J Immunol. 2016 Apr 1;196(7):3019-31.

Sema3e/Plexin D1 Modulates Immunological Synapse and Migration of Thymocytes by Rap1 Inhibition.

Ueda Y1, Kondo N1, Ozawa M1, Yasuda K2, Tomiyama T3, Kinashi T4.
1 Department of Molecular Genetics, Kansai Medical University, Osaka 573-1010, Japan;
2 Department of Urology and Andrology, Kansai Medical University, Osaka 573-1010, Japan; and.
3 Division of Gastroenterology and Hepatology, Third Department of Internal Medicine, Kansai Medical University, Osaka 573-1010, Japan.
4 Department of Molecular Genetics, Kansai Medical University, Osaka 573-1010, Japan; kinashi@hirakata.kmu.ac.jp.



Regulation of thymocyte trafficking plays an important role during thymic selection, but our understanding of the molecular mechanisms underlying these processes is limited. In this study, we demonstrated that class III semaphorin E (sema3e), a guidance molecule during neural and vascular development, directly inhibited Rap1 activation and LFA-1-dependent adhesion through the GTPase-activating protein activity of plexin D1. Sema3e inhibited Rap1 activation of thymocytes in response to chemokines and TCR stimulation, LFA-mediated adhesion, and T cell-APC interactions. Immunological synapse (IS) formation in mature thymocytes on supported lipid bilayers was also attenuated by sema3e. Impaired IS formation was associated with reduced Rap1 activation on the contact surface and cell periphery. Moreover, a significant increase of CD4(+) thymocytes was detected in the medulla of mice with T cell lineage-specific deletion of plexin D1. Two-photon live imaging of thymic explants and slices revealed enhanced Rap1 activation and migration of CD69(+) double-positive and single-positive cells with plexin D1 deficiency. Our results demonstrate that sema3e/plexin D1 modulates IS formation and Ag-scanning activities of thymocytes within thymic tissues.



Semaphorins are GPI-linked secreted proteins that have transmembrane and cysteine-rich semaphorin protein domains. Semaphorin 3E (Sema3e) and its receptor plexin D1 is known to be guidance molecules for neural cells and vascular endothelial cells. Recently Sema3e is also important for localization of immature thymocytes within the thymus. Semaphorin is known to induce actin collapse but also inhibit the binding of integrin to their ligands. However the downstream signaling important for these signaling is still intricate. Plexin D1, a putative receptor of Sema3e, has a split GTP-activating protein (GAP) domain, which has been originally reported to inhibit R-Ras or M-Ras activity. However recent biochemical study using cytoplasmic domain of plexins suggests these domain did not have Ras-GAP activity but has Rap1 GAP activity. Nonfunctional GAP mutant of plexin D1 BAC transgenic mice exhibited defective vascular and neural development, however those phenotypes were not recapitulated by R-Ras and M-Ras deficient mice, suggesting that GAP-mediated signals of plexin D1 is independent of Ras inhibition.


We have demonstrated that small-GTP Rap1 regulates integrin LFA-1 of lymphocytes in response to chemokine and TCR stimulation (1, 2). We identified RAPL (Rassf5c) and Mst1 kinase (stk4) as effectors of Rap1 (3, 4). Upon chemokine and TCR stimulation, Rap1 form complex with RAPL and Mst1 and bind to integrin αchain (3, 4). During interaction of T cells with antigen presenting cells, Rap1 signals triggered activation of NDR1 kinase via RAPL/Mst1 axis. NDR1 were associated with and recruited kindlin3 to immunological synapse, promoting high-affinity LFA-1/ICAM-1 binding and immunological synapse (IS) formation (5). Mst1 and NDR1 also required for vesicle transport regulators involved in TCR recycling/releasing machineries. Thus, Rap1 signaling is important for integrin activation as well as in vivo integrin-dependent process of lymphocytes, as we demonstrated by using knockout mouse models (6, 7, 8).


In this study, we demonstrated that sema3e/plexin D1 signals negatively regulated integrin-mediated adhesion and IS formation by mitigating Rap1 activation via the GAP domain of plexin D1 (Figure 1). Live imaging by fluorescent probe of active Rap visualized Rap1 activation of thymocytes within live thymic tissues, which were enhanced in plexin D1-defcient T cells. Enhanced Rap1 activation is associated with increased thymocyte motility. Taken together, sema3e/plexin D1 modulates antigen-scanning activities of thymocytes via regulation of Rap1 activation.


Recent studies showed that sema3e/plexinD1 is important for many pathological processes such as peripheral vascular disease, tumor progression and Type 2 diabetes mellitus. We provide Rap1 signaling as potential therapeutic target in disorder of semaphoring-mediated biological process.



Figure 1 sema3e-inhibit Rap1 activation in Ba/F3 cell lymphocyte cell lines visualized by active fluorescent probe of active Rap. Ba/F3 cells transfected with GFP-RalGDS-RBD were loaded on immobilized ICAM-1 and SDF-1, which induced Rap1 activation and adhesion to ICAM-1 in Ba/F3 cells. We also found the accumulation of GFP-RalGDS-RBD probes at leading edge (left panel). In contrast, sema3e-Fc inhibited Rap1 activation and adhesive responses with the diffused distribution of those probes.



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