Isolation of αL I domain mutants mediating firm cell adhesion using a novel flow-based sorting method

Abstract

The inserted (I) domain of αLβ2 integrin (LFA-1) contains the entire binding site of the molecule. It mediates both rolling and firm adhesion of leukocytes at sites of inflammation depending on the activation state of the integrin. The affinity change of the entire integrin can be mimicked by the I domain alone through mutations that affect the conformation of the molecule. High-affinity mutants of the I domain have been discovered previously using both rational design and directed evolution. We have found that binding affinity fails to dictate the behavior of I domain adhesion under shear flow. In order to better understand I domain adhesion, we have developed a novel panning method to separate yeast expressing a library of I domain variants on the surface by adhesion under flow. Using conditions analogous to those experienced by cells interacting with the post-capillary vascular endothelium, we have identified mutations supporting firm adhesion that are not found using typical directed evolution techniques that select for tight binding to soluble ligands. Mutants isolated using this method do not cluster with those found by sorting with soluble ligand. Furthermore, these mutants mediate shear-driven cell rolling dynamics decorrelated from binding affinity, as previously observed for I domains bearing engineered disulfide bridges to stabilize activated conformational states. Characterization of these mutants supports a greater understanding of the structure-function relationship of the αL I domain, and of the relationship between applied force and bioadhesion in a broader context.

Keywords: I domain; conformational regulation; rolling adhesion; yeast display; αL integrin.

Fig. 1.

Sorting of mock libraries by rolling adhesion. Mock libraries were created using an eGFP-expressing yeast strain surface displaying the HA I domain variant and nonfluorescent yeast expressing the wild-type I domain variant were mixed at ratios of ∼50 : 50 and 20 : 80 (HA : WT). The resulting populations, seen on the right, after one round of sorting for the firmly adherent phenotype (a), and rolling phenotype (b) using the flow-based adhesion assay in the rolling chamber.

Fig. 2.

Sorting I domain library. The I domain library, generated using error prone PCR (a) pre-sort, (b) after one round of FACS, and (c) after three rounds of sorting using rolling adhesion. Yeast were concomitantly labeled with antibodies against the c-myc epitope tag (x-axis) and ICAM-1 (y-axis).

Fig. 3.

Firmly adherent I domain mutants. Mutations found by sorting a library by rolling adhesion are found throughout the molecule, as pictured here on the two faces of I domain molecule. Black labels with an asterisk indicate mutations that were found in both the rolling and FACS sorting method, and gray labels indicate the location of novel mutations found only in the rolling adhesion method.

Fig. 4.

Relationship between rolling velocity and adhesive phenotype of I domain mutants. Video of microscopy data was analyzed using automated particle tracking to calculate rolling velocity of individual yeast cells. Yeast moving at a rate less than 0.1% of hydrodynamic velocity were characterized as firmly adherent, and overall average rolling velocity was calculated by the average of at least 30 individual yeast.