The non-receptor tyrosine kinase Lyn controls neutrophil adhesion by recruiting the CrkL–C3G complex and activating Rap1 at the leading edge

Abstract

Research Article 2153 IntroductionCell migration has been conceptualized as a cycle (Ridley et al., 2003). After a stimulus, cells polymerize actin at the front to initiate protrusions. These protrusions are stabilized by adhering to extracellular matrix (ECM) molecules (adhesion). Adhesions serve as traction sites as the cell moves forward, and are disassembled at the rear, allowing the cell to detach and retract. In general, strategies for single-cell migration can be classified into two patterns: amoeboid movement and mesenchymal migration (Friedl, 2004). Slow-moving mesenchymal cells (e.g. fibroblasts) often adopt a spindle-shaped morphology and adhere via large integrin-mediated plaque-like structures called focal adhesions. By contrast, fastmoving cells (e.g. neutrophils) use amoeboid movement associated with fast deformability and highly polarized morphology, and move more rapidly. Unlike the mesenchymal migration paradigm, large focal adhesions seen in slow moving fibroblasts and stress fibers are not detected in these cells (Friedl, 2004), suggesting fundamental differences between amoeboid and mesenchymal cell adhesions; however, integrin-mediated adhesion is necessary for persistent cell migration (Ley et al., 2007).When exposed to chemoattractants, neutrophils polarize and initiate polymerization of actin at the leading edge (pseudopod) to provide the protrusive force. Primary neutrophils and neutrophillike cells have complex signaling pathways that control formation of their front and back. A 'frontness' pathway containing Rac, the lipid products of phosphoinositide-3-kinases (PI3Ks) and polymerized actin organizes the leading edge of neutrophils, whereas the 'backness' signals, including Rho, p160-ROCK and myosin II, lead to contraction of actin-myosin complexes at the trailing edge, causing cells to de-adhere (Srinivasan et al., 2003;Wang et al., 2002;Xu et al., 2003).Despite these advances, little is known about the regulation of cell-ECM adhesion during chemotaxis. Frontness and backness responses per se do not entirely account for the persistent polarity and motility of neutrophils when they are exposed to chemoattractants. Indeed, chemoattractant stimulation of neutrophils in suspension (i.e. without cell adhesion to the substrate) only causes transient membrane extension, in sharp contrast to cells stimulated on substrates, which polarize and migrate persistently (Srinivasan et al., 2003;Wang et al., 2002;Xu et al., 2003). In circulating neutrophils 2 integrins (CD18) are highly expressed and mediate the interactions of neutrophils with endothelial cells. The importance of 2-integrin-mediated adhesion in neutrophil chemotaxis is highlighted by patients with leukocyte adhesion deficiency (LAD), who have decreased numbers of neutrophils at the sites of infection and inflammation (Bunting et al., 2002). These findings imply a crucial role of cell-substrate adhesion in the regulation of neutrophil polarity and motility.To dissect the molecular program that governs adhesion of the neutrophil lea...

Radil controls neutrophil adhesion and motility through β2-integrin activation

“…Although it has been reported that active β2-integrins localize to the leading edge of differentiated HL-60 cells (He et al ., 2011), consistent with earlier findings (Pierini et al ., 2000), we found that active β2-integrins appear in punctates that do not colocalize with F-actin and are generally enriched in the back of polarized primary human blood neutrophils and differentiated PLB-985 cells. Of importance, polarized PLB-985 cells overexpressing Radil show a significant increase in fluorescent signal of active β2-integrins at their back.…”

“…Rap1a-deficient neutrophils have been generated (He et al ., 2011) and, much like what we observed with our Radil-knockdown cells, shown to exhibit decreased adhesion on fibrinogen-coated plates. Of interest, these authors observed that Rap1a-depleted neutrophils retract slowly and show spindle-shaped morphology during chemotaxis.…”

“…In chemotaxing neutrophils, we show that the active Rap1-binding partner Radil is uniformly distributed on the plasma membrane, suggesting that active Rap1a is also uniformly distributed in these cells. Indeed, it was recently shown that the eGFP-RalGDS, a Rap1-GTP reporter (Wang et al ., 2006), is mostly uniformly distributed in differentiated HL-60 cells (He et al ., 2011). Yet we found that active β2-integrins are generally localized in the cell body and tail of polarized neutrophils.…”

Moving towards a paradigm: common mechanisms of chemotactic signaling in Dictyostelium and mammalian leukocytes

“…How the signal is transmitted from the G protein to Rap1 is not known, although several candidates have emerged. In HL-60 cells, fMLP activates a non-RTK Lyn in a G i protein-dependent manner [230]. Lyn recruits and activates the adaptor protein CrkL, which constitutively associates with a Rap1 GEF C3G.…”

Immunophilins Control T Lymphocyte Adhesion and Migration by Regulating CrkII Binding to C3G

“…The Crk-SH3N, which mediates C3G binding, plays an important role in this process, because expression of a SH3N-defective mutant inhibited cell adhesion to fibronectin (44)(45)(46). In addition, both Crk and C3G function as focal adhesion proteins and Crk-C3G complexes recruit to the leading edge of polarized cells, where they activate additional effector molecules and provide spatially instructive cues required for cell adhesion and migration (47)(48)(49)(50).…”