The XMAP215 Ortholog Alp14 Promotes Microtubule Nucleation in Fission Yeast

The organization and number of microtubules (MTs) in a cell depend on the proper regulation of MT nucleation. Currently, the mechanism of nucleation is the most poorly understood aspect of MT dynamics. XMAP215/chTOG/Alp14/Stu2 proteins are MT polymerases that stimulate MT polymerization at MT plus ends by binding and releasing tubulin dimers. Although these proteins also localize to MT organizing centers and have nucleating activity in vitro, it is not yet clear whether these proteins participate in MT nucleation in vivo. Here, we demonstrate that in the fission yeast Schizosaccharomyces pombe, the XMAP215 ortholog Alp14 is critical for efficient MT nucleation in vivo. In multiple assays, loss of Alp14 function led to reduced nucleation rate and numbers of interphase MT bundles. Conversely, activation of Alp14 led to increased nucleation frequency. Alp14 associated with Mto1 and γ-tubulin complex components, and artificially targeting Alp14 to the γ-tubulin ring complexes (γ-TuRCs) stimulated nucleation. In imaging individual nucleation events, we found that Alp14 transiently associated with a γ-tubulin particle shortly before the appearance of a new MT. The transforming acidic coiled-coil (TACC) ortholog Alp7 mediated the localization of Alp14 at nucleation sites but not plus ends, and was required for efficient nucleation but not for MT polymerization. Our findings provide the strongest evidence to date that Alp14 serves as a critical MT nucleation factor in vivo. We suggest a model in which Alp14 associates with the γ-tubulin complex in an Alp7-dependent manner to facilitate the assembly or stabilization of the nascent MT. •Alp14 (XMAP215) and Alp7 (TACC) promote microtubule nucleation in vivo•Alp14 interaction with Mto1 and the γ-tubulin complex is dependent on Alp7•Increasing the affinity of Alp14 for Mto1 increases nucleation frequency•Timed appearance of Alp14 at the nucleation site suggests a nucleation timeline Microtubules are dynamic polymers that help to organize cellular contents and divide the cell. New microtubules arise by a process of nucleation, in which tubulin subunits are stitched together to begin forming a hollow tube. Flor-Parra et al. identify XMAP215/Alp14 as a nucleation factor that facilitates the assembly of the microtubule.