Protein kinases are required for embryonic neural crest cell galvanotaxis

Abstract

Embryonic quail neural crest cells migrate towards the negative pole of an imposed dc electric field as small as 7 mV/mm (0.4 mV per average cell length). The involvement of protein kinases in the mechanism utilized by these cells to detect and respond to such imposed fields was tested through the use of several kinase inhibitors. Evidence for the involvement of protein kinase C (PKC) included: (1) inhibition of the directed motility by 1 microM sphingosine that was reversed by the addition of the phorbol ester, PMA; (2) stimulation of a faster response to the imposed field by PMA; and (3) inhibition of the directed translocation by 5 microM H-7. However, another PKC inhibitor, staurosporine, did not inhibit the directed translocation (1 nM-1 microM). We also found evidence for the involvement of either cAMP- or cGMP-dependent protein kinase. The galvanotactic response was partially inhibited by the addition of 10 microM H-9 and the response was enhanced in the presence of the phosphodiesterase inhibitor, IBMX. However, the adenylate cyclase stimulant, forskolin, had no significant influence on the directed motility, although it reduced the average cell velocity. While these experiments suggest that cAMP- or cGMP-dependent protein kinase or PKC may be involved in the galvanotaxis response, two other protein kinases appeared not to be required. The myosin light chain kinase inhibitor, ML-7, had no effect on the directed motility in an imposed field, so myosin light chain kinase may not be required for galvanotaxis. Similarly, 5 microM W-7 had no significant effect on the directed translocation, suggesting that calmodulin-dependent protein kinase is not involved. Interestingly, the continuous activity of a protein kinase is apparently not required for the directed translocation response. The addition of the PKC and cAMP-dependent protein kinase inhibitor, H-7, after the cells had been exposed to the field for 1 hour, had no effect on the subsequent directed translocation. Thus, for these inhibitors to block the directed translocation, they must be present at the same time as the initial field application. This implies that an integral step in the cellular response mechanism for galvanotaxis involves the stimulation of a protein kinase whose effect is long lasting.

Effects of Direct Current Electric Fields on Cell Migration and Actin Filament Distribution in Bovine Vascular Endothelial Cells

“…Protein kinase C phosphorylates several cytoskeletal targets, but the precise mechanism by which it induces assembly and extension is unknown. However, inhibitors of protein kinase C do prevent neural crest cells from orienting in an electric field [34]. These inhibitors have no effect on continued directed migration if administered after the cells have already polarized, suggesting that protein kinase C is required for rearrangement of the locomotive machinery, but is not necessary for the continued activity of the machinery.…”

Cellular mechanisms of direct-current electric field effects: galvanotaxis and metastatic disease

“…Measurements of [Ca 2+ ]i in mouse embryo fibroblasts, using the Ca 2+ -sensitive photoprotein aequorin, show that application of a dcEF produces a significant overall [Ca 2+ ]i increase (up to mM), which is maintained throughout exposure to the field (Onuma and Hui, 1988). Ca 2+ channel blockers, such as Co 2+ or D600, or removal of extracellular Ca 2+ , can block the dcEF-induced rise in [Ca 2+ ]i (Onuma and Hui, 1988) and, in most cases, this inhibits galvanotaxis without influencing the basic ability of the cell to move (Nuccitelli et al, 1993). dcEF stimulation also more than doubles [Ca 2+ ]i in a rat osteoblast-like cell line (Wang et al, 1998).…”

“…These include those involving protein kinases such as protein kinase C (Nuccitelli et al, 1993), cGMP-dependent protein kinase and PKA, MLCK (Pullar et al, 2001), CaMKs (Zhuang et al, 1997) and mitogen-activated protein kinase (MAPK) (Zhao et al, 2002a;Zhao et al, 2002b;McBain et al, 2003). Growth factors and their receptors are also implicated, especially epidermal growth factor (EGF), fibroblast growth factor, transforming growth factor (TGF)-β1/α (Zhao et al, 1996;Zhao et al, 2002a), hepatocyte growth factor (McBain et al, 2003) and vascular endothelial growth factor (VEGF) (Zhao et al, 2004).…”

Relocation of a Ca 2+ -Dependent Protein Kinase Activity during Pollen Tube Reorientation ���

“…Experiments with neural crest cells (Nucitelli et al, 1993) have shown that orientation is modified by inhibitors of protein kinases such as H-7. We have also found that H-7 modifies pollen tube orientation.…”