STABLE INTERMEDIATES AND HOLDPOINTS IN THE RAPID DIFFERENTIATION OF NAEGLERIA

The rapidity of the optional 90-min differentiation of Naegleria grube ri from amoebae to flagellates suggests the possibility of a free-runn ing cascade of events from initiating stimulus through gene expression to organelle assembly and cell morphogenesis, Instead our experiments reveal two points early in the differentiation at which the strength of the inducing stimulus is reevaluated by the cells. Two new physical start signals for differentiation, temperature downshift (Delta T) an d mechanical agitation, are shown to regulate differentiation synergis tically with each other and with previously defined signals. A Delta T of -10 degrees C induces complete differentiation directly in the gro wth environment, whereas smaller Delta Ts initiate differentiation and allow it to progress for a short time, after which the cells ''hold'' for up to 4 h, awaiting a stimulus to continue differentiation. Our w ork defines two ''holdpoints,'' optional points in development where p rogress can stop, awaiting a suitable signal, while cells retain whate ver intermediates represent progress. We propose that such holdpoints, which can be detected in this system because of the temporal reproduc ibility of the differentiation, are likely to be found in other differ entiating cells. (C) 1998 Academic Press.