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Synaptotagmin-mediated vesicle fusion regulates cell migration

Nature Immunology volume 11, pages 495–502 (2010)Cite this article

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Abstract

Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractant-induced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7-deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release.

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Figure 1: Screen of chemotaxis by shRNA identifies synaptotagmins SYT2, SYTL5 and SYT7.
Figure 2: Knockdown of RAB27A and RAB3A inhibits T cell chemotaxis.
Figure 3: Migration of wild-type and SYT7-deficient neutrophils ex vivo.
Figure 4: Migration of wild-type and SYT7-deficient neutrophils in vivo in a model of gout.
Figure 5: CXCR4 cell surface expression and chemokine-induced F-actin polymerization and calcium release.
Figure 6: Effect of calcium on chemotaxis, F-actin polymerization, and chemokine-induced expression of LAMP-1.
Figure 7: Localization of LysoTracker Red–stained vesicles during cell migration.

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Acknowledgements

We thank the Immune Circuits and RNAi Platform groups of the Broad Institute and W. Xu for discussions and the Imaging Core of the Ragon Institute of MGH, MIT and Harvard. Supported by the US National Institutes of Health (R01 DK074449 to A.D.L. and R01 GM064625 to N.W.A.), the Defense Advanced Research Projects Agency (W81XWH-04-C-0139 to N.H.), Fundação Luso-Americana para o Desenvolvimento (L.F.M.) and Fundação para a Ciência e a Tecnologia (L.F.M.).

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  1. Richard A Colvin

    Present address: Present address: Novartis Institute for Biomedical Research, Cambridge, Massachusetts, USA.,

Authors and Affiliations

  1. Division of Rheumatology, Center for Immunology and Inflammatory Diseases, Allergy, and Immunology, Massachusetts General Hospital (MGH) and Harvard Medical School, Boston, Massachusetts, USA

    Richard A Colvin, Terry K Means, Luis F Moita, Robert P Friday, Gabriele S V Campanella, Tabitha Abrazinski, Lindsay A Manice, Catarina Moita, Nir Hacohen & Andrew D Luster

  2. Ragon Institute of MGH, Massachusetts Institute of Technology (MIT) and Harvard, Boston, Massachusetts, USA

    Thomas J Diefenbach

  3. Cell Biology of the Immune System Unit, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Lisboa, Portugal

    Luis F Moita & Catarina Moita

  4. Division of Nephrology, Massachusetts General Hospital, Boston, Massachusetts, USA

    Sanja Sever

  5. Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA

    Norma W Andrews

  6. Yale University School of Medicine, Yale University, New Haven, Connecticut, USA.,

    Dianqing Wu

  7. Broad Institute, Cambridge, Massachusetts, USA

    Nir Hacohen

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Contributions

R.A.C. designed the experiments, did the screening, collected and analyzed data and wrote the manuscript; T.K.M. did air-pouch experiments, macrophage stimulation and GTPase assays and helped edit the manuscript; T.J.D. did confocal microscopy and analyzed lymphocytes; L.F.M. and C.M. helped with initial screens; R.P.F. assisted with air-pouch experiments; S.S. did confocal microscopy of podocytes; G.S.V.C. helped with experiments; T.A. and L.A.M. collected data; N.W.A. provided mouse strains; D.W. assisted with video migration assays of neutrophils; N.H. helped design experiments, analyzed data and helped edit the manuscript; A.D.L. helped design experiments, analyzed data and edited the manuscript; and all authors discussed results and commented on the manuscript.

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Correspondence to Andrew D Luster.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Tables 1–2 and Supplementary Methods (PDF 836 kb)

Supplementary Video 1

Bone marrow derived neutrophils from wild-type C57BL/6 mice migrating in response to fMLP. (MOV 274 kb)

Supplementary Video 2

Bone marrow derived neutrophils from Syt7−/− mice migrating in response to fMLP. (MOV 148 kb)

Supplementary Video 3

CD4+ lymphocytes obtained from wild-type C57BL/6 mice, loaded with lysotracker red, migrating in response to CXCL10. (MOV 3861 kb)

Supplementary Video 4

CD4+ lymphocytes obtained from wild-type C57BL/6 mice, loaded with lysotracker red, migrating in response to CXCL10. (MOV 1135 kb)

Supplementary Video 5

CD4+ lymphocytes obtained from Syt7−/− mice, loaded with lysotracker red, migrating in response to CXCL10. (MOV 6688 kb)

Supplementary Video 6

CD4+ lymphocytes obtained from Syt7−/− mice, loaded with lysotracker red, migrating in response to CXCL10. (MOV 626 kb)

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Colvin, R., Means, T., Diefenbach, T. et al. Synaptotagmin-mediated vesicle fusion regulates cell migration. Nat Immunol 11, 495–502 (2010). https://doi.org/10.1038/ni.1878

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