Cell shape and substrate rigidity both regulate cell stiffness

Abstract

Cells from many different tissues sense the stiffness and spatial patterning of their microenvironment to modulate their shape and cortical stiffness. It is currently unknown how substrate stiffness, cell shape, and cell stiffness modulate or interact with one another. Here, we use microcontact printing and microfabricated arrays of elastomeric posts to independently and simultaneously control cell shape and substrate stiffness. Our experiments show that cell cortical stiffness increases as a function of both substrate stiffness and spread area. For soft substrates, the influence of substrate stiffness on cell cortical stiffness is more prominent than that of cell shape, since increasing adherent area does not lead to cell stiffening. On the other hand, for cells constrained to a small area, cell shape effects are more dominant than substrate stiffness, since increasing substrate stiffness no longer affects cell stiffness. These results suggest that cell size and substrate stiffness can interact in a complex fashion to either enhance or antagonize each other's effect on cell morphology and mechanics.

Figure 1

Spread area and stiffness of hMSC as a function of substrate stiffness. (A) hMSC area increased as a function of substrate stiffness and leveled off at a saturating level of ∼6000 μm². (B) Similarly, hMSC stiffness increased as a function of substrate stiffness up to a level of ∼7 kPa. As controls, cell area and stiffness for cells grown on fibronectin-coated glass of stiffness 70 GPa are shown in A and B.

Figure 2

hMSC constrained on square adhesive islands formed by stamping fibronectin on polydimethylsiloxane microposts. Cells are stained with phalloidin (green) and vinculin (red). When spreading areas are large, hMSCs on posts of low (3.8 nN/μm) and high (18 nN/μm) spring constants develop stress fibers, whereas when spreading areas are small, no stress fibers develop. Cells grown on stiffer posts show more intense vinculin staining than those grown on soft posts. However, regardless of post stiffness, larger spread areas result in larger and more mature vinculin patches.

Figure 3

Stiffness and contractile forces of hMSCs as functions of their projected area. hMSCs are plated on microposts of different spring constants with various sizes of square islands of fibronectin. (A) Cell stiffness as measured by atomic force microscopy. For tall posts (3.8 nN/μm), cell stiffness remains constantly soft (square symbols), whereas for medium (18 nN/μm) and short (1500 nN/μm) posts, cell stiffness increases with stamped projected area (circles and triangles). (B) The total contractile forces of cells on tall posts remain small (<500 nN) but increase as cell area is increased on medium posts. Asterisks indicate that no cells of 10,000 μm² were found on tall posts.

Figure 4

hMSC stiffness is mediated through prestress generated by myosin motors. Treatment with the myosin II antagonist blebbistatin (25 μm) softens cortical stiffness for all cells (^∗). This holds true for all substrate rigidities and all constrained areas. Treatment with the tension agonist nocodazole increases cortical stiffness (+).