NCAM Expression Regulates Integrin and Actin Cytoskeletal Function in Human Natural Killer Cells

Abstract

Human natural killer (NK) cells are innate immune cells that are broadly defined as being CD56+CD3−, however a functional role for CD56 (neural cell adhesion molecule, NCAM) on NK cells has not been described. NCAM is a member of the Ig superfamily that engages in homotypic and heterotypic interactions in cis and in trans. These include NCAM-NCAM interactions and binding to heparin sulfate proteoglycans. In neuronal cells, NCAM plays a role in neurite outgrowth, cell migration and adhesion, in part through direct interactions with focal adhesion kinase (FAK). We have previously shown that CD56 is required for human NK cell migration on stromal cells, and that deletion of CD56 in primary human NK cells significantly impairs this migration. We deleted CD56 by CRISPR-Cas9 mediated gene editing in a human NK cell line and measured the effect of CD56 deletion on adhesion and migration. Using super-resolution (STED) nanoscopy, we found that activation through NK cell activating receptors and/or αLβ2 integrin leads to an increase in the frequency of actin foci >0.05 um2 in the cortical actin of CD56-knockout (KO) NK cells. This was accompanied by increased colocalization of actin and vinculin in actin asters. In wild-type NK cells, we found that CD56 co-localized with β1 integrin in the uropod of migrating cells. Using live-cell interference reflection microscopy, we showed that while adhesion to integrin ligands was not impaired in CD56-KO NK cells, integrin detachment, specifically in the uropod of migrating cells was dysfunctional. Impaired phosphorylation of the FAK homologue Pyk2 in CD56-KO NK cells in response to activation and integrin ligation further supports a novel role for CD56 in regulating integrin detachment. In summary, we have defined NCAM/CD56 as a novel regulator of human immune cell migration.