Establishing the contact between kinetochores and microtubules

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In mitosis, at the onset of division, chromosomes condense and microtubules growing from centrosomes explore the cytoplasm. Microtubules grow and shrink interchangeably, until they capture the kinetochores (see 1.1.1.1., 1.1.1.2. and 1.1.2.1.). The process is commonly known as ‘search and capture’ (Kirschner and Mitchison, 1986). The capture of kinetochores by microtubules was first visualized in newt lung cultures by Hayden et al. (1990) and Rieder and Alexander (1990). It has been proposed that the initial kinetochore-microtubule contact involves a single astral microtubule and a single kinetochore. The microtubule often extends beyond the kinetochore. Therefore, in a commonly accepted model, the initial contact between kinetochore and microtubule is by lateral surface of the microtubule (Magidson et al., 2011; Shrestha and Draviam, 2013). The chance of the chromosome encounter by microtubules is additionally increased by the presence of high RanGTP concentration in the vicinity of the chromosomes stimulating microtubule growth toward the area of chromosomes (see 1.1.2.2.; O’Connell et al., 2009). Additionally, kinetochore-derived microtubules were shown to contact centrosome-derived microtubules.

The capture of the kinetochore by the microtubule was shown to lead to a rapid pole-ward movement of the chromosome along the microtubule (Fig.6.; Rieder and Alexander, 1990; Alexander and Rieder, 1991). This kinetochore-mediated movement depends on minus end-directed motor, dynein (see 1.1.1.; Sharp, et al., 2000; Yang et al., 2007). Translocation of chromosomes toward the pole increases the chance of kinetochore encounter in microtubule-reach area (Alexander and Rieder, 1991; Hayden et al., 1990). This process also is thought to promote end-on microtubule attachmet at the kinetochore. Attachment of a kinetochore to the facing pole results in exposing the unoccupied kinetochore to microtubules emanating from the opposite pole. In the conventional model, capture of the unattached kinetochore by a microtubule from the opposite pole produces chromosome biorientation (Kops et al., 2010). Subsequently, balancing of pulling forces exerted on sister kinetochores by microtubules emanating from opposite poles produces chromosome congression, a movement toward the spindle equator.

Nevertheless, few studies reported that congression does not require bipolar attachment (Cai et al., 2009, Kapoor et al., 2006). Kapoor et al. (2006) revealed the role of Cenp-E in congression of monooriented chromosomes. Cenp-E has been shown to play a role in translocation of chromosomes, attached to microtubules emanating from a single pole towards the spindle equator, using adjactent microtubules (Fig.6.). As shown by Kapoor et al. (2006), in the absence of functional Cenp-E, kinetochores are attached to microtubules; however, chromosomes frequently remain positioned close to the poles.

Cenp-E has also been proposed to stabilize kinetochore-microtubule attachment (Fig.6.; McEwen et al., 2001; Putkey et al., 2002). McEwen et al. (2001) showed that depletion of Cenp-E reduces the number of microtubules attached to kinetochores, which is particularly prominent on uncongressed chromosomes. Cenp-E also stabilizes kinetochore-microtubule attachments of congressed and bioriented chromosomes (Putkey et al., 2002). This function is related to contribution of Cenp-E to maintenance of kinetochore association with disassembling microtubule (Lombillo et al., 1995; Gudimchuk et al., 2013). Additionally, Cenp-E has been recently implicated in tethering a kinetochore to microtubule wall and cooperating with MCAK in conversion of lateral to end-on attachments (Shrestha and Draviam, 2013).

The number of microtubules producing end-on attachments with a single kinetochore differs between organisms and ranges from one in budding yeast to around 30 in humans (Maiato et al., 2004; McEwen et al., 1997). The difference in the number of kinetochore microtubules may result from the surface on the kinetochore and/or from turnover of kinetochore microtubules (Maiato et al., 2006).

Once microtubules capture kinetochores with their plus ends, the microtubule catastrophe level is reduced in order to increase the chance of stable attachment (see 1.1.1.1. and 1.1.1.2.; Maiato et al., 2004). The exact mechanism of regulation of microtubule dynamics by kinetochore to ensure stable kinetochore-microtubule interactions is, however, not known. Simultaneously, kinetochores retain dynamicity of bound microtubules to allow chromosome mobility. Kinetochore-led movement towards the poles is associated with depolymerisation of microtubules at plus ends and transfer of chromosomes towards the spindle centre is linked to microtubule polymerization (Skibbens et al., 1993). Ability of the kinetochore to switch between pole-ward and anti-pole-ward movement is called ‘directional instability’. Directional instability takes place without loss of kinetochore-microtubule contact.

Kinetochore-associated proteins as well as microtubule plus end binding proteins have activities modulating the dynamics of kinetochore microtubules (see 1.1.1.1.). In vertebrates, MCAK regulates plus end microtubule dynamics at kinetochores and between sister kinetochores by depolymerising microtubules (Howard and Hyman, 2003; Walczak, 2003). In Drosophila, kinesins Klp10A and Klp59C have role in depolymerising microtubules (Rogers et al., 2004). To the contrary, in Drosophila, Orbit/Mast has been shown to promote polymerisation of kinetochore microtubules (Maiato et al., 2005). Studies in mammalian cells showed that EB1 associates with kinetochores attached to polymerizing microtubules, suggesting that it stabilizes kinetochore microtubules (Tirnauer et al., 2002). In humans, kinesin Kif18A has been proposed to destabilize long microtubules attached to lagging kinetochores during chromosome oscillations thus promoting chromosome congression (Gardner et al., 2008).

Movement of chromosomes requires that the stable kinetochore-microtubule association is coupled to shortening of kinetochore microtubules (Joglekar et al., 2010). The mechanism of it is not yet known. In budding yeast, Dam1 complex has been identified as an adaptor structure enabling simultaneous stable capture of kinetochore by microtubule and depolymerisation of the microtubule (Westermann et al., 2006). Dam1 complex is a multimere, in vitro forming a ring around microtubules and interacting with microtubules and Ndc80 complex (Maure et al., 2011). However, no clear evidence for the presence Dam1 ring in vivo has been reported. As Dam1 complex has not been identified beyond budding yeast and in fission yeast it is not essential, Dam1 complex may be crucial only in organism with a single kinetochore microtubule. Human Ska complex has been proposed to be a functional counterpart of Dam1 complex (Welburn et al., 2009). Structural analysis of Ska complex showed that Ska1 does not form a ring-like structure, though it may use similar mechanisms adapted for multiple kinetochore-microtubule attachments (Jeyaprakash et al., 2012).