Naegleria amoebae employ an actin-based cytoskeletal system, while flagellates use actin and microtubules. (A) This diagram illustrates the evolutionary relationships between Naegleria (top) and other eukaryotes (figure modified from Velle and Fritz-Laylin, 2019). SAR, Stramenopiles, Alveolates, and Rhizaria. (B) A representative amoeba (top) from a growing population and a flagellate (bottom) from a population of differentiated cells were fixed and stained with phalloidin to detect F-actin (green), Tubulin Tracker to visualize tubulin (magenta, γ adjustment of 0.7), and DAPI to label DNA (blue). (C) Changes in actin and microtubule cytoskeletal transcript abundance were calculated using gene expression data collected from amoeba or flagellate populations (also see Fig. S1 A and original data from Fritz-Laylin and Cande, 2010). Positive values indicate higher transcript abundance in amoebae; negative values indicate higher abundance in flagellates (AU = arbitrary units). (D) A subset of data from C highlights the expression pattern of actin assembly factors (also see Fig. S2 B). For graphs in C and D, each bar represents the average relative change in transcript abundance between amoebae and flagellates from three experiments ± SD.