Adhesive ligand tether length affects the size and length of focal adhesions and influences cell spreading and attachment

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.

FAK controls the mechanical activation of YAP, a transcriptional regulator required for durotaxis

“…Primary culture-activated human hepatic stellate cells (passage 3-6, HHStec 5300; ScienCell, Carlsbad, CA, USA) were cultured at 37°C, 5% CO 2 in culture medium containing DMEM/Nutrient Mixture F-12 Ham (D8437; Sigma-Aldrich, St. Louis, MO, USA), 10% fetal bovine serum (10270-106; Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/ streptomycin (Thermo Fisher Scientific), and 1% fungizone (15290-026; Thermo Fisher Scientific). Human foreskin fibroblasts (HFFs) were cultured in DMEM high glucose (D6429; Sigma-Aldrich) with the same supplements as previously described (16). For the polyacrylamide substrate experiments, cells were detached from culture flasks and transferred to 4, 12, 25 kPa single-rigidity or 4/25 kPa dual-rigidity polyacrylamide gels.…”

Supported lipid bilayer platforms to probe cell mechanobiology

“…al. found that human foreskin fibroblasts attached to RGD ligands on glass also preferred short tethers; increasing adhesion and cell area correlated decreasing tether length [153]. …”

Combinatorial functionalization with bisurea‐peptides and antifouling bisurea additives of a supramolecular elastomeric biomaterial

“…Several parameters in the method of covalent tethering of peptides to a biomaterial surface influence the resulting functionality of the peptide. This includes, but is not limited to, the spacing between adhesive peptides, the length of the linker that is used to attach the peptide, and the amino‐acids that flank the essential peptide sequence, which all suggest that tuning the chemical nano‐environment of peptides is of great importance for functionality of the peptides. However, a popular approach features the functionalization with both bioactive peptides, and antifouling moieties, to enhance specificity of the bioactivation and reduce adhesion of undesired entities to the functionalized biomaterial, where the chemical environment of the peptides is designed to repel any aspecific interactions.…”