Direct force probe reveals the mechanics of nuclear homeostasis in the mammalian cell.

How cells maintain nuclear shape and position against various intracellular and extracellular forces is not well understood, although defects in nuclear mechanical homeostasis are associated with a variety of human diseases. We estimated the force required to displace and deform the nucleus in adherent living cells with a technique to locally pull the nuclear surface. A minimum pulling force of a few nanonewtons--far greater than typical intracellular motor forces--was required to significantly displace and deform the nucleus. Upon force removal, the original shape and position were restored quickly within a few seconds. This stiff, elastic response required the presence of vimentin, lamin A/C, and SUN (Sad1p, UNC-84)-domain protein linkages, but not F-actin or microtubules. Although F-actin and microtubules are known to exert mechanical forces on the nuclear surface through molecular motor activity, we conclude that the intermediate filament networks maintain nuclear mechanical homeostasis against localized forces.

Actomyosin pulls to advance the nucleus in a migrating tissue cell.

The cytoskeletal forces involved in translocating the nucleus in a migrating tissue cell remain unresolved. Previous studies have variously implicated actomyosin-generated pushing or pulling forces on the nucleus, as well as pulling by nucleus-bound microtubule motors. We found that the nucleus in an isolated migrating cell can move forward without any trailing-edge detachment. When a new lamellipodium was triggered with photoactivation of Rac1, the nucleus moved toward the new lamellipodium. This forward motion required both nuclear-cytoskeletal linkages and myosin activity. Apical or basal actomyosin bundles were found not to translate with the nucleus. Although microtubules dampen fluctuations in nuclear position, they are not required for forward translocation of the nucleus during cell migration. Trailing-edge detachment and pulling with a microneedle produced motion and deformation of the nucleus suggestive of a mechanical coupling between the nucleus and the trailing edge. Significantly, decoupling the nucleus from the cytoskeleton with KASH overexpression greatly decreased the frequency of trailing-edge detachment. Collectively, these results explain how the nucleus is moved in a crawling fibroblast and raise the possibility that forces could be transmitted from the front to the back of the cell through the nucleus.

Modulating malignant epithelial tumor cell adhesion, migration and mechanics with nanorod surfaces.

The failure of tumor stents used for palliative therapy is due in part to the adhesion of tumor cells to the stent surface. It is therefore desirable to develop approaches to weaken the adhesion of malignant tumor cells to surfaces. We have previously developed SiO2 coated nanorods that resist the adhesion of normal endothelial cells and fibroblasts. The adhesion mechanisms in malignant tumor cells are significantly altered from normal cells; therefore, it is unclear if nanorods can similarly resist tumor cell adhesion. In this study, we show that the morphology of tumor epithelial cells cultured on nanorods is rounded compared to flat surfaces and associated with decreased cellular stiffness and non-muscle myosin II phosphorylation. Tumor cell viability and proliferation was unchanged on nanorods. Adherent cell numbers were significantly decreased while single tumor cell motility was increased on nanorods compared to flat surfaces. Together, these results suggest that nanorods can be used to weaken malignant tumor cell adhesion, and therefore potentially improve tumor stent performance.

Actomyosin tension exerted on the nucleus through nesprin-1 connections influences endothelial cell adhesion, migration, and cyclic strain-induced reorientation.

Endothelial cell polarization and directional migration is required for angiogenesis. Polarization and motility requires not only local cytoskeletal remodeling but also the motion of intracellular organelles such as the nucleus. However, the physiological significance of nuclear positioning in the endothelial cell has remained largely unexplored. Here, we show that siRNA knockdown of nesprin-1, a protein present in the linker of nucleus to cytoskeleton complex, abolished the reorientation of endothelial cells in response to cyclic strain. Confocal imaging revealed that the nuclear height is substantially increased in nesprin-1 depleted cells, similar to myosin inhibited cells. Nesprin-1 depletion increased the number of focal adhesions and substrate traction while decreasing the speed of cell migration; however, there was no detectable change in nonmuscle myosin II activity in nesprin-1 deficient cells. Together, these results are consistent with a model in which the nucleus balances a portion of the actomyosin tension in the cell. In the absence of nesprin-1, actomyosin tension is balanced by the substrate, leading to abnormal adhesion, migration, and cyclic strain-induced reorientation.