Ensconsin promotes Mt growth in cells and in vitro. (A) Ensconsin (Ensc) overproduction in mitotic S2 cells led to the formation of longer spindles. Ensconsin-GFP is green (gray in the panels on the right) and Mts are shown in red (gray in the middle panels). Exogenous Ensconsin-GFP is localized on the mitotic spindle during mitosis. Bar, 10 µm. (B) Analysis of mitotic spindle length in WT (n = 56) and Ensconsin-GFP–overexpressing mitotic S2 cells (n = 26). ***, P = 5.1 × 10⁻¹¹. (C) Recombinant Ensconsin purified, separated by SDS-PAGE, and stained with Coomassie blue. (D) Effect of Ensconsin on Mt polymerization in vitro. Purified porcine brain tubulin was mixed with polymerization buffer supplemented with either PBS or PBS containing Ensconsin on ice. Mt polymerization was induced by heating at 35°C. Absorbance at 350 nm was monitored to evaluate the formation of Mt polymers. After 28 min, the samples were shifted to 0°C to estimate protein aggregation. Ensconsin increased the rate of Mt polymerization and the total number of polymers in a dose-dependent manner (red curves), as shown by comparison with buffer alone (black curve).