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Quantitative dynamic footprinting microscopy reveals mechanisms of neutrophil rolling

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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.

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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.

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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.).

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Authors and Affiliations

  1. Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, La Jolla, California, USA

    Prithu Sundd, Maria K Pospieszalska, Hong Zhang & Klaus Ley

  2. Department of Physics, University of California San Diego, La Jolla, California, USA

    Edgar Gutierrez & Alexander Groisman

Authors
  1. Prithu Sundd
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  2. Edgar Gutierrez
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  3. Maria K Pospieszalska
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  4. Hong Zhang
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  5. Alexander Groisman
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  6. Klaus Ley
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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.

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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)

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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

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