Mechanism of Stiffness Sensing Revealed with a Composite Substrate

Wong et al., Proc. Natl. Acad. Sci. USA 111:17176-17181 (2014)

Our previous experiment using composite substrate, where an array of small photoresist islands are grafted on the surface of soft polyacrylamide gels, suggests that cellular stiffness sensing occurs across a distance of tens of microns rather than within single focal adhesions .  To further understand the mechanism, we simulate stiff-soft transition by placing a large, stiff island next to a matrix of small islands separated by either soft or stiff gels.  After the initial adhesion to the large island, subsequent responses of the cell may depend on how cells probe and respond to the adjacent small islands.

Cell Spreading to Cover Grafted Islands on Stiff Polyacrylamide

Control substrate consists of a stiff polyacrylamide gel grafted with stiff photoresist islands.  Cells are unable to adhere to the bare polyacrylamide surface but treat island surfaces like a stiff substrate, spreading over small islands following initial adhesion to the large island.  Recorded every 10 min.  Total duration, 9.5 hours.

Cell Spreading to Cover Printed Islands on Stiff Polyacrylamide

A second type of control substrate consists of the same island pattern printed onto the surface of a stiff polyacrylamide gel.  After initial spreading, the cell shows a response similar to that on grafted islands in the first video.  Recorded every 10 min.  Total duration, 13.2 hours.

Movement of Small Islands by Probing Forces

Testing substrate consists of a soft polyacrylamide gel grafted with the same pattern of islands.  Some small islands show minute movements, suggesting that cells apply probing forces across a distance.  Video made by alternating between two images recorded 10 min. apart.

Failure of Cells to Spread over Soft Polyacrylamide

On testing substrate, cell repeatedly extends processes across the soft gel to reach some of the small islands.  However, most such extensions fail to lead to spreading.  Images recorded 10 min. apart.

Retraction of Probing Structures in Response to Experimental Displacement

The results above show that cells on control substrate (with stiff polyacrylamide) probe the small island before spreading across polyacrylamide to cover the area.  However, the probing structure retracts if a needle is used to displace the small island toward the cell during probing, which mimics the displacement of islands on soft polyacrylamide in response to probing.  This response suggests that the cell uses the displacement in response to its probing forces to gauge the stiffness of the substrate.  Image recorded every min for a total of 9 min.

Diminished Response to Experimental Displacement after the Initiation of Spreading

Similar to the experiment above except that needle is applied after the initiation of spreading.  The manipulation causes only a partial reduction of spreading area.  Image recorded every min for a total of 25 min.

Disruption of Stiffness Sensing upon the Inhibition of Myosin II

Cells treated with 10 micromolar blebbistatin, an inhibitor of myosin II, fail to respond to the pushing needle such that probing structures persist, suggesting that myosin II is required for the retraction response.  Image recorded every min for a total of 8 min.

Disruption of Stiffness Sensing upon the Inhibition of Myosin II

Similar to the experiment above, except that the pushing needle is applied at a later time following the contact of probing structure with an island.  Cell spreading continues in spite of the manipulation.