NDR1-Dependent Regulation of Kindlin-3 Controls High-Affinity LFA-1 Binding and Immune Synapse Organization

Abstract

Antigen-specific adhesion between T cells and antigen-presenting cells (APC) during the formation of the immunological synapse (IS) is mediated by LFA-1 and ICAM-1. Here, LFA-1-ICAM-1 interactions were measured at the single-molecule level on supported lipid bilayers. High-affinity binding was detected at low frequencies in the inner peripheral supramolecular activation cluster (SMAC) zone that contained high levels of activated Rap1 and kindlin-3. Rap1 was essential for T cell attachment, whereas deficiencies of ste20-like kinases, Mst1/Mst2, diminished high-affinity binding and abrogated central SMAC (cSMAC) formation with mis-localized kindlin-3 and vesicle transport regulators involved in T cell receptor recycling/releasing machineries, resulting in impaired T cell-APC interactions. We found that NDR1 kinase, activated by the Rap1 signaling cascade through RAPL and Mst1/Mst2, associated with and recruited kindlin-3 to the IS, which was required for high-affinity LFA-1/ICAM-1 binding and cSMAC formation. Our findings reveal crucial roles for Rap1 signaling via NDR1 for recruitment of kindlin-3 and IS organization.