Probing mechanical interaction of immune receptors and cytoskeleton by membrane nanotube extraction

The role of force application in immune cell recognition is now well established, the force being transmitted between the actin cytoskeleton to the anchoring ligands through receptors such as integrins. In this chain, the mechanics of the cytoskeleton to receptor link, though clearly crucial, remains poorly understood. To probe this link, we combine mechanical extraction of membrane tubes from T cells using optical tweezers, and fitting of the resulting force curves with a viscoelastic model taking into account the cell and relevant molecules. We solicit this link using four different antibodies against various membrane bound receptors: antiCD3 to target the T Cell Receptor (TCR) complex, antiCD45 for the long sugar CD45, and two clones of antiCD11 targeting open or closed conformation of LFA1 integrins. Upon disruption of the cytoskeleton, the stiffness of the link changes for two of the receptors, exposing the existence of a receptor to cytoskeleton link-namely TCR-complex and open LFA1, and does not change for the other two where a weaker link was expected. Our integrated approach allows us to probe, for the first time, the mechanics of the intracellular receptor-cytoskeleton link in immune cells.