Cytoskeletal events underlying dendrite formation by cultured pigment cells

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Abstract
In contrast to neurite outgrowth, pigment cell dendrite formation is relatively unstudied. Keratinocyte-conditioned medium (KCM) induces a striking dendricity in human melanocytes and B16 melanoma cells that is detectable within 30 min, maximal in 24–48 hr, and quantifiable by computerized image analysis. Cyto-chalasin B (CB), known to disrupt actin microfilaments, completely blocks dendrite formation if added to cultures before or with KCM. This effect is rapidly reversible, and dendrites appear within 1 hr after refeeding with KCM alone. In contrast, CB treatment fails to disrupt existing dendrites previously induced by KCM. Agents known to cause microtubule disassembly (colchicine, nocodazole, or vinblastine) do not inhibit dendrite formation if added before or with KCM. In contrast, these agents disrupt established dendrites Inhibition of protein synthesis with cycloheximide or actinomycin D completely blocks dendrite formation, but if cultures are provided fresh KCM lacking protein synthesis inhibitors, dendrites reappear within 24 hr. Actin microfilaments visualized with a monoclonal antibody or rhodamine-phalloidin are poorly organized in untreated cells, but form numerous fibers localized along dendrites in KCM-treated cells. Microtubules visualized with a monoclonal anti-tubulin antibody are localized in the center of dendrites. These cytoskeletal changes occur without altering β actin or β tubulin mRNA levels. Taken together, these data implicate actin microfilaments in dendrite outgrowth, but not in maintenance, and conversely microtubules in dendrite maintenance but not in formation. These keratinocyte-induced changes involving β actin and β tubulin polymerization appear to require both new protein synthesis and post-translational regulation. The observed similarities between melanocytes and other neural crest-derived cells suggest that cutaneous pigment cells might serve as an alternative model for studies of neurite outgrowth.