Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration

Abstract

Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, Å-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.

Mechanosensation: A Catch Bond That Only Hooks One Way

“…Here, activation and ordering of molecules could be sensitive to force direction altering the overall geometry of interactions. Such molecular ordering in adhesions has been shown for talin and, more recently, for integrins [18–20]. Orientation of molecules could sterically limit protein-protein interactions, which in turn would alter the relative orientations between forces and sites of interactions and thus affect bond behavior.…”

Characterisation and correction of polarisation effects in fluorescently labelled fibres

“…For example, large, bulky fluorophores such as GFP will have longer • rot compared to small organic dye molecules, and shorter, stiffer linkers may yield longer • rot compared to longer, floppier linkers. This can be exploited in experimental design, for example when GFP is inserted into rigid domains of integrins for probing their orientation (3,4). Here, presence/absence of a short linker and truncation of the GFP sequence alter the constraint of GFP rotation and as a result the measured anisotropy.…”