| α2β1 integrin was identified as the primary mediator of A2058 cell response to collagen in vitro. Integrin ligation initiated a characteristic [Ca2+]i response consisting of an internal release and a receptor-mediated Ca2+ entry. Collagen-mediated Ca 2+ entry was additive to Thapsigargin-SOCR, suggesting an independent signaling mechanism. Similarly, ionophore application in a basal medium containing Ca2+ initiated a sustained influx. Elevated [Ca2+] i significantly attenuated cell migration to collagen by recruiting Ca2+/calcineurin-mediated signaling pathway. Furthermore, low [Ca2+]i induced by EGTA application in presence of ionophore fully restored cell motility to collagen. Taken together, these results suggest [Ca2+]i signaling mechanism accompanying A2058 cell response to α2β1 integrin ligation is neither necessary nor sufficient, and that elevated [Ca2+]i down-regulates cell motility via calcineurin-mediated mechanism in A2058 cell chemotaxis to collagen.; Using specific pharmacological inhibitors and a series of dominant-negative and constitutively active signaling proteins, it is shown that Ras and Rac GTPases, PI3-K, and PKC participate in cell migration mediated by β1 integrins. The specific MEK1/2 inhibitor U0126 enhances cell migration mediated by β1 integrin suggesting a potential feedback regulation mechanism. Collagen induces a time-dependent degradation of IκB-α and an increase in nuclear translocation of NF-κB which is dependent on PKC pathway. Moreover, collagen-stimulated melanoma migration directly correlated with an increase in NF-κB transactivation. Furthermore, collagen induced an increase in β1 integrin mRNA. Specific NF-κB inhibitors Helenalin and SN-50 inhibited melanoma migration to collagen, indicating a novel requirement for NF-κB transactivation in cell chemotaxis mediated by β1 integrin signals.; To study the dynamics of actin cytoskeleton rearrangement in living cells, an eukaryotic expression vector for β-actin-GFP fusion protein was generated. Expressed β-actin-GFP colocalized with endogenous cellular actin. β-actin-GFP also reorganized in response to treatments with collagen. Cells extended pseudopodial protrusions and altered their cortical structure in response to collagen stimulation. Furthermore, β-actin-GFP accumulated in areas undergoing these dynamic cytoskeleton changes, indicating that β-actin-GFP participates in actin polymerization. Findings suggest that this strategy provides an alternative to the invasive techniques currently used to study actin dynamics and permits real time visualization of actin rearrangements in response to environmental cues. |
