Identification of a cell cycle-dependent duplicating complex that assembles basal bodies de novo in Naegleria.

ABSTRACT

During the differentiation of the amoeba Naegleria pringsheimi into a flagellate, a transient complex containing γ-tubulin, pericentrin-like protein, and myosin II (GPM complex) is formed, and subsequently a pair of basal bodies is assembled from the complex. It is not understood, however, how a single GPM is formed nor how the capability to form this complex is acquired by individual cells. We hypothesized that the GPM is formed from a precursor complex and developed an antibody that recognizes Naegleria (Ng)-transacylase, a component of the precursor complex. Immunostaining of differentiating cells showed that Ng-transacylase is concentrated at a site in the amoeba and that γ-tubulin is transiently co-concentrated at the site, suggesting that the GPM is formed from a precursor, GPMp, which contains Ng-transacylase and is already present in the amoeba. Immunostaining of growing N. pringsheimi with Ng-transacylase antibody revealed the presence of one GPMp in interphase cells, but two GPMps in mitotic cells, suggesting that N. pringsheimi maintains one GPMp per cell by duplicating and segregating the complex according to its cell cycle. Our results demonstrate the existence of a cell cycle-dependent duplicating complex that provides a site for the de novo assembly of the next generation of basal bodies.