Mechanotransduction through LFA-1 controls upstream migration in the KG-1a stem cell line

ABSTRACT Immune cell recruitment into tissues requires cells to migrate on the endothelium while overcoming shear forces. Many extravasating cells, including T lymphocytes and hematopoietic stem cells, migrate upstream against the direction of shear flow on ICAM-1. Upstream migration has been shown to depend on intracellular signaling through Crk family adaptor proteins following engagement of lymphocyte function-associated antigen-1 (LFA-1). Because upstream migration is an important illustration of leukocyte mechanotransduction, we sought to identify key intracellular regulators of upstream migration using CRISPR/Cas9 gene editing and the KG-1a stem cell line. We made targeted deletions of molecules that function downstream of LFA-1 to regulate actin polymerization, link integrin signaling to the actin cytoskeleton, or form scaffolding complexes with Crk proteins. We found that CrkL, CasL (also known as HEF1 or NEDD9), L-plastin (LCP1) and RIAM (APBB1IP) are crucial for dynamic F-actin-rich lamellipodia and upstream migration. Although WASP (WAS), Hem1 (NCKAP1L), EVL and vinculin were found to be dispensable for upstream migration, pan-formin inhibition with SMIFH2 revealed a role for formins in upstream migration. This study illustrates the role of LFA-1 mechanotransduction in driving F-actin assembly at the leading edge to drive upstream migration.