Studying the role of Arp2/3 complex in cell migration

The actin cytoskeleton plays important roles in maintaining cell shape and mediating cell motility. Deregulation of actin dynamics occurs during pathological processes such as cancer metastasis, regeneration defects and developmental diseases. One specific form of actin cytoskeleton, the highly dynamic branched actin network, is critical for the formation and regulation of cell cortex and protrusions during cell migration and matrix degradation. The key nucleator for this branched actin network is the seven-protein Arp2/3 complex.;Functional studies of Arp2/3 in vivo have been severely hampered by effects on viability observed upon loss of this complex in a variety of organisms. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner.;Arp2/3-branched actin is critical for cell morphology and migration. However, perturbations and diseases affecting this network have phenotypes that cannot be fully explained by cell-autonomous effects. Using this stable Arp2/3 knockdown cell line, we also report the non-autonomous effects upon Arp2/3 depletion. We show that the main class of genes with altered expression levels was genes encoding secreted factors including chemokines, growth factors and matrix metaloproteases resembling the senescence associated secretory phenotype (SASP). These factors affect EGF chemotaxis in a non-autonomous way, resolving the recent contradictions about the role of Arp2/3 in chemotaxis. We indicate that these genes are targeted by NF-kappaB, via a CCM2-MEKK3 pathway that has been implicated in osmotic stress signaling. Thus, perturbations of Arp2/3 have potential non-autonomous effects which should be considered when evaluating diseases affecting the Arp2/3-actin cytoskeleton.;Our work has provided strong evidence that Arp2/3 complex is critical for lamellipodia and revealed the roles of Arp2/3-branched actin in cell-matrix interaction. We have also indentified and characterized novel non-cell-autonomous effects of perturbing the branched actin network which has potential clinical relevance.