The use of chlorotetracycline to localize calcium in neural crest cells

Cultured amphibian neural crest cells in the early phases of migration (1-2 days) extend broad, clear cytoplasmic processes known as lobopodia. These regions exhibit rapid changes in the contraction-relaxation cycle and in substratum adhesion. Because of the putative role of Ca2+ in both cytoskeletal function and cell attachment, its distribution in these cells was studied. Chlorotetracycline (CTC), the Ca2+-chelating fluorescent probe, was used to investigate whether there is a temporal and spatial localization of Ca2+ in cell regions concerned with movement and attachment. Differentiated neural crest cells (7 days), which contain an abundance of actin stress fibers, were also studied to determine whether these cytoskeletal elements were associated with Ca2+. The results with CTC indicate that in early migratory cells regions of membrane extension and protrusion contain higher levels of Ca2+ and that the actin stress fiber system of differentiating neural crest cells (7 days) is associated with Ca2+. It was determined that using the CTC in the fixative produced the same labeling profile as when live cells were labeled with the CTC. Use of CTC in the fixative is a decided advantage over treatment of cells with CTC while alive since it avoids the photooxidative damage which normally accompanies fluorescent observations of CTC on living cells.