Mechanotransduction through High-Affinity LFA-1 is a Minimum Requirement to Induce Kindlin-3/RACK1/OraI1 to Mediate Intracellular Calcium Flux and Outside-In Signaling

Abstract

Absence of Kindlin-3 impairs β2-integrin function on neutrophils, results in bleeding and resistance to arterial thrombosis and immunodeficiency. Kindlin-3 binds LFA-1 β-integrin tail independent of Talin-1 and is associated with integrin conversion to a high-affinity and the facilitation of micro-cluster formation that allow for long-lived shear resistance bonds with ICAM-1. However, the molecular and mechanical events that link LFA-1 to Kindlin-3 at spatially distinct sites to allow for subsequent engagement with scaffolding protein RACK1 and calcium release activated channel Orai1 have not been rigorously shown. We show that an allosteric shift of LFA-1 due to force generation on the β2-integrin cytodomain promotes interaction with Kindlin-3 F3 subdomain, and when sufficient force is transduced through this mechanical linkage induces calcium flux. We utilize real time two-line total internal reflection fluorescent and epifluorescence microscopy on neutrophil-like PLB-985 cells arrested to ICAM-1 within our microfluidic vascular mimetic we have been able to identify the stoichiometric binding rate of Kindlin-3 and subsequent RACK-1 recruitment to high-affinity LFA-1 sites in response to increasing shear profiles prior to calcium flux. In addition to outside-in signaling we have shown that force acting on LFA-1 amplifies calcium influx during CXCR1/2 ligation in a Kindlin-3 dependent manner. To assess the spatial proximity and association between Kindlin-3/RACK1 with focal adhesion clusters we are developing a FRET pair between the Kindlin-3 PH domain and RACK1 scaffolds 5 to 7 to further elucidate the spatiotemporal events and requirements for outside-in signaling and neutrophil progression to a migratory phenotype. This has allowed for us to identify key forces and proteins required for outside-in signaling during neutrophil transition from rolling to arrest which will help further glyocomimetic development to treat various disease states.