Abstract
Focal contacts act as mechanosensors allowing cells to respond to their biomechanical environment. Force transmission through newly formed contact sites is a highly dynamic process requiring a stable link between the intracellular cytoskeleton and the extracellular environment. To simultaneously investigate cellular traction forces in several individual maturing adhesion sites within the same cell, we established a custom-built multiple trap optical tweezers setup. Beads functionalized with fibronectin or RGD-peptides were placed onto the apical surface of a cell and trapped with a maximum force of 160 pN. Cells form adhesion contacts around the beads as demonstrated by vinculin accumulation and start to apply traction forces after 30 seconds. Force transmission was found to strongly depend on bead size, surface density of integrin ligands and bead location on the cell surface. Highest traction forces were measured for beads positioned on the leading edge. For mouse embryonic fibroblasts, traction forces acting on single beads are in the range of 80 pN after 5 minutes. If two beads were positioned parallel to the leading edge and with a center-to-center distance less than 10 µm, traction forces acting on single beads were reduced by 40%. This indicates a spatial and temporal coordination of force development in closely related adhesion sites. We also used our setup to compare traction forces, retrograde transport velocities, and migration velocities between two cell lines (mouse melanoma and fibroblasts) and primary chick fibroblasts. We find that maximal force development differs considerably between the three cell types with the primary cells being the strongest. In addition, we observe a linear relation between force and retrograde transport velocity: a high retrograde transport velocity is associated with strong cellular traction forces. In contrast, migration velocity is inversely related to traction forces and retrograde transport velocity.
Highlights
Cells exert forces onto their growth substrate during spreading and migration by forming adhesive contacts that connect the cellular cytoskeleton with the surrounding extracellular matrix (ECM)
Fibronectin is a dimeric protein composed of two identical 250 kDa strands connected via disulfide bonds at the C-terminus with each strand offering several motifs recognized as binding sites by the integrin family [8]
Functionalized beads act as passive sensors for cellular traction forces and do not actively apply forces to the cell
Summary
Cells exert forces onto their growth substrate during spreading and migration by forming adhesive contacts that connect the cellular cytoskeleton with the surrounding extracellular matrix (ECM). The ability of adhesive cells to spread and migrate on a 2D or 3D substrate comes with the requirement to establish cell-matrix contacts that are stable enough to withstand traction forces and dynamic enough to allow migration [4,5,6]. Fibronectin is a dimeric protein composed of two identical 250 kDa strands connected via disulfide bonds at the C-terminus with each strand offering several motifs recognized as binding sites by the integrin family [8]. The shortest amino acid sequence known to be recognized as an adhesion motif is the RGD (Arginine-Glycine-Aspartic Acid) sequence located in FN repeat III10 serving as a binding site for a5b1, a8b1, aIIbb and all av integrins [9,10]
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