerted upon integrins at the cell surface propagate through the cytoskeleton and into the nucleus, resulting in local stretching of chromatin and transcription upregulation. The mechanical integrity of the nucleus, however, is often compromised in an array of diseases ranging from cancers to laminopathies. These diseases have targeted effects on specific nuclear constituents, and in turn lead to altered cellular migration properties, increases in DNA damage and genomic instability, and compromised nuclear mechanotransduction. Consequently, the mechanobiology of the cell nucleus has garnered increasing attention over the past several decades. Studies in nuclear biomechanics primarily make use of force probes and/or light microscopy to quantify mechanical properties of nuclei and their responses to physical perturbations. In the first half of this thesis, I describe two novel methodologies for studying nuclear mechanobiology. The first is a side-view, light-sheet fluorescence microscope combined with an atomic force microscope (AFM-LS) that enables time-correlated, multi-color, 3D light sheet imaging coupled with AFM. The second method is a unique combination of light-sheet microscopy and fluorescence recovery after photobleaching (FRAP) known as SPIM-FRAP, which I used to simultaneously quantify diffusion across an entire 2D image. In the latter half of this thesis, I describe how I have used both AFM-LS and SPIM-FRAP to study nuclear mechanobiology. SPIM-FRAP is used to show how intranuclear diffusion of NLS-GFP is slowed in nucleoli, but overall uncorrelated with chromatin structure on the length scale of single fluorophores. Additionally, I use SPIM-FRAP to show that sites of DNA damage are more stable than the surrounding diffuse repair proteins. AFM-LS is used to separate of the roles of chromatin and lamin A/C in nuclear compression, regarding both the mechanical response of the nucleus as well as local nuclear curvature. I also use multi-channel, 3D AFM-LS to show how compression alone via AFM, independent of nuclear rupture, is sufficient to induce DNA damage in nuclei. This indicates a novel mechanism by which nuclei incur double-stand DNA breaks. Finally, I provide the first review of mechanical models of nuclei, along with the development of a continuum mechanical model for AFM indentation. Together, these developments in methodologies along with the coupled insights into intranuclear dynamics, nuclear mechanics, and DNA damage improve both our means of studying and our current understanding of nuclear mechanobiology.