Cells are known to respond to physical cues from their microenvironment such as matrix rigidity.
Discrete adhesive ligands within flexible strands of fibronectin connect cell surface integrins to the
broader extracellular matrix and are thought to mediate mechanosensing through the cytoskeletonintegrin-ECM
linkage. We set out to determine if adhesive ligand tether length is another physical cue
that cells can sense. Substrates were covalently modified with adhesive arginylglycylaspartic acid (RGD)
ligands coupled with short (9.5nm), medium (38.2nm) and long (318nm) length inert polyethylene
glycol tethers. The size and length of focal adhesions of human foreskin fibroblasts gradually decreased
from short to long tethers. Furthermore, we found cell adhesion varies in a linker length dependent
manner with a remarkable 75% reduction in the density of cells on the surface and a 50% reduction in
cell area between the shortest and longest linkers. We also report the interplay between RGD ligand
concentration and tether length in determining cellular spread area. Our findings show that without
varying substrate rigidity or ligand density, tether length alone can modulate cellular behaviour.