Thymocytes are highly motile cells that migrate under the influence of chemokines in distinct thymic compartments as they mature. The motility of thymocytes is tightly regulated; however, the molecular mechanisms that control thymocyte motility are not well understood. Here we report that G protein–coupled receptor kinase-interactor 2 (GIT2) was required for efficient positive selection. Notably, Git2−/− double-positive thymocytes showed greater activation of the small GTPase Rac, actin polymerization and migration toward the chemokines CXCL12 (SDF-1) and CCL25 in vitro. By two-photon laser-scanning microscopy, we found that the scanning activity of Git2−/− thymocytes was compromised in the thymic cortex, which suggests GIT2 has a key role in regulating the chemokine-mediated motility of double-positive thymocytes.
At a glance
- Thymic microenvironments for T cell differentiation and selection. Nat. Immunol. 7, 338–343 (2006). , , &
- Dynamics of thymocyte-stromal cell interactions visualized by two-photon microscopy. Science 296, 1876–1880 (2002). , , &
- Directed migration of positively selected thymocytes visualized in real time. PLoS Biol. 3, e160 (2005). , , , &
- Calcium oscillations regulate thymocyte motility during positive selection in the three-dimensional thymic environment. Nat. Immunol. 6, 143–151 (2005). , &
- Thymocyte-dendritic cell interactions near sources of CCR7 ligands in the thymic cortex. J. Immunol. 181, 7014–7023 (2008). et al.
- Pertussis toxin-sensitive signal controls the trafficking of thymocytes across the corticomedullary junction in the thymus. J. Immunol. 162, 5981–5985 (1999). et al.
- CXCR4 and CCR5 on human thymocytes: biological function and role in HIV-1 infection. J. Immunol. 161, 3103–3113 (1998). et al.
- The chemokine TECK is expressed by thymic and intestinal epithelial cells and attracts double- and single-positive thymocytes expressing the TECK receptor CCR9. Eur. J. Immunol. 30, 262–271 (2000). et al.
- Thymic T cell development and progenitor localization depend on CCR7. J. Exp. Med. 200, 481–491 (2004). et al.
- Characterization of CCR9 expression and CCL25/thymus-expressed chemokine responsiveness during T cell development: CD3highCD69+ thymocytes and gammadeltaTCR+ thymocytes preferentially respond to CCL25. J. Immunol. 168, 134–142 (2002). , , &
- PlexinD1 glycoprotein controls migration of positively selected thymocytes into the medulla. Immunity 29, 888–898 (2008). et al.
- Paxillin interactions. J. Cell Sci. 113, 4139–4140 (2000).
- The PIX-GIT complex: a G protein signaling cassette in control of cell shape. Semin. Cell Dev. Biol. 19, 234–244 (2008). &
- Arf GAPs as regulators of the actin cytoskeleton. Biol. Cell 99, 583–600 (2007). , &
- The multifunctional GIT family of proteins. J. Cell Sci. 119, 1469–1475 (2006). &
- ArfGAP family proteins in cell adhesion, migration and tumor invasion. Curr. Opin. Cell Biol. 18, 558–564 (2006). , , &
- GIT proteins, A novel family of phosphatidylinositol 3,4, 5-trisphosphate-stimulated GTPase-activating proteins for ARF6. J. Biol. Chem. 275, 13901–13906 (2000). et al.
- The GIT family of ADP-ribosylation factor GTPase-activating proteins. Functional diversity of GIT2 through alternative splicing. J. Biol. Chem. 275, 22373–22380 (2000). , , , &
- ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements. J. Cell Sci. 112, 855–866 (1999). , , &
- ARF proteins: roles in membrane traffic and beyond. Nat. Rev. Mol. Cell Biol. 7, 347–358 (2006). &
- Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D. J. Cell Biol. 154, 599–610 (2001). &
- beta2-Adrenergic receptor regulation by GIT1, a G protein-coupled receptor kinase-associated ADP ribosylation factor GTPase-activating protein. Proc. Natl. Acad. Sci. USA 95, 14082–14087 (1998). et al.
- A tyrosine-phosphorylated protein that binds to an important regulatory region on the cool family of p21-activated kinase-binding proteins. J. Biol. Chem. 274, 22393–22400 (1999). et al.
- Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling. J. Cell Biol. 145, 851–863 (1999). et al.
- GIT2 represses Crk- and Rac1-regulated cell spreading and Cdc42-mediated focal adhesion turnover. EMBO J. 25, 1848–1859 (2006). , &
- GIT1/PIX/Rac/PAK signaling module regulates spine morphogenesis and synapse formation through MLC. J. Neurosci. 25, 3379–3388 (2005). , , , &
- Neutrophil direction sensing and superoxide production linked by the GTPase-activating protein GIT2. Nat. Immunol. 7, 724–731 (2006). et al.
- An α4 integrin-paxillin-Arf-GAP complex restricts Rac activation to the leading edge of migrating cells. Nat. Cell Biol. 7, 343–352 (2005). , , &
- PAK1-PIX-PKL complex is activated by the T-cell receptor independent of Nck, Slp-76 and LAT. EMBO J. 20, 457–465 (2001). , , , &
- Dynamic recruitment of PAK1 to the immunological synapse is mediated by PIX independently of SLP-76 and Vav1. Nat. Immunol. 6, 608–617 (2005). , &
- Differential expression of the ARF GAP genes GIT1 and GIT2 in mouse tissues. J. Histochem. Cytochem. 55, 1039–1048 (2007). , , , &
- Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature 333, 742–746 (1988). , , , &
- Positive selection of antigen-specific T cells in thymus by restricting MHC molecules. Nature 335, 730–733 (1988). , , &
- CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity. J. Exp. Med. 188, 2301–2311 (1998). et al.
- Cutting edge: developmental switches in chemokine responses during T cell maturation. J. Immunol. 163, 2353–2357 (1999). , &
- Injectable dendritic cell-carrying alginate gels for immunization and immunotherapy. Biomaterials 29, 3671–3682 (2008). , , , &
- Cutting edge: hierarchy of chemokine receptor and TCR signals regulating T cell migration and proliferation. J. Immunol. 165, 15–19 (2000). , , &
- CXCR4 acts as a costimulator during thymic β-selection. Nat. Immunol. 11, 162–170 (2010). et al.
- Thymic microvascular system. Microsc. Res. Tech. 38, 287–299 (1997).
- The actin regulator coronin 1A is mutant in a thymic egress-deficient mouse strain and in a patient with severe combined immunodeficiency. Nat. Immunol. 9, 1307–1315 (2008). et al.
- Myosin-IIA and ICAM-1 regulate the interchange between two distinct modes of T cell migration. J. Immunol. 182, 2041–2050 (2009). , , , &
- Supplementary Text and Figures (4M)
Supplementary Figures 1–8, Table 1 and Methods
- Supplementary Video 1 (3M)
Directional migration of wild-type and Git2−/− thymocytes to SDF-1.
- Supplementary Video 2 (948K)
Migration of wild-type and Git2−/− thymocytes in intact thymic lobes using two-photon microscopy.
- Supplementary Video 3 (1M)
Accumulation and movement of Git2−/− thymocyte on small blood vessels.
- Supplementary Video 4 (288K)
Migratory behavior of wild-type thymocytes that move away from small blood vessels.