Until now, it has not been clear whether basal body and centriole formation occurred before or after clouds of pericentriolar material take shape. To answer that question, the researchers turned to Naegleria gruberi, which start life with amoeboid shapes but differentiate into swimming flagellates when food becomes scarce. During differentiation, the cells assemble basal bodies de novo, providing scientists with a window into the process.
The researchers knew that after the cells are transferred to a dilute buffer, γ-tubulin and pericentrin concentrate together in the cell. The percentage of cells with a concentrated region of γ-tubulin is maximal at 40 min, but no polymerized microtubules are visible. Basal bodies are assembled from the γ-tubulin concentration ∼60 min after initiation. This situation resembles what others have seen in animal cells during de novo centriole formation.Now, Kim et al. find that in vitro purified GPM from 40-min cells was competent to nucleate microtubules, but GPM from cells before or after this time was not. Phosphorylation of γ-tubulin correlated with the competency. When the team inhibited dephosphorylation of GPM in vivo, cells ended up with multiple pairs of flagella—suggesting that dephosphorylation of γ-tubulin is required to limit the number of new basal bodies.
The team doesn't yet know what regulates phosphorylation of γ-tubulin, but they are looking. They have also started using electron microscopy to study the GPM aggregates, which can reach 2 μm in diameter, to find out just what's hidden in the cloud.