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

Abstract

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.nuclear forces | cytoskeleton | nuclear positioning | nuclear mechanics | nuclear shape T he nucleus in a cultured cell such as a fibroblast is close to the center of the cell and typically has a smooth, regular shape. It is known that the migrating cell moves the nucleus by transferring cytoskeletal forces through connections between the cytoskeleton and the nuclear surface (1, 2). Even in a stationary cell, the nuclear shape and central position are stably maintained in mechanical homeostasis at defined locations in the cell, despite the fact that the dynamic cytoskeleton continues to generate constantly fluctuating forces on the nucleus (3, 4).The source of fluctuating forces on the nucleus includes nuclear-embedded microtubule motors such as dynein and kinesin (5-7) and actomyosin forces that push on and pull the nucleus (2,8,9). The nucleus is also exposed to extracellular forces, such as those applied to adhesion receptors, which can be transmitted through the cytoskeleton onto the nuclear surface (10-12). How nuclear shape and position are maintained in mechanical homeostasis despite the different types of forces that act on the nucleus is an open question. This is particularly important because deregulated positioning and irregular nuclear shapes are associated with a variety of human pathologies (reviewed in ref. 13).Here we describe a method to apply forces directly to nuclei in cultured, living, adherent cells. With this method, we estimate a minimum pulling force of a few nanonewtons-far greater than typical intracellular motor forces-is required to significantly displace and deform the nucleus. Although F-actin and microtubules are known to exert mechanical forces on the nuclear surface through molecular motor activity, we show that the intermediate filament networks maintain nuclear mechanical homeostasis. ResultsTo determine the forces that are required for moving and deforming the nucleus, and to identify the cellular components that oppose motion and deformation, we devised a method to ...

Ultradonut topology of the nuclear envelope

“…A minimal in-plane stress of ∼ − 0.0024 mN/m and an out-of plane stress of ∼ 1 Pa is sufficient to explain the geometry and the topology of the doublebilayer structure. As these stresses are much smaller than the maximum stresses membranes can endure (27)(28)(29), our findings may be of value to understanding the nanoarchitecture of the nuclear envelope. Because our model predicts a stable structure over a wide range of tensile stresses, nuclei may be able to easily adjust to the changes in nuclear stresses in a biological setting and yet acquire and maintain their geometry (30).…”

Dynamic, mechanical integration between nucleus and cell- where physics meets biology

“…Flow of fluid into the micropipette from inside the nucleus can occur through nuclear pores, which means that the actual pressure on the nuclear surface will be smaller than the suction pressure in the micropipette. However, a simple calculation shows that the resistance across the nuclear envelope to flow is 10 5 times greater than resistance to the flow in the pipette 26 . Therefore, it is a safe assumption that all the pressure drop occurs across the nuclear envelope and the actual pressure on the envelope is equal to suction pressure in the micropipette tip.…”

“…To address this limitation, we recently developed an approach 26 to apply force directly to the nuclear surface in living, adherent cells. The method is simple to implement on a commercially available Eppendorf system.…”

“…The resulting nuclear translation and deformation depends on the suction force. At low suction pressure, the pipette detaches from the nucleus without any noticeable effect on the nucleus ( Figure 1A; quantitative strain-force curves are available elsewhere 26,27 ). At higher suction pressure, the nucleus translates and deforms in the direction of the pipette.…”

Probing cytoskeletal pre-stress and nuclear mechanics in endothelial cells with spatiotemporally controlled (de-)adhesion kinetics on micropatterned substrates

“…While all of these methods have proven extremely powerful in characterizing cellular or nuclear viscoelastic properties, none of these techniques allows to probe simultaneously and non-invasively the mechanical properties of the cell and the nucleus. 12 To address this challenge, we propose to use cell-ECM adhesion and detachment (in other terms (de-)adhesion) kinetics, for characterizing combined cellular and nuclear mechanical properties. In line with work from Wildt and coworkers, who have developed surfaces composed of RGD-functionalized arrays of microscale gold strips for studying the detachment dynamics of fibroblasts, 13,14 we used culture substrates of different rigidities patterned with protein microfeatures.…”