Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Brief Communication
  • Published:

Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling

Abstract

We introduce quantitative dynamic footprinting microscopy to resolve neutrophil rolling on P-selectin. We observed that the footprint of a rolling neutrophil was fourfold larger than previously thought, and that P-selectin–PSGL-1 bonds were relaxed at the leading edge of the rolling cell, compressed under the cell center, and stretched at the trailing edge. Each rolling neutrophil formed three to four long tethers that extended up to 16 μm behind the rolling cell.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Adaptation of TIRF for qDF microscopy.
Figure 2: qDF microscopy resolves microvilli in rolling neutrophils.
Figure 3: Footprints of rolling neutrophils identify locations of stressed and compressed bonds.
Figure 4: Footprints reveal anchorage points of long tethers at the rear of rolling neutrophils.

Similar content being viewed by others

References

  1. Ley, K., Laudanna, C., Cybulsky, M.I. & Nourshargh, S. Nat. Rev. Immunol. 7, 678–689 (2007).

    Article  CAS  Google Scholar 

  2. Hattori, R., Hamilton, K., Fugate, R., McEver, R. & Sims, P. J. Biol. Chem. 264, 7768–7771 (1989).

    CAS  Google Scholar 

  3. Damiano, E.R., Westheider, J., Tozeren, A. & Ley, K. Circ. Res. 79, 1122–1130 (1996).

    Article  CAS  Google Scholar 

  4. Chesnutt, B.C. et al. Microcirculation 13, 99–109 (2006).

    Article  CAS  Google Scholar 

  5. King, M.R., Heinrich, V., Evans, E. & Hammer, D.A. Biophys. J. 88, 1676–1683 (2005).

    Article  CAS  Google Scholar 

  6. Pospieszalska, M.K. & Ley, K. Cell. Mol. Bioeng. 2, 207–217 (2009).

    Article  CAS  Google Scholar 

  7. Finger, E., Bruehl, R., Bainton, D. & Springer, T. J. Immunol. 157, 5085–5096 (1996).

    CAS  PubMed  Google Scholar 

  8. Alon, R., Hammer, D.A. & Springer, T.A. Nature 374, 539–542 (1995).

    Article  CAS  Google Scholar 

  9. Ramachandran, V., Williams, M., Yago, T., Schmidtke, D.W. & McEver, R.P. Proc. Natl. Acad. Sci. USA 101, 13519–13524 (2004).

    Article  CAS  Google Scholar 

  10. Smith, M.L., Sperandio, M., Galkina, E.V. & Ley, K. J. Leukoc. Biol. 76, 985–993 (2004).

    Article  CAS  Google Scholar 

  11. Hocdé, S.A., Hyrien, O. & Waugh, R.E. Biophys. J. 97, 379–387 (2009).

    Article  Google Scholar 

  12. Patel, K., Nollert, M. & McEver, R. J. Cell Biol. 131, 1893–1902 (1995).

    Article  CAS  Google Scholar 

  13. Shao, J.-Y., Ting-Beall, H.P. & Hochmuth, R.M. Proc. Natl. Acad. Sci. USA 95, 6797–6802 (1998).

    Article  CAS  Google Scholar 

  14. Waugh, R.E. & Hochmuth, R.M. Biophys. J. 52, 391–400 (1987).

    Article  CAS  Google Scholar 

  15. Park, E.Y.H. et al. Biophys. J. 82, 1835–1847 (2002).

    Article  CAS  Google Scholar 

  16. Tkachenko, E., Gutierrez, E., Ginsberg, M.H. & Groisman, A. Lab Chip 9, 1085–1095 (2009).

    Article  CAS  Google Scholar 

  17. Faust, N., Varas, F., Kelly, L.M., Heck, S. & Graf, T. Blood 96, 719–726 (2000).

    CAS  PubMed  Google Scholar 

  18. Stock, K. et al. J. Microsc. 211, 19–29 (2003).

    Article  CAS  Google Scholar 

  19. Truskey, G., Burmeister, J., Grapa, E. & Reichert, W. J. Cell Sci. 103, 491–499 (1992).

    PubMed  Google Scholar 

  20. Kumosinski, T.F., Brown, E.M. & Farrell, H.M. Jr. J. Dairy Sci. 76, 931–945 (1993).

    Article  CAS  Google Scholar 

  21. Sachs, L. Applied Statistics: A Handbook of Techniques. 2nd edn. (Springer-Verlag, New York, 1984).

  22. Pospieszalska, M.K., Zarbock, A., Pickard, J.E. & Ley, K. Microcirculation 16, 115–130 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by a postdoctoral fellowship (09POST2230093) from the American Heart Association (P.S.) and US National Institutes of Health grant EB02185 (K.L.).

Author information

Authors and Affiliations

Authors

Contributions

P.S. contributed to experiment design, performed all experiments and analyzed images. E.G. and A.G. designed the microfluidic device. M.K.P. performed ETMA simulations. H.Z. was involved in negative separation of mouse neutrophils from bone marrow. P.S. and K.L. wrote the manuscript. K.L. contributed to experiment design and supervised the project.

Corresponding author

Correspondence to Klaus Ley.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–15, Supplementary Table 1, Supplementary Note 1 (PDF 2065 kb)

Supplementary Video 1

Footprints of a rolling neutrophil. Rolling of a GFP-expressing neutrophil in whole mouse blood at 6 dyn cm−2. qDF illumination at 488 nm, incident angle 70°. Substrate is P-selectin, 20 molecules μm−2. The video is at half its actual speed. (MOV 1040 kb)

Supplementary Video 2

Tether anchorage points at the rear of footprints. Tether anchorage points at the rear of rolling DiI-labeled neutrophil at 8 dyn cm−2. Image was processed to reveal tether anchorage points. qDF illumination at 561 nm, incident angle 70°. Substrate is P-selectin, 20 molecules μm−2. The video is at half its actual speed. (MOV 2362 kb)

Supplementary Video 3

Unprocessed version of Supplementary Video 2. The tether anchorage points are barely visible at the rear of the rolling neutrophil. (MOV 7917 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sundd, P., Gutierrez, E., Pospieszalska, M. et al. Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling. Nat Methods 7, 821–824 (2010). https://doi.org/10.1038/nmeth.1508

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1508

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing