Mapping Cell Surface Adhesion by Rotation Tracking and Adhesion Footprinting

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.Rolling adhesion is a common process by which cells attach themselves to surfaces under shear flow, such as in the circulatory system. Leukocytes in the blood utilize this mechanism to locate inflammation sites throughout the body. During an inflammation response, endothelial cells lining the blood vessels surrounding an infection site express adhesion proteins called selectins that are specific to leukocyte surface receptors. As the first step of the leukocyte adhesion cascade, leukocytes captured via selectin-specific interactions passively roll on the blood vessel wall under blood flow toward the inflammation site in a process known as rolling adhesion [1][2][3] . Malfunction of any adhesion molecules involved in this process leads to severe immune disorders such as the leukocyte adhesion deficiencies (LAD) 4 . Rolling adhesion behavior is also exhibited by circulating tumor cells (CTCs) which is believed to enhance cancer metastasis [5][6][7][8] . Therefore, quantitative understanding of rolling adhesion is necessary to enable practical applications such as cancer screening and treatment [9][10][11] . At the molecular level, this adhesion is mediated by catch-bond-like interactions 12,13 between P-14 and E-selectins 15 expressed on endothelial cells lining blood vessels and P-selectin glycoprotein ligand-1 (PSGL-1) found at microvilli tips of leukocytes 16 . Despite our understanding of the individual components, how the molecular details of adhesion bonds scale to cell-surface adhesion and rolling behavior remains poorly understood 2,17,18 . Here, we developed a label-free method that maps the functional adhesion sites and strengths on a cell ...

Rolling Adhesion of Schizont Stage Malaria-Infected Red Blood Cells in Shear Flow

“…Although increased shear rate stretches the bond and increases the contact area, this effect is not very large for the values of spring constant and shear rates used here. This agrees with the experimental finding that cell deformation and contact area do not change much at the shear rates used here, both for leukemia cells (56) and for iRBCs (27). The maximal bond extensions in our simulations are 80 and 140 nm for shear rates 42 and 142 Hz, respectively, corresponding to forces of 40 and 70 pN, respectively.…”

“…7) and because the resulting contact area is of the same order of magnitude as the experimentally measured ones (Fig. 8) (27,56), despite the fact that we do not model deformable cells, for which similar contact areas have been found in simulations (47). However, we also note that the peak force of 70 pN reported in our adhesive dynamics simulations already comes close to the range 150-250 pN, which has been reported for unfolding of immunoglobulin domains as they are also present in the PfEMP-1 binding partners ICAM-1 and CD36 (76).…”

“…3). By tracking not only translational, but also rotational velocity (55,56), which in our case can be easily realized by using a fluorescent stain for the parasite, we can compare better with theoretical predictions and conclude that schizont rolling might be just at the onset of stable rolling adhesion. The adhesive dynamics approach is computationally very efficient and allows us to assemble state diagrams representing a complete picture of spherical iRBC rolling adhesion (Fig.…”

“…7 shows that with these values, we get good agreement between our computer simulations and the experimentally measured values, suggesting that we have achieved a reasonable estimate for the molecular parameters of the schizont stage and that the used ranges for ligand distances and on-rates bracket the real values. We finally investigate the bond dynamics and the adhesive footprint predicted by the adhesive dynamics simulations for the schizonts, a question that in the future could be approached experimentally with fluorescent sensors (56). In Fig.…”

“…We therefore have conducted flow chamber experiments for iRBCs moving over endothelial monolayers, in which we track not only translational velocity, as commonly done for WBCs in flow chambers, but also make use of the presence of the parasite to track rotational velocity, which before has been done only for Janus particles (55) and leukemia cells with intracellular granular structures (56). Using this approach, we can demonstrate experimentally that cell movement in the trophozoite stage corresponds more to flipping than to rolling, as suggested before by computer simulations for deformable cells (31,53,54), and that smooth rolling emerges in the schizont stage.…”