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Confinement-optimized three-dimensional T cell amoeboid motility is modulated via myosin IIA–regulated adhesions

Nature Immunology volume 11, pages 953–961 (2010)Cite this article

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Abstract

During trafficking through tissues, T cells fine-tune their motility to balance the extent and duration of cell-surface contacts versus the need to traverse an entire organ. Here we show that in vivo, myosin IIA–deficient T cells had a triad of defects, including overadherence to high-endothelial venules, less interstitial migration and inefficient completion of recirculation through lymph nodes. Spatiotemporal analysis of three-dimensional motility in microchannels showed that the degree of confinement and myosin IIA function, rather than integrin adhesion (as proposed by the haptokinetic model), optimized motility rate. This motility occurred via a myosin IIA–dependent rapid 'walking' mode with multiple small and simultaneous adhesions to the substrate, which prevented spurious and prolonged adhesions. Adhesion discrimination provided by myosin IIA is thus necessary for the optimization of motility through complex tissues.

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Figure 1: Deletion of myosin IIA in T cells from Myh9flox/flox–Lck-Cre mice.
Figure 2: MyoIIA-cKO T cells have more contact with HEVs and adhesion to integrin substrates.
Figure 3: Naive myosin IIA–deficient T cells have less intra–lymph node migration.
Figure 4: Naive myosin IIA–deficient T cells have trafficking defects in vivo due to retention in the lymph nodes.
Figure 5: T cell migration efficiency in confined environments is controlled by myosin IIA.
Figure 6: Myosin IIA regulates the crawling mode of T cells under confinement.
Figure 7: Deregulated surface adhesion of T cells lacking myosin IIA.

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References

  1. Dustin, M.L. Stop and go traffic to tune T cell responses. Immunity 21, 305–314 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. von Andrian, U.H. & Mempel, T.R. Homing and cellular traffic in lymph nodes. Nat. Rev. Immunol. 3, 867–878 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Cahalan, M.D. & Parker, I. Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs. Annu. Rev. Immunol. 26, 585–626 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bajenoff, M. et al. Highways, byways and breadcrumbs: directing lymphocyte traffic in the lymph node. Trends Immunol. 28, 346–352 (2007).

    Article  CAS  PubMed  Google Scholar 

  5. DiMilla, P.A., Barbee, K. & Lauffenburger, D.A. Mathematical model for the effects of adhesion and mechanics on cell migration speed. Biophys. J. 60, 15–37 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Palecek, S.P., Loftus, J.C., Ginsberg, M.H., Lauffenburger, D.A. & Horwitz, A.F. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 385, 537–540 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Lammermann, T. et al. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature 453, 51–55 (2008).

    Article  PubMed  Google Scholar 

  8. Woolf, E. et al. Lymph node chemokines promote sustained T lymphocyte motility without triggering stable integrin adhesiveness in the absence of shear forces. Nat. Immunol. 8, 1076–1085 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Friedl, P., Entschladen, F., Conrad, C., Niggemann, B. & Zanker, K.S. CD4+ T lymphocytes migrating in three-dimensional collagen lattices lack focal adhesions and utilize β1 integrin-independent strategies for polarization, interaction with collagen fibers and locomotion. Eur. J. Immunol. 28, 2331–2343 (1998).

    Article  CAS  PubMed  Google Scholar 

  10. Vicente-Manzanares, M., Ma, X., Adelstein, R.S. & Horwitz, A.R. Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat. Rev. Mol. Cell Biol. 10, 778–790 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lammermann, T. & Sixt, M. Mechanical modes of 'amoeboid' cell migration. Curr. Opin. Cell Biol. 21, 636–644 (2009).

    Article  PubMed  Google Scholar 

  12. Pollard, T.D. & Borisy, G.G. Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Jacobelli, J., Chmura, S.A., Buxton, D.B., Davis, M.M. & Krummel, M.F. A single class II myosin modulates T cell motility and stopping, but not synapse formation. Nat. Immunol. 5, 531–538 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Jacobelli, J., Bennett, F.C., Pandurangi, P., Tooley, A.J. & Krummel, M.F. Myosin-IIA and ICAM-1 regulate the interchange between two distinct modes of T cell migration. J. Immunol. 182, 2041–2050 (2009).

    Article  CAS  PubMed  Google Scholar 

  15. Morin, N.A. et al. Nonmuscle myosin heavy chain IIA mediates integrin LFA-1 de-adhesion during T lymphocyte migration. J. Exp. Med. 205, 195–205 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Smith, A., Bracke, M., Leitinger, B., Porter, J.C. & Hogg, N. LFA-1-induced T cell migration on ICAM-1 involves regulation of MLCK-mediated attachment and ROCK-dependent detachment. J. Cell Sci. 116, 3123–3133 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Conti, M.A., Even-Ram, S., Liu, C., Yamada, K.M. & Adelstein, R.S. Defects in cell adhesion and the visceral endoderm following ablation of nonmuscle myosin heavy chain II-A in mice. J. Biol. Chem. 279, 41263–41266 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Zhang, D.J. et al. Selective expression of the Cre recombinase in late-stage thymocytes using the distal promoter of the Lck gene. J. Immunol. 174, 6725–6731 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Srinivas, S. et al. Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 1–8 (2001).

    Article  Google Scholar 

  20. Sumen, C., Mempel, T.R., Mazo, I.B. & von Andrian, U.H. Intravital microscopy: visualizing immunity in context. Immunity 21, 315–329 (2004).

    CAS  PubMed  Google Scholar 

  21. Faure-Andre, G. et al. Regulation of dendritic cell migration by CD74, the MHC class II-associated invariant chain. Science 322, 1705–1710 (2008).

    Article  CAS  PubMed  Google Scholar 

  22. Straight, A.F. et al. Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor. Science 299, 1743–1747 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Andrew, N. & Insall, R.H. Chemotaxis in shallow gradients is mediated independently of PtdIns 3-kinase by biased choices between random protrusions. Nat. Cell Biol. 9, 193–200 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Bajenoff, M. et al. Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25, 989–1001 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hara, T. et al. A transmembrane chemokine, CXC chemokine ligand 16, expressed by lymph node fibroblastic reticular cells has the potential to regulate T cell migration and adhesion. Int. Immunol. 18, 301–311 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Katakai, T., Hara, T., Sugai, M., Gonda, H. & Shimizu, A. Lymph node fibroblastic reticular cells construct the stromal reticulum via contact with lymphocytes. J. Exp. Med. 200, 783–795 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Snapper, S.B. et al. WASP deficiency leads to global defects of directed leukocyte migration in vitro and in vivo. J. Leukoc. Biol. 77, 993–998 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Nombela-Arrieta, C. et al. A central role for DOCK2 during interstitial lymphocyte motility and sphingosine-1-phosphate-mediated egress. J. Exp. Med. 204, 497–510 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Shiow, L.R. et al. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Foger, N., Rangell, L., Danilenko, D.M. & Chan, A.C. Requirement for coronin 1 in T lymphocyte trafficking and cellular homeostasis. Science 313, 839–842 (2006).

    Article  PubMed  Google Scholar 

  31. Renkawitz, J. et al. Adaptive force transmission in amoeboid cell migration. Nat. Cell Biol. 11, 1438–1443 (2009).

    Article  CAS  PubMed  Google Scholar 

  32. Malawista, S.E. & de Boisfleury Chevance, A. Random locomotion and chemotaxis of human blood polymorphonuclear leukocytes (PMN) in the presence of EDTA: PMN in close quarters require neither leukocyte integrins nor external divalent cations. Proc. Natl. Acad. Sci. USA 94, 11577–11582 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Fukui, Y. & Inoue, S. Amoeboid movement anchored by eupodia, new actin-rich knobby feet in Dictyostelium. Cell Motil. Cytoskeleton 36, 339–354 (1997).

    Article  CAS  PubMed  Google Scholar 

  34. Jay, P.Y., Pham, P.A., Wong, S.A. & Elson, E.L. A mechanical function of myosin II in cell motility. J. Cell Sci. 108, 387–393 (1995).

    CAS  PubMed  Google Scholar 

  35. Wilson, C.A. et al. Myosin II contributes to cell-scale actin network treadmilling through network disassembly. Nature 465, 373–377 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Shulman, Z. et al. Lymphocyte crawling and transendothelial migration require chemokine triggering of high-affinity LFA-1 integrin. Immunity 30, 384–396 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Xu, J. et al. Divergent signals and cytoskeletal assemblies regulate self-organizing polarity in neutrophils. Cell 114, 201–214 (2003).

    Article  CAS  PubMed  Google Scholar 

  38. Dulyaninova, N.G., Malashkevich, V.N., Almo, S.C. & Bresnick, A.R. Regulation of myosin-IIA assembly and Mts1 binding by heavy chain phosphorylation. Biochemistry 44, 6867–6876 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Bullen, A., Friedman, R.S. & Krummel, M.F. Two-photon imaging of the immune system: a custom technology platform for high-speed, multicolor tissue imaging of immune responses. Curr. Top. Microbiol. Immunol. 334, 1–29 (2009).

    PubMed  Google Scholar 

  40. Kolega, J. Phototoxicity and photoinactivation of blebbistatin in UV and visible light. Biochem. Biophys. Res. Commun. 320, 1020–1025 (2004).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank P. Beemiller for help with two-photon data analysis with Imaris and Matlab software; S. Peck for assistance in maintenance of microscopes; M. Tang (Stanford Microfabrication Center) for Silicon Masters; S. Jiang for technical assistance with cell sorting; O. Khan and M. Werner for help with mouse genotyping; M. Heuze for assistance in setting up the microchannel system; and B. Sauer (Stowers Institute for Medical Research) for the pBS479 vector. Supported by the National Institutes of Health (AI52116 to M.F.K.) and the Larry L. Hillblom Foundation (R.S.F.).

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Authors and Affiliations

  1. Department of Pathology, University of California San Francisco, San Francisco, California, USA

    Jordan Jacobelli, Rachel S Friedman, Caitlin M Sorensen & Matthew F Krummel

  2. Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA

    Mary Anne Conti & Robert S Adelstein

  3. Institut National de la Santé et de la Recherche Médicale U653, Institut Curie, Paris, France

    Ana-Maria Lennon-Dumenil & Matthieu Piel

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Contributions

J.J. designed, did and analyzed all experiments and wrote the manuscript; R.S.F. provided assistance with in vivo experiments and participated in two-photon experiments; M.A.C. and R.S.A. generated the Myh9flox/flox mice and provided reagents; A.-M.L-D. and M.P. provided assistance in establishing the microchannel fabrication technique; C.M.S. helped with tissue sectioning and staining and with mouse genotyping; and M.F.K. coordinated the project and participated in the conception and execution of experiments and in writing the manuscript.

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Correspondence to Matthew F Krummel.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 (PDF 5480 kb)

Supplementary Video 1

Naïve MyoIIA-deficient T cells have reduced intra-lymph node migration. Representative movie of interstitial migration in vivo of control T cells (green) and MyoIIA cKO T cells (red). Naïve CD8+ T cells were purified by negative selection from control and MyoIIA cKO mice, then labeled with either CFSE or CMTMR, mixed at a 1:1 ratio and injected intravenously into recipient mice. Popliteal, axillary and inguinal lymph nodes were isolated 18h after transfer and imaged by time-lapse 2-photon laser scanning microscopy. The duration of the timelapse is 30 min at 20 sec intervals between frames. The tracks were obtained using Imaris software and the latest trailing 20 frames are shown. The grid spacing is at 20 μm. (MOV 1254 kb)

Supplementary Video 2

Representative behavior of T cells in 4, 8 and 20 μm wide microchannels. CD8+ T cells 4-5 days post-activation were injected into fabricated microchannels allowed to enter the channels for 2h and then imaged. Timelapse imaging was done using a 20X objective at a 30 sec intervals for 2h. Representative T cells crawling within microchannels of different width are shown to highlight the different average speeds of T cells as a function of confinement. (MOV 2951 kb)

Supplementary Video 3

Representative 'weaving' behavior of a control T cell in a 20 μm wide microchannel. CD8+ T cells 4-5 days post-activation were injected into fabricated microchannels in the presence of vehicle control and then imaged. Imaging was done using a 10X Phase contrast objective at 1.5 min intervals for a minimum of 2h. A representative control T cell is shown rapidly 'weaving' between the two side walls of the microchannel. (MOV 911 kb)

Supplementary Video 4

Blebbistatin treated T cells show increased adhesion and reduced 'weaving' behavior in 20 μm wide microchannels. CD8+ T cells 4-5 days post-activation were injected into fabricated microchannels in the presence of 100 μm blebbistatin (racemic mix) and then imaged. Imaging was done using a 10X Phase contrast objective at 1.5 min intervals for a minimum of 2h. A representative blebbistatin treated T cell is shown displaying increased dwell-time adhering to walls and reduced 'weaving' between the two side walls of the microchannel. (MOV 660 kb)

Supplementary Video 5

Representative 'walking' mode of control T cells in microchannels. CD8+ T cells 4-5 days post-activation were labeled with CMTMR and then treated with vehicle control and injected into variable size microchannels. Representative movie of the behavior of a 'walking' control T cell in a microchannel over time. Brightfield and TIRF images were taken at 5 sec intervals for 5 min with a 100x TIRF objective. The brightfield images were overlaid with the TIRF adhesion area images (green). White arrows highlight the presence of simultaneous distinct adhesion zones arising in the direction of motion. (MOV 3632 kb)

Supplementary Video 6

Representative 'sliding' mode of blebbistatin treated T cells in microchannels. CD8+ T cells 4-5 days post-activation were labeled with CMTMR and then treated with 100 μm blebbistatin (racemic mix) and injected into variable size microchannels. Representative movie of the behavior of a 'sliding' blebbistatin treated T cell in a microchannel over time. Brightfield and TIRF images were taken at 5 sec intervals for 5 min with a 100x TIRF objective. The brightfield images were overlaid with the TIRF adhesion area images (green). As opposed to the 'walking' mode, the 'sliding' blebbistatin treated T cell shows only one main adhesion zone and has extended contact with a microchannel side wall. (MOV 4581 kb)

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Jacobelli, J., Friedman, R., Conti, M. et al. Confinement-optimized three-dimensional T cell amoeboid motility is modulated via myosin IIA–regulated adhesions. Nat Immunol 11, 953–961 (2010). https://doi.org/10.1038/ni.1936

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