Abstract
Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell’s surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level.
Highlights
Rolling adhesion is a common process by which cells attach themselves to surfaces under shear flow, such as in the circulatory system
Unlike phase-contrast or fluorescence imaging, which have typically been used for whole cell identification and tracking[19,24] (Fig. 1a,b), dark-field microscopy reveals μm to sub-μm-sized, highly scattering spots inside most HL-60 cells (Fig. 1c)
Control experiments using 6-μmpolystyrene beads coated with P-selectin glycoprotein ligand-1 (PSGL-1) show extremely uniform rolling velocity on P-selectin-coated surfaces, as compared to HL-60 cells
Summary
Rolling adhesion is a common process by which cells attach themselves to surfaces under shear flow, such as in the circulatory system. Endothelial cells lining the blood vessels surrounding an infection site express adhesion proteins called selectins that are specific to leukocyte surface receptors. We developed a label-free method that maps the functional adhesion sites and strengths on a cell surface as it rolls across a surface coated uniformly with adhesion receptors. Constructing the adhesion map from the instantaneous angular velocity reveals that the adhesion profile along the rolling circumference is inhomogeneous. We corroborated these findings by obtaining fluorescent footprints of molecular adhesion events using probes derived from recently developed DNA-based molecular force sensors[20]. Our methods will enable researchers to generate significantly richer data when studying the rolling adhesion of immune cells and circulating tumor cells
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