JCB_201608122 1609..1622

To maintain genetic integrity, eukaryotic cells must segregate their chromosomes to opposite spindle poles during anaphase before the cell division. Chromosome segregation is mainly driven by microtubules (MTs) that attach to kinetochores (KTs). The KT–MT interaction is established in a stepwise manner during the early stage of mitosis (prometaphase; Tanaka, 2010; Cheerambathur and Desai, 2014): KTs are initially loaded on the lateral side of an MT extending from a spindle pole (a spindle MT; Rieder and Alexander, 1990; Tanaka et al., 2005) and then tethered to the MT end (Maure et al., 2011; Shrestha and Draviam, 2013). Subsequently, sister KTs interact with MTs from opposite spindle poles (biorientation) after aberrant KT– MT interactions are removed (Tanaka et al., 2002; Kitamura et al., 2007). A delay in any of these steps could compromise the efficiency in establishing a proper KT–MT interaction. In particular, the initial KT–MT encounter often becomes a ratelimiting step in this process. Several mechanisms are thought to facilitate the initial KT encounter with MTs extending from a spindle pole (Fig. S1 A): spindle MTs extend in various directions as if they are “searching” for KTs (search and capture; Kirschner and Mitchison, 1986). However, a random search-and-capture process does not completely explain the efficiency of the KT encounter with spindle MTs (Wollman et al., 2005). This is at least partly explained by the effect of the small GTPase Ran bound by GTP (Ran-GTP). Ran-GTP forms a concentration gradient around chromosomes, which “guides” the extension of a spindle MT toward a chromosome (Clarke and Zhang, 2008). These are thought to be long distance effects in the KT–MT encounter (Fig. S1 A). For example, a Ran-GTP gradient can only be formed over a long range (>5 μm) because of its rapid diffusion, and it cannot be formed in cells undergoing closed mitosis, e.g., budding yeast, whose nuclei contain a uniformly high Ran-GTP concentration (Carazo-Salas and Karsenti, 2003; Kaláb et al., 2006; Athale et al., 2008). Over a short distance (up to 2–3 μm), KT encounter with spindle MTs is facilitated by KT diffusion as well as MT angular diffusion (pivoting) in which KTs and MTs move randomly and vigorously (Fig. S1 A; Wollman et al., 2005; Kitamura et al., 2007; Brun, 2011; Kalinina et al., 2013). The extent of KT and MT diffusion is subject to temperature and other aspects of the extracellular environment (Kalinina et al., 2013). Meanwhile, MTs can be generated not only at spindle poles but also at KTs (Khodjakov et al., 2003; Maiato et al., 2004; Kitamura et al., 2010). Such KT-derived MTs, which are short in both length and lifetime, grow and shrink in various directions. KT-derived MTs could subsequently interact with a spindle MT, and this event may facilitate KT encounter with a spindle MT. Indeed, the appearance of KT-derived MTs is correlated with a more efficient KT encounter with spindle MTs (KT capture; Kitamura et al., 2010). So far, however, there has been no evidence that KT-derived MTs promote KT capture because it has not been possible to specifically remove KT-derived MTs. For example, The initial kinetochore (KT) encounter with a spindle microtubule (MT; KT capture) is one of the rate-limiting steps in establishing proper KT–MT interaction during mitosis. KT capture is facilitated by multiple factors, such as MT extension in various directions, KT diffusion, and MT pivoting. In addition, KTs generate short MTs, which subsequently interact with a spindle MT. KT-derived MTs may facilitate KT capture, but their contribution is elusive. In this study, we find that Stu1 recruits Stu2 to budding yeast KTs, which promotes MT generation there. By removing Stu2 specifically from KTs, we show that KT-derived MTs shorten the half-life of noncaptured KTs from 48–49 s to 28–34 s. Using computational simulation, we found that multiple factors facilitate KT capture redundantly or synergistically. In particular, KT-derived MTs play important roles both by making a significant contribution on their own and by synergistically enhancing the effects of KT diffusion and MT pivoting. Our study reveals fundamental mechanisms facilitating the initial KT encounter with spindle MTs. Molecular mechanisms facilitating the initial kinetochore encounter with spindle microtubules