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Stressed microvilli and long tethers in rolling, tight adhesion zones and aft trunks in arresting neutrophils revealed using Total Internal Reflection Fluorescence Microscopy (TIRFM)
Author(s) -
Sundd Prithu,
Gutierrez Edgar,
Pospieszalska Maria,
Groisman Alexander,
Ley Klaus
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.590.2
Subject(s) - total internal reflection fluorescence microscope , microvillus , chemistry , biophysics , substrate (aquarium) , fluorescence microscope , pseudopodia , adhesion , actin , fluorescence , biology , optics , ecology , biochemistry , physics , organic chemistry , membrane
Neutrophils are covered with microvilli. P‐selectin‐PSGL‐1 bonds are predicted to form when the microvillus tip approaches the P‐selectin substrate to within 70 nm. A microfluidic device with a substrate coated with P‐selectin/ICAM‐1 was perfused with blood from mice expressing GFP in neutrophils. Rolling interactions were studied at realistic wall shear stresses ( τ w = 6–8 dynes/cm 2 ) using TIRFM. Following bond formation, microvilli undergo compression approaching the substrate to within 25 nm near the cell center. At the trailing edge, the P‐selectin‐PSGL‐1 bonds stretch to 125–150 nm before they dissociate. Rolling is stabilized by the formation of 3–4 long (up to 16 μm) tethers (life time~1–10s). Adding the chemokine CXCL1 to the substrate induces neutrophil arrest and formation of a trunk‐like structure that stretches up to 10 μm behind the cell. The closest contact (area~1–3 μm 2 ) between the arrested neutrophil and the substrate is always found near the cell center. Its distance from the substrate (44 nm) corresponds to the length of the ICAM‐1‐LFA‐1 bond. These results identify the molecular and cellular dimensions of neutrophils during rolling and transition to arrest, thus providing a framework for the biomechanical analysis of this fundamental process. This work was supported by 09POST2230093 from American Heart Association (P.S) and NIH EB 02185 (K.L).