A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

Summary

In this protocol, we describe a micropipette method to directly apply a controlled force to the nucleus in a living cell. This assay allows interrogation of nuclear mechanical properties in the living, adherent cell.

Abstract

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous with the cytoskeleton, methods are needed to probe its mechanical behavior in adherent cells. Here, we discuss the direct force probe (DFP) as a tool to apply force directly to the nucleus in a living adherent cell. We attach a narrow micropipette to the nuclear surface with suction. The micropipette is translated away from the nucleus, which causes the nucleus to deform and translate. When the restoring force is equal to the suction force, the nucleus detaches and elastically relaxes. Because the suction pressure is precisely known, the force on the nuclear surface is known. This method has revealed that nano-scale forces are sufficient to deform and translate the nucleus in adherent cells, and identified cytoskeletal elements that enable the nucleus to resist forces. The DFP can be used to dissect the contributions of cellular and nuclear components to nuclear mechanical properties in living cells.

Introduction

Pathologies such as cancer involve alterations to nuclear shape and structure^1,2, which are generally accompanied by a 'softening' of the nucleus^3,4. Nuclear resistance to mechanical deformation has been generally characterized by applying a force to isolated nuclei⁵.

The nucleus in cells is molecularly connected to the cytoskeleton by the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex^6,7,8<....

Protocol

1. Preparing Cells for Imaging

NOTE: The direct force probe (DFP) can be used for any adherent cell type. Here, NIH 3T3 mouse fibroblasts are used as the model cell line for this protocol.

1. Culture NIH 3T3 fibroblast cells in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% donor bovine serum and 1% Penicillin-Streptomycin on a 35-mm glass bottom dish until desired confluency. Maintain cells at 37 °C and 5% CO₂.
   1. Be sure to coat all 35-mm glass bottom dishes with 5 µg/mL of fibronectin (or similar ECM protein), before seeding NIH 3T3 cells for imaging.
      NOTE: The cells must be fully sprea....

Results

Figure 2A shows the forcing of an NIH 3T3 mouse fibroblast nucleus. As the micropipette tip is translated to the right, the nucleus deforms and eventually detaches from the micropipette tip. The length strain of the nucleus is seen to increase with increasing suction force (Figure 2B). The front edge of the nucleus (micropipette pulling edge) forms a nuclear protrusion and the trailing edge is displaced from its original position.......

Discussion

Measuring the mechanical integration of the nucleus with the cytoskeleton is a challenge for most current methods, such as micropipette aspiration¹⁶, because they require either isolated nuclei (where the nucleus is decoupled from the cytoskeleton) or nuclei in suspended cells (where extracellular forces, such as traction forces, are absent). Force has been applied to the nucleus by applying biaxial strain to cells adherent to a membrane^17,18

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
FluoroDish	WPI	FD35
SYTO 59	ThermoFisher Scientific	S11341
Femtotips	Eppendorf	930000043
InjectMan NI2	Eppendorf	NA	discontinued, current equivalent model: InjectMan 4
FemtoJet	Eppendorf	NA	Current model FemtoJet 4i
Plan Fluor oil immersion 40x	Nikon	NA
Apo TIRF oil immersion 60x	Nikon	NA
Donor Bovine Serum (DBS)	ThermoFisher Scientific	16030074	NIH 3T3 serum
Dulbecco's Modification of Eagle's (DMEM)	Mediatech cellgro	MT10013CVRF	NIH 3T3 medium
Penicillin-Streptomycin	Mediatech	MT30004CIRF	NIH 3T3 medium supplement
Immersion Oil Type LDF Non-Fluorescing	Nikon	77007	Immersion oil for objective lens