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 mislocalized 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.

KEYWORDS:

• ICAM-1
• immunological synapse
• LFA-1
• NDR1
• Rap1
• single-molecule measurement
• vesicular transport

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Supplemental material for this article may be found at https://doi.org/10.1128/MCB.00424-16.

ACKNOWLEDGMENTS

We thank R. Hamaguchi for technical assistance, M. Ishibashi and M. Ueda for advice and help with the TIRF microscope settings, T. Katakai for advice on lymph node slice preparation and its two-photon imaging, H. Miyoshi and A. Miyawaki for lentivirus vectors, and H. Mizuno for NDR plasmids.

This study was supported by CREST, Japan Science Technology Agency, and KAKENHI from the Ministry of Education, Science, Sport and Culture of Japan (a Grant-in-Aid for Scientific Research, a Grant-in-Aid on Innovative Areas, and a Grant-in-Aid for Young Scientists) (15K21524, 22111003, and 25291047).