Naegleria's mitotic spindles are built from unique tubulins and associated microtubule binding proteins

Naegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis. We and others have shown that Naegleria amoebae express two α- and two β-tubulins during mitosis. These mitotic tubulins are evolutionarily divergent relative to typical α- and β-tubulins, contain residues that suggest distinct microtubule properties, and may represent drug targets for the “brain-eating amoeba” Naegleria fowleri. As a stress response, Naegleria can exit the cell cycle and differentiate to a secondary, flagellated cell type. This differentiation involves assembly of flagella and cytoplasmic microtubule arrays that are assembled from distinct and conserved flagellar tubulins. Naegleria, therefore, provides a unique opportunity to study the evolution and functional specificity of tubulins and microtubule structures. While its flagella closely resemble those of other eukaryotic species, Naegleria's mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles that twists from end-to-end. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers, and secondary bundles function as bridging fibers. To test this, and other hypotheses regarding Naegleria spindle dynamics, we have optimized a previously developed mitotic synchrony protocol and have collected and analyzed RNA from various cell cycle time points. These data have revealed a mitotic microtubule protein network that is distinct from that used during assembly of flagellar microtubule arrays, highlighting the contrast between Naegleria's divergent microtubule spindle and its highly conserved flagellar microtubule arrays.