NUCLEOCYTOSKELETAL CONNECTIONS IN MECHANOTRANSDUCTION

Cells continuously sense and respond to their physical surroundings through their cytoskeleton. The perinuclear actin cap (or actin cap) is a recently-characterized cytoskeletal organelle composed of thick, parallel, and highly contractile actomyosin filaments that are specifically anchored to the apical surface of the interphase nucleus. The actin cap is present in many types of adherent eukaryotic cells but is disrupted in several human disease models, including laminopathies and cancer. Through its large terminating focal adhesions and anchorage to the nuclear lamina and nuclear envelope through LINC (Linkers of the Nucleoskeleton to the Cytoskeleton) complexes, the perinuclear actin cap plays a critical role in mechanotransduction, the ability of cells to sense and respond to mechanical forces. In this work, I demonstrate that only fibers of the actin cap, not conventional basal actin stress fibers, form in response to low physiological mechanical stresses in adherent fibroblasts. While conventional basal stress fibers form only past a threshold shear stress of 0.5 dyn/cm2, actin-cap fibers form at shear stresses 50 times lower and orders-of-magnitude faster than biochemical stimulation. This fast differential response is uniquely mediated by focal adhesion protein zyxin at low shear stress and actomyosin contractility of the actin cap. I identify additional roles for lamin A/C of the nuclear lamina and LINC molecules nesprin2giant and nesprin3, which anchor actin cap fibers to the nucleus. I briefly explore mechanotransduction of interstitial fluid flow within a three-dimensional culture system. Next, I seek to characterize the extent of mechanotransduction in the nucleus through a novel microscopy assay that rapidly quantifies global acetylation on histone H3 and measures several cell and nuclear properties, including cell and nuclear morphology descriptors, cell-cycle phase, and filamentous-actin content of thousands of cells simultaneously, without cell detachment from the substrate, at single-cell resolution. These measurements reveal that isogenic, isotypic cells of identical DNA content and the same cell-cycle phase can still display large variations in H3 acetylation and that these variations correlate with specific phenotypic variations, in particular, nuclear size and actin cytoskeleton content, but not cell shape. The dependence of cell and nuclear properties on cell-cycle phase is assessed without artifact-prone cell synchronization. To further demonstrate the versatility of this assay, I quantify the complex interplay among cell cycle, epigenetic modifications, and phenotypic variations following pharmacological treatments targeting DNA integrity, cell cycle, and chromatin-modifying enzymes. Finally, recent literature suggests that the actin filament network regulates epigenetics that determine DNA packing in the nucleus and ultimately gene transcription, especially by way of histone modifications. However, a molecular mechanism underlying these cytoskeleton-based histone modifications is missing. Here, I hypothesize that the LINC complex proteins that physically connect the cytoskeleton to the nuclear lamina at the nuclear envelope are key mediators of histone modifications. Using the newly established high-throughput single-cell phenotyping method, I quantitatively examine actin filament content and organization in the cytoplasm, nuclear morphology, nuclear lamina and LINC protein expression and organization at the nuclear membrane, and histone acetylation and methylation of specific residues in the same individual cells simultaneously. I conclude that LINC complex proteins nesprin2giant and nesprin3, as well as lamin A/C of the nuclear lamina, regulate these histone modifications in a very complex manner. Taken together, all of these results suggest an interconnected pathway for mechanotransduction that physically connects the extracellular milieu to the nucleus