Insulin regulates lymphocyte traction on fibronectin-coated compliant substrate in a calcium-dependent manner

Lymphocyte–extracellular matrix (ECM) interactions occur intermittently throughout the lymphocyte’s life cycle. Alterations in blood insulin levels following feeding modulates naïve lymphocyte trafficking and adhesion to fibronectin via a pathway involving insulin-like growth factor-1 receptor (IGF-1R), phospholipase C gamma 1 (PLC-γ1) and β ₂ integrin activation. Lymphocytes exert traction forces, on the ECM during the process of extravasation. While these forces are essential for several homeostatic processes, the role of insulin in modulating lymphocyte-derived traction forces upon ECM adhesion is unknown. The aim of the current study was to investigate the effect of insulin on the traction generated by lymphocytes when adhered onto a fibronectin-coated substrate. Jurkat T-cells were placed on a fibronectin layer (50µg/ml, ⁓100µm thickness) coated on polyacrylamide gels of stiffness ⁓ 400Pa with red fluorescence beads as fiduciary markers. The cellular force generated by Jurkat T-cells was mapped using traction force microscopy. To elucidate the role of PLC-γ1 in cellular force generation, the traction of Jurkat T-cells lacking PLC-γ1, as well as those of a knockout cell where PLC-γ1 was restored were quantified and compared with wild-type Jurkat T-cells. Lack of PLC-γ1 attenuated adhesion when compared to wild-type Jurkat T-cells. Additionally, the traction force generated by each cell type decreased with increasing concentration of extracellular calcium. Treatment of adherent Jurkat T-cells with insulin increased traction in lower extracellular calcium condition while a dip was observed when a high extracellular calcium was present, in comparison to the untreated cells. However, the effect of insulin treatment was lost in the case of Jurkat T-cells lacking PLC-γ1. Together these results indicate that insulin regulates traction force generated by adherent Jurkat T-cells via a process involving PLC-γ1, in a calcium dependent manner.