In the 1960s my lab set out to use genetics to dissect cell differentiation. We chose as research organism a remarkable unicellular eukaryote that lives in freshwater, Naegleria gruberi, which can alternate between walking amoebae and swimming flagellates. When amoebae are transferred from their growth environment to a nutrient-free aqueous environment, they undergo synchronous differentiation to streamlined flagellates, a conversion completed within 60 to 100 minutes. We found that these differentiating cells undergo a yin-yang change in utilization of two eukaryotic motility systems. Amoebae walk using an actin-based motility system, which becomes latent in the flagellates, and the actin mRNA of amoebae is rapidly destroyed during differentiation. The flagellates swim using a tubulin-based motility system, but the tubulin that makes up the flagellar and basal body microtubules is entirely synthesized as a programmed event of differentiation. Evidence that this differentiation is regulated in part by changes in intracellular free calcium ions led us to calmodulin, the conserved calcium-binding protein that in all eukaryotes transduces the calcium signal to diverse effector proteins. Two calmodulins were found in Naegleria flagellates, and they are neatly segregated in the cells, with the major one (CaM-1) localized in the flagella and the other (CaM-2) in the flagellate cell body. During differentiation the mRNAs for the two calmodulins increase in abundance and then rapidly decrease concurrently with those for α- and β-tubulins (Fulton et al., 1995). We also have studied the formation of the centriolar 9-triplet basal bodies, and have shown that the two basal bodies of flagellates form in a two-step process by self-assembly of the first and then mentored assembly of the second basal body. In collaboration with Lillian Fritz-Laylin, we have shown that these two modes of centriole assembly are ancient and have been conserved through the evolution of eukaryotes.

Cell Differentiation, Centrioles, and Cell Death. Epigenetic regulation of gene expression in Naegleria