Traction force microscopy with elastic pillars for quantification of forces during cell apoptosis

Apoptosis is an integral process within cellular homeostasis, and the balance between cell death and cell survival is lost in cancer. Oncogenic mutations, such as downregulation of p53, are often involved in abrogation of apoptosis and therefore higher cell survival and malignancy. The apoptotic process is characterised by morphological changes, including rounding up of the cell and membrane blebbing [1]. These morphological changes involve contractile forces mediated by the actin–myosin cytoskeleton, which is also necessary for localising fragmented DNA into apoptotic bodies [2]. An understanding of the forces present within apoptosis will facilitate further comprehension of the process and may lead to new directions for therapeutics.

Here we present a method for determination of traction forces during apoptosis, which we demonstrate by inducing apoptosis in mouse embryonic fibroblasts (MEFs). Arrays of elastic pillars are created [3], formed by mixing polydimethylsiloxane (PDMS) with a curing agent (Sylgard 184), pouring this mixture into a mould with holes of variable depth and 1 µm width, and curing at 70 °C overnight. The depth of holes in the mould determines the height of pillars, affecting the spring constant of the pillars that the cell is in contact with. Pillars are then coated with fibronectin to allow cell attachment, and cells seeded on top. Cells are given 2–3 h to spread in normal media, before changing their media for serum-free media for overnight starvation. The next day, media is changed for serum-free media containing the apoptosis inducing agents with cell death and contraction occurring over the following few hours.

As contraction occurs, the forces generated by the cells deflect the pillars, which is recorded using brightfield microscopy, with images taken every 2 s over a 10 min time period. The video produced is analysed by a custom Matlab code to calculate the forces applied to the pillars. The maximum displacement of each pillar covered by the cell is multiplied by the spring constant of the pillars to determine the maximum force applied by the cell using Hooke's law. An average of the maximum forces is taken to give a single value readout of mean maximum force. A force map is also generated, showing the direction of the maximum force for each pillar, and can demonstrate how during contraction, force is applied to push pillars away from the cell edge that contracts inwards, as well as how forces may differ in the cell body and cell extensions during the contraction process.

Figure 1 shows an example of a cell with quantification of its apoptotic force generation on 5 µm high pillars (spring constant  =  2.35 nN µm⁻¹). The contraction of a mouse embryonic fibroblast is observed over 10 min following overnight serum starvation, and subsequent exposure to 50 ng ml⁻¹ TNF-α and 10 µg ml⁻¹ cycloheximide. Figure 1(A) shows the maximum force exerted on each individual pillar covered by the cell and their directions, as well as average of these maximum force magnitudes, and figure 1(B) shows brightfield images of the cell at t  =  0 mins and t  =  10 min, displaying cell contraction and rounding up, as well as blebbing behaviour. The highest force generation is seen in the centre of the cell body, as well as force being applied to the pillars to push them away by the cell extensions during the contraction.

Zoom In Zoom Out Reset image size

This assay has potential for future development into an in vitro drug toxicity assay for chemotherapeutic drugs. For drugs that are expected to induce apoptosis, force generation during cell death could be a marker for efficacy, and differences in force generation a marker for potency. Quantification of contractility could also differentiate between drugs that affect the actin cytoskeleton and those that initiate extrinsic or intrinsic apoptotic pathways, and show how force is generated either within the cell body or cell extensions such as filopodia. This assay may also be able to differentiate between apoptosis, necrosis, and anoikis, i.e. cell death induced by detachment from an extracellular matrix, as well as assess the effect of chemotherapeutic drugs on healthy cells.

This work was supported by the European Research Council (ERC grant agreement 282051) and an Imperial College PhD Scholarship.