The nucleus has a smooth, regular appearance in normal cells, and its shape is greatly altered in human pathologies. Yet, how the cell establishes nuclear shape is not well understood. We imaged the dynamics of nuclear shaping in NIH3T3 fibroblasts. Nuclei translated toward the substratum and began flattening during the early stages of cell spreading. Initially, nuclear height and width correlated with the degree of cell spreading, but over time, reached steady-state values even as the cell continued to spread. Actomyosin activity, actomyosin bundles, microtubules, and intermediate filaments, as well as the LINC complex, were all dispensable for nuclear flattening as long as the cell could spread. Inhibition of actin polymerization as well as myosin light chain kinase with the drug ML7 limited both the initial spreading of cells and flattening of nuclei, and for well-spread cells, inhibition of myosin-II ATPase with the drug blebbistatin decreased cell spreading with associated nuclear rounding. Together, these results show that cell spreading is necessary and sufficient to drive nuclear flattening under a wide range of conditions, including in the presence or absence of myosin activity. To explain this observation, we propose a computational model for nuclear and cell mechanics that shows how frictional transmission of stress from the moving cell boundaries to the nuclear surface shapes the nucleus during early cell spreading. Our results point to a surprisingly simple mechanical system in cells for establishing nuclear shapes.

The nucleus is mechanically coupled to the three cytoskeletal elements in the cell via linkages maintained by the LINC complex (for Linker of Nucleoskeleton to Cyto-skeleton). It has been shown that mechanical forces from the extracellular matrix (ECM) can be transmitted through the cytoskeleton to the nuclear surface. Here we quantified nuclear shape in NIH 3T3 fibroblasts on polyacrylamide gels with a controlled degree of cross-linking. On soft substrates with a Young's modulus of 0.4 kPa, the nucleus appeared rounded in its vertical cross-section, while on stiff substrates (308 kPa), the nucleus appears more flattened. Over-expression of dominant negative Klarsicht ANC-1 Syne Homology (KASH) domains, which disrupts the LINC complex, eliminated the sensitivity of nuclear shape to substrate rigidity; myosin inhibition had similar effects. GFP-KASH4 over-expression altered the rigidity dependence of cell motility and cell spreading. Taken together, our results suggest that nuclear shape is modulated by substrate rigidity-induced changes in actomyosin tension, and that a mechanically integrated nucleus-cytoskeleton is required for rigidity sensing. These results are significant because they suggest that substrate rigidity can potentially control nuclear function and hence cell function.

Nuclear positioning is a crucial cell function, but how a migrating cell positions its nucleus is not understood. Using traction-force microscopy, we found that the position of the nucleus in migrating fibroblasts closely coincided with the center point of the traction-force balance, called the point of maximum tension (PMT). Positioning of the nucleus close to the PMT required nucleus-cytoskeleton connections through linker of nucleoskeleton-to-cytoskeleton (LINC) complexes. Although the nucleus briefly lagged behind the PMT following spontaneous detachment of the uropod during migration, the nucleus quickly repositioned to the PMT within a few minutes. Moreover, tractiongenerating spontaneous protrusions deformed the nearby nucleus surface to pull the nuclear centroid toward the new PMT, and subsequent retraction of these protrusions relaxed the nuclear deformation and restored the nucleus to its original position. We propose that the protruding or retracting cell boundary transmits a force to the surface of the nucleus through the intervening cytoskeletal network connected by the LINC complexes, and that these forces help to position the nucleus centrally and allow the nucleus to efficiently propagate traction forces across the length of the cell during migration.

Cells respond to mechanical signals, but the subcellular mechanisms are not well understood. The nucleus has recently emerged as an important mechanosensory organelle in the cell, as it is intimately connected to the cytoskeleton. Mechanical forces applied to cells that act on membrane-embedded receptors are transmitted through the cytoskeleton to the nuclear surface. Interfering with linkers of the nucleus to the cytoskeleton causes defects in cell mechanosensing and cell function. In this chapter, we discuss recent work in this area, highlighting the role that the nuclear linkages with the cytoskeleton play in cellular mechano-transduction.