Novel automated tracking analysis of particles subjected to shear flow: kindlin-3 role in B cells.

Shear flow assays are used to mimic the influence of physiological shear force in diverse situations such as leukocyte rolling and arrest on the vasculature, capture of nanoparticles, and bacterial adhesion. Analysis of such assays usually involves manual counting, is labor-intensive, and is subject to bias. We have developed the Leukotrack program that incorporates a novel (to our knowledge) segmentation routine capable of reliable detection of cells in phase contrast images. The program also automatically tracks rolling cells in addition to those that are more firmly attached and migrating in random directions. We demonstrate its use in the analysis of lymphocyte arrest mediated by one or more active conformations of the integrin LFA-1. Activation of LFA-1 is a multistep process that depends on several proteins including kindlin-3, the protein that is mutated in leukocyte adhesion deficiency-III patients. We find that the very first stage of LFA-1-mediated attaching is unable to proceed in the absence of kindlin-3. Our evidence indicates that kindlin-3-mediated high-affinity LFA-1 controls both the early transient integrin-dependent adhesions in addition to the final stable adhesions made under flow conditions.