Chromosome-associated spindle microtubule nucleation

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Since the discovery of centrosomes, it had been long thought that centrosomes are indispensable for spindle formation. However, more recent studies have shown that mitotic spindle microtubules can also be generated independently of centrosomes. Laser ablation or microsurgical removal of centrosomes from mitotic animal cells did not greatly affect the structure of the spindle (Khodjakov et al., 2000; Hinchcliffe et al., 2001). Mutant flies without centrioles have no developmental abnormalities (Basto et al., 2006). Furthermore, acentriolar spindle formation has been observed in plant mitosis (Wadsworth and Khodjakov, 2004). Chromosomes are another site enabling initiation of spindle microtubule assembly. While in cells with centrosomes the relative contribution of centrosomes and chromosomes to nucleation of spindle microtubule is not clear, chromosomes are the only cue for spindle microtubule assembly in cells without centrosomes.

The ability of kinetochores to generate microtubules has been first demonstrated in studies with microtubule recovery from microtubule depolymerising drugs (Witt et al., 1980; De Brabander et al., 1981). Microtubule polymerization from kinetochores has also been observed in physiological conditions in mammalian and Drosophila cells (Khodjakov et al., 2003; Maiato et al., 2004). However, kinetochores are more likely to be a source of a short range signal promoting nucleation in their vicinity, rather than possess nucleation activity (Tulu et al., 2006). This is due to the fact that kinetochores are attached by the plus ends of microtubules, which would be contradictory to nucleating from minus ends (see 1.1.1.1.). Close vicinity of nucleation sites to kinetochores ensures easy capture of microtubules by the kinetochore. Nevertheless, nucleation directly from a kinetochore cannot be excluded, given that γ-TuRC components were found in association with kinetochore components, together promoting microtubule formation (Mishra et al., 2010). In either case, following microtubule plus end polymerization promoted by the kinetochore (see 1.1.3.3.) accounts for kinetochore-mediated assembly of kinetochore fibers (Maiato et al., 2004; Mishra et al., 2010).

In cultured cells, microtubules growing from chromosomes were observed primarily close to kinetochores. Nevertheless, the bulk chromatin has also been shown to participate in spindle microtubule assembly. Introduction of beads covered with random DNA into Xenopus egg extracts in the absence of centrosomes induced spindle formation (Heald et al., 1996). As the chromatin was lacking centromeric region and thus was unable to assemble the kinetochore (see 1.1.3.1.), this experiment suggested that chromosome arms may largely contribute to the spindle formation.

In vitro studies in Xenopus egg extract showed the role of RanGTP gradient and the chromosome passenger complex (CPC) in the chromatin-mediated spindle microtubule assembly (Kahana and Cleveland, 1999; Kelly et al., 2007). These pathways operate by regulating multiple spindle assembly factors (SAFs), including those stabilizing and de-stabilizing microtubules around chromosomes (Kalab and Heald, 2008; Meunier and Vernos, 2012).

RanGTP gradient is the most well characterized pathway of chromosome-induced spindle assembly (Kahana and Cleveland, 1999; Kalab and Heald, 2008). It is based on Ran protein existing in two forms, RanGTP and RanGDP. The guanine nucleotide exchange factor (Ran-GEF), RCC1, is a protein bound to chromatin where it exchanges GDP into GTP-bound state of Ran in the vicinity of chromatin. Opposite exchange is propelled by GTPase activating protein (GAP) in the cytoplasm. Originally, RanGTP/GDP exchange by RCC1 has been linked to release of importin cargos during nuclear trafficking in interphase (Pamberton and Paschal, 2005). It has been proposed that after nuclear envelope breakdown (NEB) at mitosis onset, a diffusion-limited gradient of RanGTP/GDP is generated (Fig.4.; Kalab and Heald, 2008). High concentration of RanGTP locally activates SAFs, by outcompeting them from importin (Gruss et al., 2001; Nachury et al., 2001; Wiese et al., 2001). In turn, local concentration of RanGTP-activated SAFs, such as TPX2, HURP, NuMa or Nup107-160 promotes spindle microtubule nucleation and assembly (Gruss et al., 2002; Schatz et al., 2003; Nachury et al., 2001; Koffa et al., 2006; Sillje et al., 2006; Mishra et al., 2010). RanGTP gradient steeply decreases away from chromosomes, however, the pathway activates a wider downstream gradient of factors stimulating spindle assembly and stabilizing microtubules growing towards chromosomes (Caudron et al., 2005; Dogterom et al., 1996, Carazo-Salas and Karsenti, 2003). Importance of RanGTP has been demonstrated when manipulated Ran levels differently modulated formation of microtubule asters in Xenopus egg extract (Kahana and Cleveland, 1999). Additionally, manipulation of RanGTP level in mammalian cells and in syncytial Drosophila embryos leads to abnormal spindle assembly (Kalab et al., 2006; Silverman-Gavrila and Wilde, 2006).

The second proposed pathway of chromosome-induced spindle assembly is dependent on CPC, Chomosome Passenger Complex (Sampath et al., 2004). The complex is composed of Aurora B kinase, Incenp, Borealin and Survivin/Deterin (Carmena et al., 2012). The CPC localizes to chromosomes and, is enriched at the centromeric region between sister chromatids (see 1.1.3.5.). In Xenopus egg extract, interaction of CPC with chromosomes activates Aurora B, which is then targeted to microtubules via Incenp (Kelly et al., 2007; Tseng et al., 2010). Interaction of CPC with chromosomes and microtubules in confined space has been proposed to promote local spindle assembly (Tseng et al., 2010). Aurora B, has been shown to stimulate microtubule assembly in the vicinity of chromosomes in Xenopus egg extracts by locally inhibiting factors promoting microtubule depolymerization, like MCAK (mitotic centromere-associated kinesin; see 1.1.3.5.) and Op18/Stathmin (Andrews et al., 2004; Lan et al., 2004; Sampath et al., 2004; Gadea and Ruderman, 2006; Kelly et al., 2007).

It is widely agreed that CPC and RanGTP pathways function independently but jointly in spindle assembly (Sampath et al, 2004; Kelly et al., 2007; Kalab and Heald, 2008). While RanGTP pathway is known to account for spindle assembly from the bulk chromatin as well as from kinetochores, localization of CPC function is unclear (Heald et al., 1996; Torosantucci et al., 2008). Sampath et al. (2004) proposed the role of CPC in the process is dependent on the bulk chromatin. To the contrary, Maresca et al. (2009) postulated that CPC activity in spindle formation is generated from the centromeric region and is required for spindle assembly in the absence of RanGTP gradient.