Microtubule structure and dynamics

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The basic functional unit of the spindle is the microtubule. Microtubule is a hollow tube composed of the polymerized αβ-tubulin heterodimers (Fig.1.; Nogales 2000). The αβ-tubulin heterodimers polymerize in a head-to-tail manner and thus they form a linear structure, called the protofilament. In vivo, 13 protofilaments associate side-by-side and adopt a cylindrical 25 nm diameter structure of the microtubule (Evans et al., 1985; Desai and Mitchison, 1998). Adjacent protofilaments contact each other laterally through α-α and β-β interactions, except in one pair of protofilaments where the interactions are between α- and β-tubulin subunits (Mandelkow et al., 1986; Kikkawa et al., 1994). This difference in the contact mode of protofilaments leads to discontinuity of the microtubule lattice, called the seam.

Head-to-tail association of the αβ-tubulin heterodimers determines the polarity of the microtubule and affects polymerization kinetics on both ends of the microtubule. Polymerization occurs fast, at the side of β-tubulin, and this end of microtubule is called the plus-end (Mitchison et al., 1993). Plus end can also de-polymerize (Mitchison and Kirschner, 1984). The minus end, which exposes α-tubulin, is less dynamic (Fan et al., 1996). In vitro minus ends can grow at a slow rate, however, in vivo they are usually stabilized or they depolymerise (Jiang and Akhmanova, 2011).

Growth of microtubule occurs by adding GTP-bound tubulin dimers to the microtubule lattice. Addition of new dimers is accompanied by hydrolysis of GTP to GDP on β-tubulin, to which the dimer is added (David- Pfeuty et al., 1977; Howard and Hyman, 2003). A layer of the last added GTP-tubulin forms a GTP-cap at the end of microtubule, which stabilizes microtubule and promotes polymerization (Drechsel and Kirschner, 1994). If the exposed GTP in microtubule undergoes hydrolysis to GDP, tubulin changes its conformation, which results in peeling of protofilaments at the microtubule tips (Mandelkow et al., 1991; Wang and Nogales, 2005). This destabilizes microtubule structure and leads to microtubule depolymerization. Shift between GTP/GDP-bound tubulin dimers at the microtubule is the basis of microtubule dynamic instability, a term introduced by Mitchison and Kirschner (1984). Switch from growing to shortening state of microtubules is called the catastrophe and switch from shortening to growing state is called the rescue (Desai and Mitchison, 1997).

Reorganization of the interphase microtubule network into mitotic spindle is associated with destabilization of microtubules and microtubule re-formation. Transition between interphase and metaphase involves shift of microtubule dynamics towards catastrophes and reduction of rescue events (Rusan et al., 2001). Next, highly dynamic spindle microtubules are nucleated by centrosomes and their plus ends switch between phases of growth and shrinkage before they disassemble or become selectively stabilized (see 1.1.2.1.; Kirschner and Mitchison, 1986; Mitchison and Kirschner, 1985; Hayden et al., 1990). Further, the spindle assembles owing to intrinsic microtubule dynamics but also to regulators of microtubule dynamics, which belong to microtubule associated proteins (MAPs) (Wittmann et al., 2001; Akhmanova and Steinmetz, 2008). Regulation of microtubule dynamics by MAPs can occur via different ways. They can increase or decrease rescue and catastrophe frequencies, as well as reduce or accelerate polymerisation or de-polymerization rates. MAPs can also promote or inhibit nucleation of microtubules, sequester free tubulin or severe microtubules.

MAPs accumulating and functioning at the plus ends of microtubules, the plus end tracking proteins (+TIPS), have a particular contribution to microtubule dynamics (Howard and Hyman, 2007; Akhmanova and Steinmetz, 2008). EB1 protein is a highly conserved protein, regarded as the main regulator of microtubule growth (Tirnauer and Bierrer, 2000; Morrison, 2007). It is present at all growing ends of microtubules, thus it is considered a marker of dynamic ends of microtubules. EB1 interacts directly with growing microtubules and has been proposed to recruit many cargoes to microtubule plus ends, which then promote polymerization or depolymerization of microtubules (Akhmanova and Steinmetz, 2008; Jiang et al., 2012). Among many recruited MAPs, XMAP215 acts as microtubule polymerase in Xenopus (Brouhard et al., 2008). In Xenopus, it can also promote microtubule shrinkage (Shirasu-Hiza et al., 2003). Microtubule dynamics is reduced after depletion of XMAP215 homologue in Drosophila, Msps (Brittle and Ohkura, 2005). Kinesin 13 family members promote microtubule catastrophes and CLASPs, CLIP (cytoplasmic linker protein)-associated proteins decrease catastrophe rate and increase microtubule rescue frequency (Desai et al., 1999; Moore and Wordeman, 2004; Al-Bassam et al., 2010).

Genome-wide RNAi screen in Drosophila cells led to identification of Sentin, a plus end EB1 cargo protein (Li et al., 2011). Sentin depletion phenocopies EB1 and Msps depletion, as it produces shorter spindle and, similarly to EB1 and Msps, Sentin promotes dynamics of microtubule (Li et al., 2011; Rogers et al., 2002; Goshima et al., 2005). Furthermore, fusion of Sentin with EB1 lacking domain responsible for binding its cargoes was shown to rescue EB1 phenotype (Li et al., 2011). Thus, Sentin has been proposed to be the dominant EB1 cargo in promoting microtubule dynamics. Additionally, Sentin is responsible for accumulation of Msps to the ends of growing microtubules. However, Sentin recruited by EB1, independently of Msps, stimulates microtubule growth and promotes catastrophes in Drosophila (Li et al., 2012). It has been proposed that the combined activities of Msps and Sentin, both recruited by EB1, are required for generation of dynamic microtubules in cells.