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Regulation of thymocyte positive selection and motility by GIT2

Nature Immunology volume 11, pages 503–511 (2010)Cite this article

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Abstract

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.

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Figure 1: Impaired generation of CD4SP thymocytes in DO11.10+ Git2−/− mice.
Figure 2: Fewer CD4SP thymocytes in TCR-transgenic Git2−/− mice because of impaired positive selection, not more apoptosis or negative selection.
Figure 3: The defect in the positive selection of TCR-transgenic Git2−/− thymocytes is intrinsic to hematopoietic cells.
Figure 4: Greater migratory activity of Git2−/− DP thymocytes in response to SDF-1 or CCL25.
Figure 5: Greater directional and random motility of Git2−/− thymocytes.
Figure 6: More Rac1 activation and actin polymerization in Git2−/− thymocytes in response to SDF-1.
Figure 7: More Git2−/− DP thymocytes overcome TCR-mediated stop signals in the presence of SDF-1.
Figure 8: Altered motility of Git2−/− thymocytes in the cortex.

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Acknowledgements

We thank the Transgenic/Targeted Mutagenesis Core Facility of the University of California San Francisco Comprehensive Cancer Center for creating Git2−/− mice; the Biological Imaging Development Center of the University of California San Francisco for Imaris software support; and E. Manser (Institute of Molecular and Cell Biology, Singapore) for monoclonal and polyclonal antibodies that recognize the α- and β-isoforms of PIX. Supported by the Leukemia and Lymphoma Society (H.P.) and the National Institutes of Health (R01AI064227 to E.R.).

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Authors and Affiliations

  1. Department of Medicine, Division of Rheumatology, Rosalind Russell Medical Research Center for Arthritis, University of California San Francisco, San Francisco, California, USA

    Hyewon Phee, Marianne Mollenauer & Arthur Weiss

  2. Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California Berkeley, Berkeley, California, USA

    Ivan Dzhagalov & Ellen Robey

  3. Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California, USA

    Marianne Mollenauer & Arthur Weiss

  4. Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Yana Wang

  5. Department of Biological Engineering and Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Darrell J Irvine

  6. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA

    Darrell J Irvine

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Contributions

H.P. designed the study, did experiments, analyzed data and wrote the manuscript; M.M. did experiments; I.D. did the two-photon experiments and analyzed data; Y.W. and D.J.I. supplied alginate beads; E.R. supervised the two-photon experiments and discussed data; and A.W. designed the study, supervised the research and revised the manuscript.

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Correspondence to Arthur Weiss.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Table 1 and Methods (PDF 4120 kb)

Supplementary Video 1

Directional migration of wild-type and Git2−/− thymocytes to SDF-1. (AVI 3025 kb)

Supplementary Video 2

Migration of wild-type and Git2−/− thymocytes in intact thymic lobes using two-photon microscopy. (AVI 947 kb)

Supplementary Video 3

Accumulation and movement of Git2−/− thymocyte on small blood vessels. (AVI 1062 kb)

Supplementary Video 4

Migratory behavior of wild-type thymocytes that move away from small blood vessels. (AVI 284 kb)

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Phee, H., Dzhagalov, I., Mollenauer, M. et al. Regulation of thymocyte positive selection and motility by GIT2. Nat Immunol 11, 503–511 (2010). https://doi.org/10.1038/ni.1868

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