Abstract
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 cytoskeleton-integrin-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 (9.5 nm), medium (38.2 nm) and long (318 nm) 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.
Highlights
Cells are known to respond to physical cues from their microenvironment such as matrix rigidity
The density of extracellular matrix (ECM) proteins such as collagen and fibronectin play a major role in determining cell behaviour[9,15,16,17]
We considered the possibility that cells are receptive to ECM rigidity and adhesive ligand density and to the length of the local tether to which the ligand attaches to the broader ECM microenvironment
Summary
Functionalization and validation of surfaces with tethers of different lengths. In order to investigate the effect of tether length on cellular behaviour, we covalently coupled RGD peptides to glass surfaces via inert polyethylene glycol tether molecules. Consistent with the size and length of focal adhesions, cells seeded on RGD surfaces with the shortest tether were well spread and displayed a greater density of cells attached to the surface, comparable to cells seeded for the same time on glass surfaces with a fibronectin coating (Fig. 3b) This confirmed that the RGD peptide is acting as an effective adhesive ligand for the cells. Cell spread area and cell attachment decrease with increasing tether length; Lower row: Vehicle control (VC) surfaces with short, medium and long length tethers but lacking the RGD adhesive ligand. It is unclear whether the extreme range of length scales studied here are physiologically relevant to a cell in its native environment, the experiments provide key insights into understanding the mechanism of cellular mechanosensing This is illustrated by the tether length-dependent effects on cell surface density and spread area. By determining the length scale over which cells are able to sense their environment we provide constraints for those models trying to understand the mechanism of mechanosensing
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